Clinical trial NCT04228445
Conditions: Ureter Injury
Sponsor: Prove pharm
Start date: 2020-02-13; Completion date: 2021-06-03
Enrollment: 121 participants; Allocation: RANDOMIZED; Intervention model: SEQUENTIAL

Brief summary:
To determine whether the use of 3,3'-Dioxo-2,2'-Bisindolylidene-5,5'-Disulfonate Disodium 0.8% Solution for injection provides a visualization advantage compared to saline when used as an aid in the determination of ureteral patency

Detailed description:
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 3,3'-Dioxo-2,2'-bisindolylidene-5,5'-disulfonate disodium 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.

Arms: arm 1: 48 subjects randomly treated with 5 mL of drug arm 2: 48 subjects randomly treated with 2.5 mL of drug arm 3: 96 subjects treated with 5 ml of saline than crossover to treatment arm
Interventions: intervention 1: 3,3'-Dioxo-2,2'-bisindolylidene-5,5'-disulfonate disodium 0.8% intervention 2: Saline spray
Intervention descriptions: intervention 1: Experimental contrast dye that is commonly used as a visualization aid in pelvic and abdominal surgeries and for various diagnostic procedures in medical practice. intervention 2: Placebo

Protocol documents:
SAP_006.pdf: 
STATISTICAL ANALYSIS PLAN 
Protocol Number: 
PVP-19IC01 
Protocol Title:  
 
An Open-Label, Randomized, Multicenter 
Study to Evaluate the Safety, Efficacy, 
Pharmacokinetics and Physician Satisfaction 
of Two Different Doses of 3,3'-Dioxo-2,2'-
bisindolylidene-5,5'-disulfonate disodium 
0.8% Solution When Used as an Aid in the 
Determination of Ureteral Patency 
Date of Original Protocol 
01 July 2019    
Administrative Change 1 Date: 18 October 2019 
Amendment 1 
10 December 2019 
Amendment 2 
02 September 2019 
Date of Original SAP: 
11 September 2019 
SAP Amendment 1 Date: 
02 September 2020 
SAP Amendment 2 Date: 
26 June 2021 
NCT#: 04228445




 
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TABLE OF CONTENTS 
SIGNATURES .................................................................................................................................2 
TABLE OF CONTENTS .................................................................................................................3 
IN-TEXT TABLES ..........................................................................................................................5 
LIST OF FIGURES .........................................................................................................................6 
LIST OF ABBREVIATIONS AND PHRASES .............................................................................7 
1. 
INTRODUCTION AND SCOPE .................................................................................9 
2. 
OVERVIEW OF STUDY OBJECTIVES AND ASSESSMENTS............................10 
2.1. 
Study Endpoints ..........................................................................................................14 
2.1.1. 
Efficacy Endpoints ......................................................................................................14 
2.1.2. 
Safety Endpoints .........................................................................................................14 
3. 
GENERAL CONSIDERATIONS ..............................................................................16 
3.1. 
Sample Size Determination ........................................................................................16 
3.2. 
Interim Analysis ..........................................................................................................17 
3.3. 
Analysis Population ....................................................................................................20 
3.4. 
Test Hypothesis and P Value Justification .................................................................20 
3.5. 
Procedures for Handling Missing Data .......................................................................21 
3.6. 
Analysis Center ...........................................................................................................21 
3.7. 
Treatment Group vs Randomization Group ...............................................................21 
3.8. 
Definitions and Derived Variables .............................................................................22 
3.8.1. 
Study Period Definitions.............................................................................................22 
3.8.2. 
IC Treatment Responders ...........................................................................................23 
3.8.3. 
Time to Visualization (TTV) of Blue Color in the Ureteral Jets After IC 
Administration ............................................................................................................23 
3.8.4. 
Physician Satisfaction Agreement Scale ....................................................................25 
3.8.5. 
Concordance in Conspicuity Assessments between the Surgeons and the 
Blinded Central Readers .............................................................................................25 
4. 
STUDY POPULATION SUMMARIES ....................................................................27 


 
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4.1. 
Disposition ..................................................................................................................27 
4.2. 
Demographics and Baseline Characteristics ...............................................................28 
4.3. 
Medical History ..........................................................................................................28 
4.4. 
Protocol Deviations ....................................................................................................28 
4.5. 
Treatment Compliance ................................................................................................29 
4.6. 
Prior and Concomitant Medications ...........................................................................29 
5. 
EFFICACY ANALYSIS ............................................................................................30 
5.1. 
Conspicuity Score from the Central Readers ..............................................................30 
5.1.1. 
Missing Data Handling Procedure ..............................................................................32 
5.2. 
PSAS with IC Treatment ............................................................................................33 
5.3. 
IC Treatment Responders ...........................................................................................34 
5.4. 
Time to Visualization .................................................................................................35 
5.5. 
Concordance Assessments ..........................................................................................37 
5.5.1. 
Treatment Effect on Conspicuity per Surgeons Review .............................................37 
5.5.2. 
Covariate Analysis ......................................................................................................37 
5.5.3. 
Dichotomization of the Difference in Conspicuity .....................................................38 
5.6. 
Covariate Analysis and Subgroup Analysis ...............................................................39 
6. 
PHARMACOKINETICS EVALUATIONS ..............................................................40 
7. 
SAFETY AND TOLERABILITY EVALUATIONS ................................................41 
7.1. 
Total Exposure ............................................................................................................41 
7.2. 
Adverse Events ...........................................................................................................41 
7.3. 
Changes in Laboratory Tests ......................................................................................42 
7.4. 
Changes in Vital Signs................................................................................................42 
7.5. 
Changes in 12-Lead ECG ...........................................................................................43 
8. 
TABLE OF CONTENTS OF PLANNED DATA SUMMARIES .............................44 
8.1. 
Summary Tables .........................................................................................................44 
8.2. 
Figures ........................................................................................................................46 
8.3. 
Data Listings ...............................................................................................................46 
 


 
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IN-TEXT TABLES 
 
Table 1:  Schedule of Assessments ................................................................................................13 
Table 2:  Distribution of Each Conspicuity Score and IC Treatment Effect Size .........................17 
Table 3:  Interim Analysis Conditional Power and Decision Principle .........................................19 
 
 
 


 
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LIST OF FIGURES 
Figure 1:  Study Scheme ................................................................................................................23 
 
 


 
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LIST OF ABBREVIATIONS AND PHRASES 
Abbreviation 
Definition 
AE 
adverse event 
AEOSI 
Adverse event of special interest 
ALP 
Alkaline Phosphatase 
ALT 
Alanine Aminotransferase 
AST 
Aspartate Aminotransferase 
ATC 
Anatomical Therapeutic Chemical 
BMI 
body mass index 
CMH 
Cochran-Mantel-Haenszel  
CP 
Conditional Power 
CV 
Coefficient of Variation 
DMC 
Data Monitoring Committee 
ECG 
Electrocardiogram 
eCRF 
Electronic case report form 
ES 
Effect Size 
GEE 
Generalized Estimating Equation 
IC 
Indigo Carmine 
ICF 
Informed Consent Form 
ICH 
International Conference on Harmonization 
IM 
Intramuscular 
IVRS/IWRS 
Interactive Voice Response System/Interactive Web Response System 
ITT 
Intent to Treat  
IV 
Intravenous 
Kg 
Kilogram 
m2 
Meters Squared 
MedDRA 
Medical Dictionary for Regulatory Activities 
mITT 
Modified Intent to Treat  
mL 
Milliliter 
OR 
Odds Ratio 


 
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Abbreviation 
Definition 
PP 
Per Protocol 
PH 
Proportional Hazards 
PK 
Pharmacokinetic 
PSAS 
Physician Satisfaction Agreement Scale 
PVP 
Provepharm, Inc. 
RBC 
Red Blood Cell 
RGM 
Ratio of Geometric Means 
SAE 
Serious Adverse Event 
SAP 
Statistical Analysis Plan 
SD 
Standard deviation 
TEAE 
Treatment Emergent Adverse Event 
TTV 
Time from Drug Administration to Visualization of Blue Color 
µg 
Microgram 
µL 
Microliter 
UOVS 
Ureter Orifice Visualization Scale 
VS 
Vital Signs 
WBC 
White Blood Cell count 
 
 
 


 
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1. 
INTRODUCTION AND SCOPE 
Protocol PVP-19IC01 is an Open-Label, Phase 3, 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.  
This Statistical Analysis Plan (SAP) is intended to provide a more technical and detailed 
elaboration of the principal statistical features stated in the protocols.  The objective of the SAP 
is to reasonably assure that the statistical methodologies to be used for analysis are complete and 
accurate.   
In the development of this SAP, the following documents were used: 
• Protocol PVP-19IC01: Amendment 02, 02 September 2020  
• PVP-19IC01 eCRF:  Version 31. (draft), 28 August 2020 
 
The principles in the following guidance documents are followed in preparation of this SAP: 
• ICH E3 (1995): Structure and Content of Clinical Study Reports 
• ICH E6 (1996): Guideline for Good Clinical Practice 
• ICH E9 (1998): Statistical Principles for Clinical Trials 
In the event that a discrepancy is found between the descriptions in the statistical section of 
the protocol and this document, the description in this document supersedes the 
descriptions in the statistical section of the protocol. 
 
 


 
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2. 
OVERVIEW OF STUDY OBJECTIVES AND ASSESSMENTS   
The primary objective of this study is 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 
5-point conspicuity score as follows:  
1 = No jet observed 
2 = Weak jet, little color contrast 
3 = Color contrast or significant jet flow 
4 = Strong jet flow with good color contrast 
5 = Strong jet flow with striking contrast in color 
 
Secondary objectives are to assess: 
1. 
To evaluate the safety profile of IC when used as an aid in the determination of 
ureteral patency.  
2. 
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).    
3. 
To determine proportion of responders. To determine proportion of subjects meeting 
the responder definition in conspicuity score following IC treatment based on the 
blinded central reviewer’s assessment.  A subject is a responder when there is >=1 
point difference in the conspicuity score following the IC vs saline treatment (IC – 
Saline >=1) and the conspicuity score following the IC treatment is (3, 4, or 5). The 
responder criteria will be assessed separately for each ureter for each subject. 


 
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4. 
To describe the time to visualization (TTV) of blue color in the ureteral jets flow 
following IC treatment when used as a visualization aid during urological and 
gynecological surgical procedures  
5. 
To determine the IC pharmacokinetic profile in a subset of subjects from 2 
investigational sites. 
6. 
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. 
The study will enroll approximately 116 subjects from approximately 10 – 20 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. Any procedure done for 
the purpose of the surgery before the date of signing the informed consent may be used as long 
as it was within 30 days of the scheduled surgery date.  
On the day of surgery (Day 1) the subject 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). Each randomized subject 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.   
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 and video recorded for 10 mins.  The 
time period that will be captured on video is from injection to 10 minutes post injection. If both 
ureters cannot be visualized simultaneously, then alternating 15-30 second images of each ureter 
or ureteral orifice will be obtained. The process will be repeated in the same patient for the IC 
dose. Hence, each subject will have 2 videos and each will be about 10 minutes in length. 
The videos will be sent to a central imaging group that will pool and blind the videos. Videos 
will then be assessed by 2 blinded central reviewers for assessment of ureteral patency using the 
5-point conspicuity score criteria.  The 2 independent central reviewers will provide 2 
conspicuity scores (1 for left ureter and 1 for right ureter) for each video.  The consistency 
between the two reviewers will be checked; the two reviewers will be considered consistent if 
their scores for the corresponding ureter is (+/-) 1 point of a given video.  In this case, the 
average score across the two reviewers will be the final score for efficacy analysis.  Otherwise, a 
third reviewer (a judicator) will step in and will review this video. The scores from this third 
reviewer will be the final score for this video for efficacy analysis.        
After the procedure, the surgeon performing the procedure will provide his/her overall 
satisfaction assessment by rating his/her agreement with the statement “Compared to the saline 


 
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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. 
In addition, after the procedure the surgeon will also review the videos of his/her patients and 
will provide the conspicuity scores for each ureter after each treatment using the same 5-point 
conspicuity score criteria.  The surgeons score will be utilized for conspicuity score concordance 
analysis. 
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 
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. The first post-op stool will also be collected for analysis in this PK group. 
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. 
Protocol Schedule of Assessment is included here for the convenience of review. 


 
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Table 1:  Schedule of Assessments  
 


 
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2.1. 
Study Endpoints  
2.1.1. 
Efficacy Endpoints 
The primary efficacy endpoint is conspicuity score after each treatment provided by the central 
readers:   
1 = No jet observed 
2 = Weak jet, little color contrast 
3 = Color contrast or significant jet flow 
4 = Strong jet flow with good color contrast 
5 = Strong jet flow with striking contrast in color 
 
The secondary efficacy endpoints include: 
1. 
Proportion of physicians who agree that compared to saline, IC treatment improves 
visualization as an aid for the assessment of ureteral patency  
2. 
To determine proportion of IC treatment responders per the blinded central reviewers’ 
assessment.  A subject is a responder to the IC treatment when there is >=1 point 
difference in the conspicuity scores between the IC and saline treatment (IC – Saline 
>=1) and the conspicuity score following the IC treatment is (3, 4, or 5). The 
responder criteria will be assessed separately for each ureter for each subject. 
3. 
Time (minutes) from start of drug administration to visualization (TTV) of the blue 
color in the ureteral jets flow after IC administration 
Other efficacy endpoints will include: 
1. 
Conspicuity scores assessed by the surgeons 
2. 
Concordance of the Conspicuity scores between the surgeons’ assessments and the 
blinded central reviewers’ assessments. 
 
2.1.2. 
Safety Endpoints 
The safety endpoints include  
1. Treatment emergent adverse events 


 
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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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(1988) as summarized by professor Gary Kock in 2003 at Drug Information Association (DIA) 
10th Annual Biostatistics Workshop in Japan, August 29, 2003 in Tokyo, Japan1.     
The prediction of the conditional power for a statistically significant result from the final analysis 
(at the two-sided 0.05 level) given the observed difference between treatments at a formal 
interim analysis can be based on the estimated treatment effect (equation 2); 2-sided 95% 
confidence intervals for the CP will also be calculated. 
 
Where T_hat = estimate of treatment effect at interim and SE = standard error of the treatment effect. In 
this study, T-hat will be the estimate of odds ratio of treatment and SE will be the standard error of the 
estimate, both are in the log scale. P=proportion of study completed (=0.5), alpha (α)=0.05/2, 
gamma (γ)=0.05 
 
Following Mehta et al (2011, page 6)2 adaptive approach the interim analysis results will be 
classified into three zones based on the estimated CP and decision to be made in each zone is 
specified as follows. 
 
 
 
1 Page 8 “Statistical Considerations for the Conduct and Reporting of Interim Analyses”. (GGK 8/11/2003)  
2 Adaptive increase in sample size when interim results are promising: A practical guide with examples. Cyrus R. 
Mehta and Stuart J. Pocock.  Statist. Med. 2011, 30 3267--3284 
 


 
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Table 3:  Interim Analysis Conditional Power and Decision Principle        
Conditional Power 
Decision 
Rationale 
 CP <0.20 
Stop the study because the evidence for 
unfavorable efficacy is sufficiently 
convincing that no additional efficacy data is 
necessary.  
An unfavorable zone: the conditional 
power (CP) is fairly low, suggesting that 
the final result is not likely to achieve 
significance, hence, the study has fairly 
low probability to reject the null 
hypothesis.  This zone is defined to be CP 
< 40% 
0.2 <=CP <0.4 
No sample size re-estimate because the 
evidence for unfavorable efficacy. Complete 
the study per current protocol.  
0.4 <=CP <=0.8 
Re-estimate sample size, study continues to 
complete the enrollment per re-estimated 
sample size 
A Promising zone: the conditional power 
is in between 40% to 80%, suggesting that 
the interim analysis result is promising, 
but the study has to have an increased 
sample size to have at least 80% power to 
reject the null hypothesis 
CP > 0.8 
No sample size re-estimate. Complete the 
study per current protocol. 
A favorable zone: the conditional power 
is fairly high, suggesting that the interim 
result is as good or better than the 
expectation, the study has fairly high 
probability to reject the null hypothesis as 
is.  This zone is defined to be CP > 80%. 
 
To protect the data integrity, an independent statistician/programmer who is not involved with 
the study will be responsible to perform the analysis.  A Data Monitoring Committee (DMC) will 
be formed to review the interim analysis results.  All members of the DMC will not be involved 
with the conduct of the study. To perform the interim analysis, the independent statistician will 
receive the conspicuity score assessed by the central reviewers and the randomization code for 
each video/subject.  It is worthy to point out that randomization is only pertained to the treatment 
of IC vs Saline but the dose of IC will not be revealed for interim analysis.  
The DMC may choose to share with the study team the aggregated results/data (e.g., summary 
tables) but the individual subject data will not be shared with the study team.  All 
documentations for the interim analysis, including but not limited to analysis data, associated 
programs, summary tables and/or listings, and DMC communications, will be securely stored 
away until the study database is locked.    


 
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3.3. 
Analysis Population 
The following analysis sets will be identified for this study. 
Enrolled Analysis Set: All subjects who sign the ICF for the study are considered to have 
enrolled.  
Intent-to-Treat (ITT) Analysis Set:  The ITT Analysis Set will include all randomized subjects.  
This dataset may also be referenced as the ‘Randomized Set’.  The ITT subjects may or may not 
receive randomized treatment.   All ITT subjects will be included in the randomized treatment 
group even if a dosing error occurred.  See additional discussion in Section 3.7.  
Safety Analysis Set:  The safety set will include all ITT Analysis Set subjects who are treated 
with any study drug (Saline or IC) and will be used for safety and tolerability assessments.  In 
the safety analysis set all subjects will be included in the actual treatment group received if a 
dosing error occurred.  See additional discussion in Section 3.7.  
Efficacy Analysis Set:  The efficacy analysis set includes all Safety Analysis Set subjects who 
have a surgical procedure to assess ureteral patency and who have received both study drugs 
(Saline and IC dose) and a video approximately 10 minutes in length is available after each 
treatment.  The 21 patients who were randomized and treated prior to the development of 
the conspicuity score will be excluded from the efficacy population.  This will be the primary 
population for efficacy assessments.  This is also referenced as the modified intent-to-treat 
(mITT) Analysis Set.  All efficacy evaluations will be based on the mITT population.  In the 
mITT analysis set all subjects will be included in the actual treatment group received if a dosing 
error occurred.  See additional discussion in Section 3.7.  
Per Protocol (PP) Analysis Set: The PP set includes 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. In the PP analysis set all subjects will be included in the actual treatment group 
received if a dosing error occurred.  See additional discussion in Section 3.7.  
 
3.4. 
Test Hypothesis and P Value Justification 
The study has a planned interim analysis to review the effect size assumption used to determine 
the sample size.  To control for the overall alpha for the primary efficacy analysis, an alpha of 
0.0001 will be used for the interim analysis, and alpha of 0.0499 will be used for the final 
analysis.  The details of the interim analysis, including scope, method, and decision rules, will be 
described in the Data Monitoring Committee charter.   
In the final analysis, there are two null hypotheses for the primary efficacy endpoint conspicuity. 
1. 
there is no difference between the IC high dose and the normal saline 


 
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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 0.0499, the two p-values will be 
ordered from large to small. When both nominal p-values are less or equal to 0.0499, 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 the larger nominal p-value is 
greater than 0.0499 but the smaller p-value is less or equal to 0.025, the null hypothesis 
associated with the larger p-value is accepted and the null hypothesis associated with the smaller 
nominal p-value will be rejected. If the smaller nominal p-value is greater 0.025, the null 
hypothesis associated with this p-value is also accepted.  The Overall Type I error control will 
apply to the primary endpoint conspicuity only.  
3.5. 
Procedures for Handling Missing Data  
Missing or partial data associated with efficacy assessments will be discussed specifically within 
each section. No missing data imputation will be performed for safety parameters.  However, 
AEs with missing severity assessments will be tabulated as “severe,” and AEs with missing 
relationship assessments will be tabulated as “related” for the purpose of analysis; and the 
missing data will be presented in data listing as is. 
3.6. 
Analysis Center  
This is a multicenter study; however investigative center will not be included in the analysis 
model as a covariate due to expected small sample size per center, additionally, the primary 
efficacy conspicuity score data will come from central readers whereas the conspicuity score 
from the local readers (surgeons) will be used for confirmatory analysis. Potential center effect 
will be assessed if data suggested. 
3.7. 
Treatment Group vs Randomization Group  
All qualified subjects will be randomized to either a dose of 2.5 mL IC (referred to as the Low 
dose) or a dose of 5.0 mL IC (referred to as the High dose).  All randomized subjects will receive 
a dose of normal saline before receiving the randomized IC dose.   
Hence, there are 2 randomization groups, namely IC High Dose and IC Low Dose and 3 
treatment groups (IC High dose, IC Low dose, and Saline) in this study. 
If a dosing error occurred (e.g., a subject was randomized to the IC High dose but actually 
received IC Low dose, and vice versa), the subject will be included in the actual treatment group 
received for efficacy, PK, and safety analyses; this will be an important protocol deviation and 
the incident will be clearly documented in the study report and in the database.   


 
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Data presentation for study population, physician overall satisfaction, safety lab, and 12-ECG 
will be based on the 2 randomization groups; whereas the data presentation for conspicuity, 
TTV, adverse events, and vital signs will be based on the 3 treatment groups.  A Study Overall 
group may be included in certain data presentations (e.g., demographics) if data warrant.  The 
Saline group will include all subjects who had saline dose, hence, the number of subjects in this 
treatment group will expect to be twice the size of IC Low dose and IC High dose.  If data 
warrants, the Saline group may also be split by IC dose group.  That is, subjects received the IC 
Low dose vs subjects received the IC High dose.      
 
3.8. 
Definitions and Derived Variables 
3.8.1. 
Study Period Definitions  
The study duration for each subject is divided into 3 periods  
1. Screening Period (Visit 1): The duration from signing informed consent until before 
receiving the first dose of study drug will be described as the screening period.  The last 
measurement taken prior to receiving the first dose of study drug (saline injection) 
is the Baseline measurement. Hence, Baseline measure could be either a scheduled 
assessment or an unscheduled assessment. 
2. Treatment period (Visit 2): Study day 1 (procedure day) is the treatment period.  
Although the actual date/time for some PK samples may occur on study day 2 or day 3, 
all PK analysis will be labelled as treatment period because PK period starts on Study 
Day 1.  
3. Post treatment follow-up period (Visit 3): this period starts the day after procedure 
through end of study.   Subjects who completed Visit 3 final follow-up are considered 
complete the study. 
 
A study scheme is provided in Figure 1. 
 


 
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Figure 1:  Study Scheme 
 
3.8.2. 
IC Treatment Responders 
A responder (Yes/No) variable will be derived for each subject by comparing the conspicuity 
score following IC treatment vs the conspicuity score after saline treatment.  When there is >=1 
point improvement (IC – Saline) in the conspicuity score and the conspicuity score following the 
IC treatment is (3, 4, or 5) for a subject, this subject is considered a responder of the IC 
treatment.  The responder assessment will be performed for each ureter separately. 
3.8.3. 
Time to Visualization (TTV) of Blue Color in the Ureteral Jets After IC 
Administration  
Each randomized subject is expected to receive 1 dose of saline and 1 dose of IC; time of each 
injection, including start time and completion time, will be recorded as HHMMSS (hour, minute, 
and second). TTV event is assessed only after IC treatment within 10 minutes after the 
injection start. Time when this event occurred after IC injection will be captured as HHMMSS.  
Therefore, TTV following each injection will be derived as follows. 
TTV (minutes) = (time when adequate visualization occurs - start time of injection) / 60 


 
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If the adequate visualization of blue color in the ureter jet flow at an ureteral orifice 
following IC injection for a subject did not occur for any reason, this subject will be 
censored for this analysis.   The censored time will be set to 10 minutes. 
TTV will be derived for each subject following IC injection for each ureter separately. 
3.8.3.1. 
Missing Data Imputation Rules for TTV 
In order to calculate TTV, the injection start time and time when blue color is adequate 
visualized in the ureter jet flow must be present. Although the clinical team will make the best 
effort to eliminate missing or partial time, mistakes could occur. The following rules will be 
implemented should a subject had missing or partial injection time or time when blue color in the 
ureter jet flow is adequately visualized.  
1. When the injection start time is missing or partial 
1) 
If injection start time is missing but the completion time is present, the missing start 
time will be set to be the same as the injection completion, and vice versa.  
2) 
If the hour and minutes are non-missing but the seconds are missing for injection start 
time, the first second (01) will be assigned to the injection time.  
3) 
Otherwise the median elapsed time (defined as time between surgery start time and 
injection start time) in the study across all subjects who did not have missing data will 
be used to impute the missing time.  That is, the missing injection start time will be 
set as (Surgery start time + median elapsed time).  
The rules will be implemented based on this order. 
 
2. When event occurred but the time is missing or partial 
A subject is considered to have had this event when the conspicuity score is 4 or higher per 
central reader score for this ureter.  When a subject has this event but the time when the event 
occurred is missing, the following rules will be applied. 
1) 
If the time is partial (i.e., only the seconds are missing), the first second (01) will be 
assigned. 
2) 
Is the time is missing, the subject will not be censored for this endpoint. The missing 
time, however, will be assigned to 10 minutes after the injection start time. 
3) 
This event will be considered as an important protocol deviation. The subject will be 
excluded from the efficacy PP analysis set. A sensitivity analysis may be performed 
for the TTV endpoint based on the efficacy PP analysis set.  
If a subject had issue(s) with a ureter initially this subject will be censored for this analysis 
regardless if the issue(s) is or is not fixed during the procedure.       


 
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3.8.4. 
Physician Satisfaction Agreement Scale  
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 
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. 
Overall Satisfaction =Yes if PSAS is 1 or 2, otherwise 
Overall Satisfaction = No 
Physician who did not provide rating (missing data) will be grouped in the ‘No’ group for overall 
satisfaction analysis.  
 
3.8.5. 
Concordance in Conspicuity Assessments between the Surgeons and the 
Blinded Central Readers   
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. In addition, each qualified subject must have 2 kidneys, resulting 4 observations of 
conspicuity score per subject from the surgery and four observations from the blinded central 
readers.  
The following notation will be adapted for each conspicuity score for a given subject: 
• CSSL is the conspicuity score from the left ureter after saline injection 
• CSSR is the conspicuity score from the right ureter after saline injection 
• CSIL is the conspicuity score from the left ureter after IC injection 
• CSIR is the conspicuity score from the right ureter after IC injection 


 
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After the videography is read by the central blinded reader, each subject will then have 4 pairs of 
conspicuity scores. For example, CSSL from the surgeon and the CSSL from the central reader. 
Because the conspicuity has value of 1, 2, 3, 4, or 5, the difference in each pair of conspicuity 
scores between the surgeon and the blinded central readers will be derived and it will have a 
possible value ranging from (-4 to +4).  Based on the difference in each pair of conspicuity score, 
a new dichotomized concordance response variable will be derived as  
 Concordance Response = Yes if the difference in conspicuity score is within +/- 1 
point. That is, the difference is ranged from (-1 to +1), inclusive.  
 Concordance Response = No if the difference in conspicuity is more than 1 point 
 
 
 
 
 


 
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4. 
STUDY POPULATION SUMMARIES 
All study population summaries will include 2 randomization groups plus an ‘Overall’ column to 
display the summary statistics across all subjects. 
Study population data listing by individual subject will be prepared for all study population 
endpoints, including the observed value and derived variables.  
 
4.1. 
Disposition 
A summary table (Table 14.1.1.1) will provide frequency counts for subject disposition, 
including all enrolled subjects, randomized subjects, treated subjects (Saline or IC), subjects who 
completed study, subjects who did not complete study, and reason for not receiving Saline 
injection or IC injection, and reasons for study discontinuation.    
Primary reason for early termination of study will include 
1) Adverse Event 
2) Study Non-compliance  
3) Lost to Follow-Up 
4) Physician Decision 
5) Sponsor Decision 
6) Subject Decision 
7) Other 
If a subject who receives a dose of normal saline injection and fails to receive a dose of IC, the 
subject will be considered to have discontinued study drug treatment.  Reason for treatment 
discontinuation will be captured and tabulated. Moreover, the disposition table will identify 
number (%) subjects included for safety analysis set, efficacy analysis set (mITT), and efficacy 
per protocol (PP) analysis set. 
Another summary table (Table 14.1.1.2) will be produced to provide number of subjects in the 
safety, mITT, and efficacy PP analysis set by investigator and will display the center with the 
highest enrollment first. 
Disposition summary will be based on the ITT analysis set.  


 
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4.2. 
Demographics and Baseline Characteristics 
The demographic summary (Table 14.1.2.1) will include descriptive statistics for age, age group 
(age <65, age >=65, >=75 years), sex, race, ethnicity, weight, height, and body mass index 
(BMI) at baseline for overall and by treatment group.  
Baseline characteristics and patient population characteristics will also include the following 
study surgery information: 
1) Surgery duration (hours), method of surgery (cystoscopic, robotic, open, 
laparoscopic, other), and surgeon’s specialty 
2) Requiring (yes/no) fluid to be instilled into the bladder 
a. If Yes, the amount and type of fluids  
3) Type, amount, and timing (relative to the start time of saline injection) of hydration 
4) Use of any diuretics during the procedure (yes/no)   
Demographics and baseline characteristics will be tabulated for the safety analysis set with no 
formal inferential tests.  The same demographics table will also be prepared for the mITT 
analysis set (Table 14.1.2.2).   
 
4.3. 
Medical History 
All medical history data captured will be mapped using MedDRA (version 22).  Subjects with 
medical history by SOC and preferred term will be tabulated by randomization group and study 
overall (Table 14.1.3) based on the safety analysis set. 
 
4.4. 
Protocol Deviations 
All protocol deviations will be identified and will be classified as either an ‘Important Protocol 
Deviation’ or ‘Protocol Deviation’. 
Important Protocol Deviation: An Important Protocol Deviation is a protocol deviation that may 
significantly impact the completeness, accuracy, and/or reliability of the study data or that may 
significantly affect a subject’s rights, safety, or well-being. Examples may include: 
 Failure to meet all entry criteria; 
 Non-compliant with study drug treatment regimen; 
 Did not receive randomized treatment due to dosing error; 
 Use of prohibited concomitant medications; 


 
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Protocol Deviation: Any alteration/modification, divergence or departure from the IRB-approved 
protocol.  A protocol deviation is an unanticipated or unintentional divergence or departure from 
the expected conduct of an approved study that is not consistent with the current research 
protocol, or consent document. 
All protocol deviations will be tabulated by protocol type, protocol deviation category for each 
treatment group and study overall (Table 14.1.4) based on the safety analysis set. 
4.5. 
Treatment Compliance 
Doses of study medication (Saline and IC) will be administered to the study subjects under the 
observation of study personnel while confined to the study site.  The exact time of administration 
of study medication will be documented within each subject’s eCRF.   
No formal Compliance data will be tabulated for the study report. 
4.6. 
Prior and Concomitant Medications 
All prior and concomitant medications will be mapped using WHO drug (version September 
2019).  Subjects with prior and concomitant medications will be tabulated by medication class 
and drug standardized name. The medication class will be the WHO Drug ATC level 3 text if 
available, otherwise, ATC level 2 text will be the medication class (Table 14.1.5.1).  The prior 
and concomitant medications summary will be based on the safety analysis set. 
All efforts will be made to ensure the capture of onset/stop dates of a medication.  If at the end 
there are missing dates in the concomitant medication dataset, the following rules will be 
implemented for the purpose of analysis. 
Concomitant meds will include medications with end-date that on or after the surgery date when 
present or if ongoing is checked.  When ongoing is not checked and the end-date is partial the 
following rules will be used to determine if a medication is concomitant.  
a) EXCLUDE a medication if the year is before the surgery 
b) EXCLUDE a medication if the year is the same year but the month is before the surgery;  
c) INCLUDE a medication if the year is the same year as the surgery but the month is 
missing  
When end-date is completely missing, the med will be accounted as concomitant medication. 
 
Concomitant other procedures will be mapped using MedDRA version 22.  All other procedures 
performed during the study will be tabulated by system organ class and preferred term (Table 
14.1.5.2). 


 
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5. 
EFFICACY ANALYSIS 
Efficacy data will be displayed by treatment group except PSAS and TTV.  
All efficacy analyses will be based on the Efficacy analysis set (mITT Population).  Although the 
PP analysis set is defined in Section 3.3 efficacy analysis based on the PP analysis set will not be 
routinely provided.  That is, when number of mITT subjects excluded from the efficacy PP 
analysis set is more than 10% of the mITT analysis set, then efficacy analysis will also be 
performed based on the efficacy PP analysis set to evaluate the consistency and the robustness of 
the efficacy conclusion drawn from the mITT analysis set.    
Efficacy data listing by individual subject will be prepared for all efficacy endpoints, including 
the observed value and derived variables.  
 
5.1. 
Conspicuity Score from the Central Readers 
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 conspicuity endpoint. The recording 
videography will be read by the surgeon (who is unblinded for treatment and blinded for IC 
dose) and by two center readers who is blinded to the treatment (Saline vs IC) and IC dose.  The 
score from the central readers will be the source for the primary endpoint analysis and the 
conspicuity score provided by the surgeons will be utilized for concordance analysis (see Section 
5.5 for details).   
There will be 2 central reviewers to watch each video after the videos are blinded.  The ratings 
are acceptable for consistency if the scores from the two independent reviewers for a ureter 
under the same treatment are within (+/-) 1 point.  In this case, the average score across the two 
reviewers will be the final score from the central reader.  Otherwise, a third independent 
reviewer (a judicator) will review the same video and this judicator’s score will be the final score 
from the central reader. 
As indicated earlier in Section 3.7 the summary table for this endpoint will include 3 treatment 
groups, namely IC High Dose, IC Low Dose, and Saline Group.  It is expected that the sample 
size (n) for the Saline group will be twice the size of the sample size for the IC Hight dose and 
IC Low Dose. 
The treatment effect on the conspicuity score will be evaluated using a Generalized Estimating 
Equation (GEE) for Repeated Measures to control for the intra-subject correlation by fitting a 
proportional odds model to evaluate the treatment effect. A full model (Model 1) will provide 
the chi-square tests (Score Statistics for Type 3 GEE Analysis) for the effects from treatment (IC 
High dose / IC Low dose, Saline), ureter (left side ureter vs right side ureter), and 
treatment*ureter interaction on the response based on multi-normal distribution for an ordinal 


 
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response.  The model estimates the cumulative logits of better outcome (conspicuity=5) to the 
poor outcome (conspicuity=1).  Since it is expected that the interaction effect will not be 
statistically significant (p >0.10), a reduced model (Model 2) will also be used in which the 
interaction effect will be dropped.  The goodness of fit of the models (Full model vs the Reduced 
model) 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 (treatment, ureter, treatment*ureter interaction in full model) 
will be determined based on the Score Statistics for Type 3 GEE Analysis.   
For the primary endpoint analysis, GEE models will be fitted separately for each comparison, 
namely IC Hight dose vs Saline, and IC Low dose vs Saline, and IC High dose vs IC Low dose.   
An example of SAS syntax is showing below for this analysis, in which the conspicuity is treated 
as an ordinal response variable and a proportional odds model is used model the better response 
compared to the poorer response.  Stokes et al. (2000)3 recommended that the null hypothesis of 
treatment effect would be based on the score statistics for Type 3 GEE analysis, whereas the 
parameter will be the GEE parameter estimates with the empirical standard error because the Z 
and the Wald statistic generally produce more liberal p-value than the score statistic.  
In this example, the treatment is a binary code; IC High dose=1 and Saline=0.  For analysis of IC 
Low dose vs Saline, IC Low dose=1 and Saline=0; when comparing IC High dose vs IC Low 
dose, IC High dose=1 and IC Low dose=0. 
PROC GENMOD DATA=respH descending; 
CLASS USUBJID URETER(ref=’Right’) TRT (ref=’Saline’); 
MODEL conspicuity = trt ureter trt*ureter  
 
(Model 1)  
/LINK=clogit DIST=mult TYPE3; 
REPEATED subject = Usubjid / type=ind; 
ESTIMATE ‘OR: IC vs Saline’ trt 1 -1/ exp; 
 
 
ods output Estimates=OR1 GEEEmpPEst=est1 Type3=pval1; 
Run;  
 
PROC GENMOD DATA=respH descending; 
CLASS USUBJID URETER(ref=’Right’) TRT (ref=’Saline’); 
MODEL conspicuity = trt ureter  
 
 
 
(Model 2)  
/Link=clogit DIST=mult TYPE3; 
REPEATED SUBJECT = Usubjid / type=ind; 
ESTIMATE ‘OR: IC vs Saline’ trt 1 -1/ exp; 
 
 
ods output Estimates=OR2 GEEEmpPEst=est2 Type3=pval2; 
Run;  
 
Where:  
 
3 Stokes ME, David CS, Koch GG (2000). Categorical Data Analysis using the SAS System 2nd Edition (page 485).  


 
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1. Dataset respH include subjects from IC High dose only. 
2. The ‘descending’ option indicates that the ordered values are listed in descending order.  
3. Conspicuity score (1, 2, 3, 4, or 5) 
4. ‘TRT’ is Treatment (coded to IC or Saline)  
5. ‘URETER’ is coded to ‘Left’ or ‘Right’ 
6. ‘USUBJID’ is the unique Subject Identification 
7.  REPEATED statement controls the intra-subject correlation due to repeated measures. In this 
example, independent correlation matrix is used. 
8. Three output datasets:  
a. OR=estimated odds ratio (IC /Saline) 
b. EST= Analysis of GEE Parameter Estimates based on Empirical Standard Error 
Estimates 
c. PVAL= P-value for each effect in the model per Score Statistics For Type 3 GEE 
Analysis     
The analysis summary table (Table 14.2.1.1) will include distribution of conspicuity score in 
each treatment group by ureter (left vs right and overall), p-value based on score statistics for 
Type 3 GEE analysis, odds ratio estimates and standard error, and 95% confidence intervals for 
the odds ratio. Three treatment group comparisons will be provided, namely difference between 
IC High dose vs Saline, IC Low dose vs Saline and IC High dose vs IC Low dose (an exploratory 
analysis). 
The summary table will display the nominal p-values from each comparison (High dose vs 
Saline, Low dose vs Saline, and High dose vs Low dose).  However, the statistical significance 
of the normal nominal p-value between the IC High doe vs Saline and between the IC Low dose 
vs Saline will follow the rule defined in Section 3.4 in order to control for family-wide type I 
error for the primary endpoint.  
5.1.1. 
Missing Data Handling Procedure 
When conspicuity assessment cannot be performed, conspicuity score will be missing.  Reason 
for each Not Assessed cases will be documented in the database.  
• For the primary analysis (Table 14.2.1.1), missing data will not be imputed.  Subjects will 
be included in the input dataset as is.  This analysis will be referred to as the Observed 
Data Analysis.  
• However, the impact of missing data will be evaluated by performing 3 sensitivity 
analyses. The sensitivity analysis will use the same model specified for the primary 
analysis; nominal p-values from each sensitivity analysis will be reported as. Those 3 
sensitivity analyses are described below: 
o Sensitivity Analysis 1 – This analysis will include all MITT subjects who have no 
missing data. That is, all subjects in this analysis will have 4 conspicuity scores as 
planned (Table 14.2.1.2). 


 
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o Sensitivity Analysis 2 – This analysis will exclude all subjects who are not in the 
PP population and subjects who have ≥ 1 missing data.  This would represent the 
best outcome for the study since all potential confounded issues will be removed 
(Table 14.2.1.3).     
o Sensitivity Analysis 3 – This analysis will impute the missing data that creates the 
worst scenario for the study (Table 14.2.1.4). 
 Missing data for Saline Treatment: Assign missing value for saline 
treatment to be the best value from this patient if scores are available. If no 
score is available for this patient, then assign the best score of 5 to missing 
data. 
 Missing score for IC Treatment: Assign missing value for IC treatment to 
the worst score the patient had if scores are available. If none is available, 
assign the worst score of ‘1’ to missing data.  
 
5.2. 
PSAS with IC Treatment 
Each subject will have 1 PSAS score.  Ratings in the PSAS with the IC treatment will be 
tabulated (Table 14.2.2) by treatment (IC High dose, IC Low dose, and overall (pooled across 
high dose and low dose) with number (%) subjects in each rating category and overall 
satisfactory.  
Two-sided 95% confidence intervals on proportion of physicians who are satisfied (with ratings 
1 or 2) will be derived using the Wilson confidence limits for the binomial proportion.  This is 
also known as score confidence limits.  The Wilson interval has been shown to have better 
performance than the Wald interval and the exact (Clopper-Pearson) interval (SAS Users’ 
manual). 
A binominal 1-way proportion approach will be used to examine that ≥ 50% of the physicians 
are satisfied with the IC treatment (a superiority test) as an aid for the assessment of ureter 
patency comparing to the saline treatment.  Below is an example of SAS syntax to implement 
this analysis. 
 
 


 
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DATA psas; 
Set PSASscore; 
If score in (1, 2) then psasC=’1Yes’; else psasC=’2No’; 
Run; 
PROC SORT data=psas; BY trt psasC; 
PROC FREQ DATA=psas ORDER=DATA; 
   TABLES psasC / BINOMIAL(SUP P=0.45 MARGIN=0.05 CL=WILSON) alpha=.05; 
   BY trt; 
   ODS OUTPUT BinomialCLs=bCL 
 
 
BinomialSup=pval 
 
; 
run; 
This syntax makes the ‘Yes’ response to be the first category so the binomial test will examine 
the proportion of response in the Yes category.  The null proportion of 0.45 and margin of 0.05 
makes the point estimate of superiority limit to be 0.50 (superiority limit = the null proportion + 
margin).  The ‘TRT’ is coded to ‘High’, ‘Low’ or ‘All’. ‘All’ includes subjects from both IC 
High and IC Low doses. 
An exploratory analysis to examine any difference in PSAS with the use of IC High dose vs the 
IC Low dose will be performed via a Cochran-Mantel-Haenszel (CMH) test for ‘Row Mean 
Scores Differ’.  Below is an example of SAS syntax to implement this analysis   
PROC FREQ DATA=psas; 
   TABLES dose*psas / noprint CMH2 alpha=.05; 
   ODS OUTPUT CMH=pval 
 
; 
run; 
where ‘dose’ is coded to Low or Hight; the ‘PSAS’ is the satisfaction rating score (1, 2, 3, 4, or 
5).   
Normal p-value for each analysis will be reported as is without controlling for multiple 
comparisons. 
 
5.3. 
IC Treatment Responders 
In this study each subject is served as his/her control when the IC treatment is given as a 
visualization aid when determining ureter patency, IC treatment responder, hence, could be 
defined based on the conspicuity score following IC and saline.  
To determine proportion of subjects meeting the responder definition in conspicuity score 
following IC treatment the blinded central reviewer’s assessment will be used.  A subject is a 
responder when there is >=1 point improvement in the conspicuity scores following the IC vs 
saline treatment (IC – Saline >=1) and the conspicuity score following the IC treatment is (3, 4, 
or 5). The responder criteria will be assessed separately for each ureter for each subject. 


 
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Since the responder definition will be defined for each ureter per subject, when a subject has 
missing data, the corresponding responder variable cannot be defined.  The primary analysis will 
exclude all subjects that the responder definition cannot be derived due to missing data; this will 
be referred to as the Observed Analysis.   
To assess the impact of missing data, a sensitivity analysis will be conducted. In this sensitivity 
analysis, the responder variable will be set to ‘No’ for those that cannot be derived due to 
missing data.  This analysis will represent the worst-case scenario for this analysis. 
Summary tables (Tables 14.2.3.1 and 14.2.3.2) will provide number (%) subjects meeting the 
responder definition in the following categories: overall (left or right), left ureter, right ureter, or 
both ureters for each IC dose and overall; 95% confidence intervals based on normal 
approximation will also be provided, along with the difference in proportion of responders 
between the IC high dose vs IC low dose.   
 
5.4. 
Time to Visualization  
In this study each subject will contribute 2 TTV observations, one for left and one for right 
ureter. Each time to event observation will be considered an independent observation.   
As indicated earlier in Section 3.7 the summary table for this endpoint will include 2 treatment 
groups, namely IC High Dose, IC Low Dose. Time (minutes) to visualization (TTV) of blue 
color in the ureter jet flow will be evaluated using the Kaplan-Meier Survival Analysis approach. 
Descriptive summary, including number of subjects (%) subjects censored; time to event 
percent-tiles (25%, 50%, and 75%) and 95% confidence intervals and Kaplan-Meier mean (SE) 
will be estimated for each ureter. The following is an example SAS syntax for this analysis. 
ods output CensoredSummary=Ncensor  
 
 
   ProductLimitEstimates=PL 
 
 
 
   Means=KMmeans 
 
 
   Quartiles=quart 
 
 
   Survivalplot=SurvivalPlotData 
 
 
; 
 
PROC LIFETEST DATA=ttv METHOD=PL ATRISK 
 
 
PLOTS=survival(ATRISK(ATRISKTICK)= 0 2 4 6 8 10); 
       
 
TIME aval*Cnsr(1); 
 
 
 
*---- stratified test ---*; 
 
 
 
STRATA ureter / GROUP=trt; 
 
 
 
; 
run; 
where TRT is coded to HighDose vs LowDose 
In additional, KM-survival analysis descriptive summary will also be provided for the overall.  In 
this analysis, number of observations will not be the same as number of subjects. 


 
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Using data ‘SurvivalPlotData’ Kaplan-Meier survival curves will be presented for each treatment 
group along with number of subjects at risk at 0, 2, 4, 6, 8, and 10 minutes.  The KM-survival 
plots will be prepared for each ureter and overall.   
If data warrants, a Cox Proportional hazards (PH) model will be fitted to estimate hazards ratios 
(HR) between IC High dose vs IC Low dose using a stratified Cox PH model that includes main 
effect of treatment and stratified by ureter (left vs right).  The HR estimate and 95% CI for the 
HR will be based on the Score Statistics.  The p-value for treatment effect will be based on the 
Score Statistics because the p-value from the score statistics produced by PROC PHREG will be 
the same as the stratified log-rank test produced by PROC LIFETEST. In order to obtain HR 
95% CI using the score statistics, an external SAS macro developed by based on Lin (2016)4 will 
be used (because the current SAS 9.4 does not have this computation option).  Note the option of 
TIES=DISCRETE should be specified for SAS procedure PROC PHREG. 
A summary table (Table 14.2.4) will include the following. 
• number (%) subjects with and without (censored) event from the left ureter and from the 
right ureter. This will allow each subject to be accounted once per treatment,  
• Kaplan-Meier product limit estimates of quartiles of time to first event (25%, 50%, and 
75% tiles and 95% CIs) and  
• KM means (SE) of time to first event  
• Hazard ratio (IC High/IC Low) and 95% Cis and p-value based on score test.   
To compute the KM means and its standard error, in case a subject is censored at 10 minutes the 
option TIMELIM will be set to the maximum observed time in minutes when SAS procedure 
PROC LIFETEST is used to perform KM survival analysis. Hence, the calculated mean survival 
time will be the area under the Kaplan-Meier survival curve from 0 to this maximum observed 
time.  
Below is an example of SAS syntax to estimate the difference between treatment groups IC High 
dose vs IC Lower dose using the external SAS macro HazardRatio.  This parameter will not 
applicable for Saline treatment. 
DATA CoxHS; 
 
set ttv; 
 
 
If CNSR=0 THEN EVENTYN=1;  *-- YES ---*; 
 
 
If CNSR=1 THEN EVENTYN=0;  *-- NO ---*; 
 
TIME=AVAL; 
if trt=’IC High’ then Treatment=1; else Treatment=0; 
 
run; 
 
4 Lin DY, Dai L, Cheng G, Sailer MO: On confidence intervals for the hazard ratio in randomized clinical 
trials. Biometrics, 75: 1098-1102, 2016.  http://dlin.web.unc.edu/software/HazardRatio/ 


 
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*---- use the macro to get Score HR 95% CI ****************; 
%HazardRatio (Data = CoxHS, Result=HR HL, Time = Time, Status = 
EventYN, Ties=DISCRETE, Stratum = Ureter, Treatment = Treatment, Alpha 
= 0.05, Accuracy = 0.00001); 
 
data Result1; 
 
set HR_HL; if method='Score'; 
HR=exp(estimate); 
HR Lower=exp(Lower); 
HR_Upper=exp(Upper); 
 
Run; 
 
 
5.5. 
Concordance Assessments 
The concordance between the surgeon’s conspicuity and the blinded reader’s conspicuity will be 
assessed in three ways. 
5.5.1. 
Treatment Effect on Conspicuity per Surgeons Review 
The first approach is to evaluate the treatment effect based on the surgeons conspicuity score.  
The objective for this analysis is to check if the conclusion of treatment effect shown from the 
central readers rating can be confirmed by the surgeons reading.  If the answer is a ‘Yes’ then 
one can conclude that concordance is achieved.  Hence, this analysis serves as a confirmatory 
analysis of the primary analysis.  In additional, we can also compare the magnitude of treatment 
effect (expressed as the odds ratio) between the primary analysis and confirmatory analysis.  All 
subjects with ≥ 1 missing data from surgeon will be excluded from this analysis. 
Procedure and summary table (Table 14.2.5.1) for this confirmatory analysis will follow the 
same GEE approach described in Section 5.1, however, the nominal p-values will be displayed 
as is without control for multiplicity.   
5.5.2. 
Covariate Analysis  
The second approach is to pool the conspicuity scores from both surgeon and blinded reader into 
a single dataset and add a new covariate (evaluator) into the GEE model; the purpose is to assess 
if the effect of evaluator is or is not a statistically different covariate.   A summary table (Table 
14.2.5.2) will include the results from the following covariate analysis.  All subjects with ≥ 1 
missing data from either surgeon or central reader will be excluded from this analysis. 
To perform this analysis, a new variable (rater: surgeon vs central reader) and interactions will be 
added to the full model (Model 3) and Reduced model (Model 4). Score statistics for Type 3 
GEE analysis will be used to determine if the rater is statistically significant factor. 


 
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Amendment 2 
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Page 38 of 47 
 
PROC GENMOD DATA=resp descending; 
CLASS usubjid ureter(ref=’Right’) Rater(ref=’Surgeon’) 
 TRT(ref=’Saline’); 
MODEL conspicuity = trt ureter rater 
 
 
    (Model 3)  
 
 
trt*ureter rater*ureter rater*trt 
 
 
Rater*ureter*trt 
/LINK=clogit DIST=mult TYPE3; 
REPEATED subject = usubjid / type=ind; 
Run;  
 
PROC GENMOD DATA=resp descending; 
CLASS usubjid ureter(ref=’Right’) Rater(ref=’Surgeon’) 
 TRT (ref=’Saline’); 
MODEL conspicuity = trt ureter rater  
 
 
     (Model 4)  
/Link=clogit DIST=mult TYPE3; 
REPEATED SUBJECT = usubjid / type=ind; 
Run; 
Where rater will be coded as (central vs surgeon).  
This covariate analysis will be performed only for the comparisons between the IC treatment vs 
the Saline treatment.  Each GEE model will be fitted separately for each comparison. 
 
5.5.3. 
Dichotomization of the Difference in Conspicuity 
The third approach is to dichotomize the difference in conspicuity between the surgeon and the 
blinded reader.  The four scores from the surgeon will be paired with the four scores from the 
blinded central reader based on treatment and ureter for a given subject.  Based on the difference 
in the paired conspicuity (see Section 3.8.5) a concordance response variable will be derived for 
each pair of the conspicuity: 
a. the ratings are in agreement when the difference in score is (+1, 0, -1), otherwise   
b. the ratings are not in agreement when the difference is more than +/- 1 point 
The association of this concordance response variable (agree vs not agree) with treatment and 
ureter will also be evaluated using the GEE model based on the concept of logistic regression for 
dichotomized response.   In case of missing data that prevent from forming a pair of observations 
to derive this concordance response variable, the data will be excluded from this analysis.  
The score statistics will be used to determine if the treatment is statistically associated with this 
dichotomized response. The following is an example of SAS syntax for this analysis between the 
IC Hight dose vs Saline. This analysis will be performed separately for each comparison (IC 
High dose vs Saline, IC Low dose vs Saline, and IC High dose vs IC Low dose).  All subjects 
with ≥ 1 missing data from either surgeon or central reader will be excluded from this analysis. 
 
 


 
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Statistical Analysis Plan 
Amendment 2 
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Page 39 of 47 
 
 
PROC GENMOD DATA=dischotH descending; 
CLASS usubjid ureter(ref=’Right’) TRT(ref=’Saline’); 
MODEL response (event=’Y’) = trt ureter trt*ureter                                              (Model 5) 
/LINK=LOGIT DIST=BINOMIAL TYPE3; 
REPEATED subject = usubjid / type=EXCH; 
ESTIMATE ‘OR: IC vs Saline’ trt 1 -1 / exp; 
Run;  
 
PROC GENMOD DATA= dischotH descending; 
CLASS usubjid ureter(ref=’Right’) TRT(ref=’Saline’); 
MODEL response (event=’Y’) = trt ureter                                                      
     (Model 6) 
/LINK=LOGIT DIST=BINOMINAL TYPE3; 
REPEATED SUBJECT = usubjid / TYPE=EXCH; 
ESTIMATE ‘OR: IC vs Saline’ trt 1 -1 / exp; 
 
Where:  
1. ‘TRT’ is coded to IC or Saline 
2. ‘URETER’ is coded to ‘Left’ or ‘Right’ 
3. ‘USUBJID’ I the unique Subject Identification 
4.  REPEATED statement controls the intra-subject correction due to repeated measures. In this 
example, exchangeable working correlation structure is used (TYPE=EXCH). 
The analysis summary table (Table 14.2.5.3) will include distribution of number (%) of pairs 
that are in agreement vs not in agreement in each treatment group by ureter (left vs right), score 
statistics for Type 3 GEE analysis, odds ratio estimates, and 95% confidence intervals for the 
odds ratio between IC High Dose vs Saline, IC Low dose vs Saline and IC High dose vs IC Low 
dose (an exploratory analysis).  All nominal p-values will be displayed as is without control for 
multiplicity.   
 
5.6. 
Covariate Analysis and Subgroup Analysis 
The impact of some potential covariates on the primary efficacy endpoint will be evaluated if 
data warrants. The potential covariates include but not limited to 
1. Surgery type 
2. Surgeons specialty type 
3. Age and age group, sex, race group 
Each variable will be assessed individually by adding the variable to the primary efficacy models 
described in Section 5.1. 
Similarly, if data warrants, subgroup analyses defined by the above variables will be performed. 
All those analyses, if performed, will be exploratory/ confirmatory.  


 
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6. 
PHARMACOKINETICS EVALUATIONS 
PK portion of the study are dropped. Hence, all previously defined PK analyses are no longer 
applicable. 


 
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Amendment 2 
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Page 41 of 47 
 
7. 
SAFETY AND TOLERABILITY EVALUATIONS 
Safety data summaries will be displayed by randomization group for clinical safety laboratory 
test and ECG; adverse events and vital signs will be tabulated by treatment groups.  Safety data 
listing by individual subject will be prepared for all safety endpoints.  
7.1. 
Total Exposure 
All subjects are expected to receive a dose of normal saline and a dose of IC.  Hence, no formal 
total exposure in terms of number of doses or treatment duration will be applied. 
7.2. 
Adverse Events 
Adverse events reported from Day 1 beginning at the Saline injection through the final follow-up 
will be considered treatment emergent adverse events (TEAEs).   Adverse event reported after 
Saline injection and prior to IC injection will be mapped to Saline treatment, all adverse 
events after IC injection, including events reported during the follow period will be 
mapped to IC treatment.  For all AEs, if the onset date is missing/partial, the AE is TEAE 
and will be mapped to IC study unless the available portion of the date/time indicates 
otherwise.   
The Medical Dictionary for Regulatory Activities (Version 22) will be used to classify all AEs 
with respect to system organ class and preferred term. 
The following summary tables will be produced for the TEAEs. All data summaries will provide 
number (%) subjects as well as total number of events in each category by treatment group (IC 
High, IC Low, and Overall). 
1. a topline summary of TEAEs (Table 14.3.2.1) 
2. a summary table by preferred term in descending order of total incidence (Table 14.3.2.2) 
3.  a summary table by system organ class and preferred term (Table 14.3.2.3) 
4. a detailed summary table by system organ class, preferred term and severity 
(Table 14.3.2.4.1) 
5. a detailed summary table by system organ class, preferred term and relationship 
(Table 14.3.2.4.2) 
6. a table of serious TEAEs by system organ class and preferred term (Table 14.3.2.5) 
7. TEAE leading to study discontinuation (Table 14.3.2.6) 
In addition, adverse event of special interest (Table 14.3.2.7) will be identified and summarized 
if data warrant.  Adverse event of special interest (AEOSI) could include but not limit to 
transient elevation of blood pressure and reflex bradycardia. For example, TEAE of tachycardia, 
hypotension, hypertension, rash or erythema, respiratory symptoms, such as dyspnea or 


 
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Amendment 2 
26 June 2021 
 
 
Page 42 of 47 
 
bronchospasm will be flagged as AEOSI.  All adverse events will be reviewed before database 
lock to identify all AEOSI in the study. 
 
7.3. 
Changes in Laboratory Tests 
Safety laboratory tests are scheduled to be performed during screening and first follow-up post 
procedure.  Additional unscheduled laboratory tests would be performed as clinically indicated.  
Local lab will provide the results to each center. 
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. 
Lab results will be tabulated based on local normal ranges [Table 14.3.3.1.1 (hematology), 
Table 14.3.3.1.2 (chemistry)].  That is, the number of subjects with normal, low, or high lab 
results at screening (Baseline) and end of study will be cross-tabulated for IC High dose, IC Low 
dose and Study Overall without any inferential statistics.  This analysis will include only the 
scheduled lab tests. 
Number (%) subjects with abnormal lab results, either clinically significant, or not clinically 
significant (scheduled and unscheduled lab) determined by the investigator, will be tabulated by 
IC dose group and study overall without inferential statistics [Table 14.3.3.2.1 (hematology), 
Table 14.3.3.2.2 (chemistry)].   
7.4. 
Changes in Vital Signs 
During screening and follow-up visits body temperature, pulse rate, pulse oximeter (%) 
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 
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. 
Investigator will determine each vital sign as normal or abnormal, and if it is abnormal, the 


 
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Amendment 2 
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Page 43 of 47 
 
investigator will also determine if the abnormal vital signs is or is not clinically significant. Any 
clinically significant changes to vital signs or heart rhythm will be reported as an adverse event. 
Vital signs data summary will be provided by treatment groups (IC High, IC Low, and Saline). 
Changes in vital signs will be derived for each subject at each timepoint as  
Change = Post baseline – baseline  
Vital signs collected on Day 1 prior to Saline injection will be the baseline vital signs if not 
missing; otherwise, measurements taken at screening visit will served as the baseline. 
The summary table (Table 14.3.4.1) will display descriptive summary of vital signs and change 
from baseline (sample size, mean, median, and standard deviation, range) without inferential 
statistics.   
Number (%) subjects with abnormal vital signs, either clinically significant or not clinically 
significant from all vital signs assessments (scheduled and unscheduled) will be tabulated by 
treatment groups (IC High, IC Low, and Saline) without inferential statistics (Table 14.3.4.2). 
 
7.5. 
Changes in 12-Lead ECG 
During screening and first post procedure follow-up visit, 12-lead ECG will be assessed. ECG 
interpretation will include normal, abnormal but not clinically significant; abnormal and 
clinically significant.   
Number (%) subjects in each results interpretation category at each time point will be tabulated 
by randomization group (IC High, IC Low) and study overall, including shift from normal to 
abnormal at end of study without any inferential statistics (Table 14.3.5).   
Other 12-Lead ECG related parameters, including PR interval, RR interval, QRS duration, QT 
interval, and QTc interval, will also be collected.  Those parameters will not be tabulated, but 
data listings will be provided.   
 
 









Prot_003.pdf: 



Prot_005.pdf: 
Protocol PVP-19IC01 Clarification Letter #2, dated 18 Dec 2020 
Page 1 of 2 
To:   PVP-19IC01 Principal Investigators  
From:  Provepharm PVP-19IC01 Study Team 
RE: 
Removal of Pharmacokinetic sub study from protocol PVP-19IC01 amendment #2: 
An Open-Label, Randomized, Multicenter Study to Evaluate the Safety, Efficacy, 
Pharmacokinetics and Physician Satisfaction of Two Different Doses of 3,3'-Dioxo-2,2'-
bisindolylidene-5,5'-disulfonate disodium 0.8% Solution When Used as an Aid in the 
Determination of Ureteral Patency. 
This Clarification Letter is to remove the pharmacokinetic (PK) sub-study from the PVP-19IC01 
protocol, Amendment # 2 dated 02 September 2020.  
This protocol specifies that PK samples of plasma, urine and stool will be collected from a subset 
of approximately 16 subjects from 2 participating PK sites. 
Zero patients have been enrolled into the PK sub study due to the difficulty in recruiting patients 
for the clinical trial during the COVID-19 emergency.   Additionally, the logistical complexity 
involved with conducting the PK portion has made it challenging for the 2 sites to recruit 
consenting subjects.  Therefore, the recruitment of patients for the PK sub study of the trial will 
be discontinued.  A separate PK study will be conducted to obtain the necessary PK data.  
Additionally, it was noted that the header of the approved protocol amendment 2, starting at page 
19 through the end of the protocol, references the year 2019.  The correct year should be 2020. 
Please sign and date in the acknowledgment section below, return a copy of the signed document 
to Provepharm, Inc. and file the original in the current protocol section of your investigational 
site file. 
NCT#: 04228445


Protocol PVP-19IC01 Clarification Letter #2, dated 18 Dec 2020 
Page 2 of 2 
Approvals: 
Sponsor 
 
Director, Clinical Operations 
Signature and Date 
Medical Monitor 
 
 
Signature and Date 
Acknowledged: 
Principal Investigator: 
Printed Name 
Signature and Date 

Prot_002.pdf: 
Clinical Study Protocol 
An Open-Label, Randomized, Multicenter Study to Evaluate the 
Safety, Efficacy, Pharmacokinetics and Physician Satisfaction of Two 
Different Doses of 3,3'-Dioxo-2,2'-bisindolylidene-5,5'-disulfonate 
disodium 0.8% Solution When Used as an Aid in the Determination of 
Ureteral Patency. 
PVP-19IC01 
Version 2.0 
Amendment 1 
Development Phase: 
3 
Investigational Product: 
3,3'-Dioxo-2,2'-bisindolylidene-5,5'-disulfonate 
disodium (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: 
01 July 2019 
Administrative Change 1 Date: 
18 October 2019 
Amendment 1 Date: 
10 December 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. 
NCT# 04228445


Provepharm Inc. 
 
Indigo Carmine 0.8%  
Protocol PVP-19IC01 
CONFIDENTIAL 
Version 2.0, dated 10 December 2019 
Page 2 of 68 
 
CONFIDENTIAL INFORMATION 
This document is the sole property of Provepharm Inc. This document and any and all 
information contained herein have 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. 




Provepharm Inc. 
 
Indigo Carmine 0.8%  
Protocol PVP-19IC01 
CONFIDENTIAL 
Version 2.0, dated 10 December 2019 
Page 4 of 68 
 
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 
EDC 
Electronic Data Capture 
eCRF 
Electronic Case Report Form 
FDA 
Food and Drug Administration 
FPFV 
First Patient First Visit 


Provepharm Inc. 
 
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Protocol PVP-19IC01 
CONFIDENTIAL 
Version 2.0, dated 10 December 2019 
Page 5 of 68 
 
GCP 
Good Clinical Practices 
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 
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 


Provepharm Inc. 
 
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Protocol PVP-19IC01 
CONFIDENTIAL 
Version 2.0, dated 10 December 2019 
Page 6 of 68 
 
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 


Provepharm Inc. 
 
Indigo Carmine 0.8%  
Protocol PVP-19IC01 
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Version 2.0, dated 10 December 2019 
Page 7 of 68 
 
Table of Contents 
Sponsor Signature Page .................................................................................................................3 
ABBREVIATIONS ........................................................................................................................4 
1 
PROTOCOL SUMMARY ...........................................................................................10 
1.1. 
Protocol Synopsis ...........................................................................................................10 
1.2. 
Study Schema .................................................................................................................17 
1.3. 
Schedule of Assessments (SoA) .....................................................................................18 
2. 
INTRODUCTION ........................................................................................................19 
2.1. 
Study Rationale ...............................................................................................................19 
2.2. 
Background .....................................................................................................................20 
3. 
OBJECTIVES AND ENDPOINTS .............................................................................21 
3.1. 
Objectives .......................................................................................................................21 
3.1.1. 
Primary Objective ...........................................................................................................21 
3.1.2. 
Secondary Objectives......................................................................................................21 
3.2. 
Endpoints ........................................................................................................................22 
3.2.1. 
Efficacy Endpoints ..........................................................................................................22 
3.2.2. 
Safety Endpoints .............................................................................................................22 
4. 
STUDY DESIGN...........................................................................................................23 
4.1. 
Overall Design ................................................................................................................23 
4.2. 
Scientific Rationale for Study Design ............................................................................23 
4.3. 
Justification for Dose ......................................................................................................24 
4.4. 
End of Study Definition ..................................................................................................24 
5. 
STUDY POPULATION ...............................................................................................25 
5.1. 
Inclusion Criteria ............................................................................................................25 
5.2. 
Exclusion Criteria ...........................................................................................................25 
5.3. 
Screen Failures ................................................................................................................26 
6. 
STUDY DRUG ..............................................................................................................27 
6.1. 
Study Drug(s) Administered ...........................................................................................27 
6.2. 
Preparation/Handling/Storage/Accountability ................................................................28 
6.3. 
Measures to Minimize Bias: Randomization and Blinding ............................................28 
6.4. 
Study Drug Compliance..................................................................................................29 
6.5  
Concomitant Therapy .....................................................................................................29 
6.6 
Prohibited Concomitant Medications/Foods/Supplements .............................................30 
7. 
DISCONTINUATION OF STUDY DRUG AND SUBJECT 
DISCONTINUATION/WITHDRAWAL ...................................................................31 
7.1. 
Subject Discontinuation/Withdrawal from the Study .....................................................31 
7.2. 
Lost to Follow up ............................................................................................................31 
8. 
STUDY ASSESSMENTS AND PROCEDURES .......................................................33 
8.1. 
Efficacy Assessments .....................................................................................................35 
8.1.1. 
Ureter Assessment ..........................................................................................................35 
8.1.2. 
Physician Satisfaction Agreement Scale (PSAS) ...........................................................35 
8.1.3. 
Videography specifics .....................................................................................................36 


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Version 2.0, dated 10 December 2019 
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8.2. 
Safety Assessments .........................................................................................................36 
8.2.1. 
Physical Examinations ....................................................................................................36 
8.2.2. 
Vital Signs .......................................................................................................................36 
8.2.3. 
Electrocardiograms .........................................................................................................37 
8.2.4. 
Clinical Safety Laboratory Assessments ........................................................................37 
8.3. 
Adverse Events and Serious Adverse Events .................................................................38 
8.3.1. 
Time Period and Frequency for Collecting AE and SAE 
Information .....................................................................................................................38 
8.3.2. 
Method of Detecting AEs and SAEs ..............................................................................39 
8.3.3. 
Follow-up of AEs and SAEs ...........................................................................................39 
8.3.4. 
Regulatory Reporting Requirements for SAEs ...............................................................39 
8.3.5. 
Pregnancy ........................................................................................................................39 
8.4. 
Pharmacokinetics ............................................................................................................40 
8.4.1. 
Plasma samples ...............................................................................................................40 
9. 
STATISTICAL CONSIDERATIONS ........................................................................42 
9.1. 
Statistical Hypotheses .....................................................................................................42 
9.2. 
Sample Size Determination ............................................................................................42 
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...............................................................................................................................42 
9.2.2. 
Table 2  Number of Subjects Per Group Using Two-group Chi-
square Test Comparing Proportions in 3 Categories [1, 2] .............................................42 
9.3. 
Populations for Analyses ................................................................................................43 
9.4. 
Statistical Analyses .........................................................................................................44 
9.4.1. 
Type 1 Error Control for the Primary Efficacy Endpoint ...............................................44 
9.4.2. 
Treatment Effect Based on the Operating Surgeon’s UOVS 
Scores ..............................................................................................................................44 
9.4.3. 
Treatment Effect on UOVS Based on Central Rater’s 
Assessment ......................................................................................................................45 
9.4.4. 
Treatment Effect on Time to Visualization ....................................................................45 
9.4.5. 
Assessment of PSAS with IC Treatment ........................................................................45 
9.4.6. 
Assessment of Covariates ...............................................................................................46 
9.4.7. 
Safety Analysis ...............................................................................................................46 
9.4.8. 
PK Analysis ....................................................................................................................46 
10. 
SUPPORTING DOCUMENTATION AND OPERATIONAL 
CONSIDERATIONS ....................................................................................................47 
10.1. 
Regulatory, Ethical, and Study Oversight Considerations .............................................47 
10.1.1. Regulatory and Ethical Considerations ...........................................................................47 
10.1.2. Financial Disclosure........................................................................................................47 
10.1.3. Informed Consent Process ..............................................................................................48 
10.1.4. Institutional Review Board/Independent Ethics Committee (IRB) ................................48 
10.1.5. Data Protection................................................................................................................49 
10.1.6. Dissemination of Clinical Study Data.............................................................................49 
10.1.7. Data Quality Assurance ..................................................................................................50 
10.1.8. Source Documents ..........................................................................................................50 
10.1.9. Study and Site Start and Closure ....................................................................................51 


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10.1.10. Publication Policy ...........................................................................................................51 
10.2. 
Adverse Events: Definitions and Procedures for Recording, 
Evaluating, Follow-up, and Reporting ............................................................................52 
10.2.1. Definition of AE .............................................................................................................52 
10.2.2. Definition of SAE ...........................................................................................................53 
10.2.3. Recording and Follow-Up of AE and/or SAE ................................................................54 
11. 
REFERENCES..............................................................................................................58 
Appendix 1: 
   Administrative Change #1 Summary of Changes ......................................60 
Appendix 2: 
   Amendment # 1 Summary of Changes ........................................................61 
 


Provepharm Inc. 
 
Indigo Carmine 0.8%  
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Version 2.0, dated 10 December 2019 
Page 10 of 68 
 
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 3,3'-Dioxo-2,2'-bisindolylidene-5,5'-disulfonate disodium 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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Protocol PVP-19IC01 
CONFIDENTIAL 
Version 2.0, dated 10 December 2019 
Page 11 of 68 
 
5. An exploratory comparison will be performed to assess 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 
3,3'-Dioxo-2,2'-bisindolylidene-5,5'-disulfonate disodium commonly 
referred to as 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 in a 1:1 ratio 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. 


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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. 
In a subset of subjects from 2 sites (approximately 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 Drug 
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 
approximately 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 
The study plans to randomize  96 subjects ; 48 subjects to 2.5 mL IC and 48 
subjects 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% (about 20) subjects 


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may be enrolled to account for protocol deviations, withdrawal of consent, 
etc., if necessary. 
A subgroup of subjects at 2 sites (Approximate N=16, at least 8 from each 
IC dose level) will be evaluated for pharmacokinetics/metabolites. 
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  


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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 
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 include 
1. Incidence of treatment emergent adverse events 
2. Changes in safety laboratory tests and vital signs 
3. Changes in 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-∞  
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 model will be used to 
calculate PK parameters using the observed concentrations and actual 
sampling times.  All bioanalytical analyses will be completed to GLP 


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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 
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. 


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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 the first bowel movement 
post-surgery in this same subgroup. 
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). 




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1.3. 
Schedule of Assessments (SoA) 
 
 
Screening 
period 
Day -30 to -
11 
Randomization/ 
Treatment 
Day 1 
 
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 
 
 
 
 
Vital Signs 
X 
X3 
 
X 
 
12 Lead ECG 
X 
 
 
X 
 
Randomization 
 
X 
 
 
 
Concomitant Medications 
X 
X 
 
X 
X 
AE/SAE 
 
X 
 
X 
X 
Blood Safety Labs4 
X 
 
 
X5 
 
PK Blood6 
 
X 
 
 
 
PK Urine7 
 
X 
 
 
 
Bowel Prep8 
X (Day -1) 
 
 
 
 
Stool collection for assay9 (PK group) 
 
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 approximately 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 
(Voelcker 1903) 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 (Chi 2016, Cohen 2018).  
 
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 (Ibeanu 2009).  
 
 


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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 (Grimes 2017). In the US, approximately 
600,000 hysterectomies, 226,000 prolapse repairs, and 135,000 stress incontinence operations 
are performed annually (Vakili 2005; 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 from approximately 16 subjects from 2 
investigational sites. 
5. An exploratory comparison will be performed to assess 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 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 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. 
E09. Subjects who are pregnant or breast-feeding 


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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 and eligibility 
criteria. 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.  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 
 
Randomization 
All subjects will be centrally assigned to randomized study drug using the randomization feature of 
the electronic data capture (EDC) system.  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 the EDC system 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. 
 
 


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6.6 
Prohibited Concomitant Medications/Foods/Supplements 
Prohibited medications, foods, and/or supplements are anything that could discolor urine. The 
length of time required from last dose to study randomization is at the discretion of the 
investigator and should be based on the known duration of the effect on urine coloration and 
documented in the source documentation. Examples of prohibited medications are: 
 
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 the EDC system 
• 
Randomization/Treatment (Day 1/Day of Surgery/Treatment) 
o After a subject is confirmed to be eligible, the subject will be randomized via 
the EDC system. Randomization will be a 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 post 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 the 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.  
c. The surgeon will document the UOVS score following the use of saline in 
the assessment of the patency of the ureters.  
 


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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.   
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. Urine 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. 
 


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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: 
• 
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 


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3 = Neither Agree nor Disagree 
4 = Disagree 
5 = Strongly disagree 
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 and weight.  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 


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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 
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, 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 


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return to normal or baseline or are no longer considered clinically significant by the investigator 
or medical monitor. 
• 
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 After IC Treatment 
Subjects in whom ureteral patency was not visualized or was poorly visualized after IC treatment 
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 AEs and 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 


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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. 
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 or female partners of male 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. 


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Abnormal pregnancy outcomes (e.g., spontaneous abortion, fetal death, stillbirth, congenital 
anomalies, ectopic pregnancy) are considered SAEs. 
 
8.4. 
Pharmacokinetics 
PK samples of plasma, urine, and stool will be collected from approximately 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 of plasma (4 ml of whole blood) 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. 
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-∞  
2. 
AUCextr, AUC%extr  
3. 
Cmax, Clast, Tmax, and Tlast  
4. 
λz: the apparent plasma terminal phase rate constant. 


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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 model 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 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 was 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 expected 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 (approximate 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.  
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 be 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 performed to assess 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.  
In this 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 site will 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 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 model 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 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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• 
Initial notification via telephone does not replace the need for the investigator to 
complete and sign the SAE CRF pages within the designated reporting time frames. 
• 
Contacts for SAE reporting can be found in Study Contact List. 


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11. 
REFERENCES 
 
1. 
AAGL Practice Committee.  AAGL Practice Report: Practice guidelines for 
intraoperative cystoscopy in laparoscopic hysterectomy. J Minim Invasive Gynecol 
2012;19:407-411 
 
2. 
American Regent Package Insert. Indigo Carmine Injection American Regent, Inc. 
NDC:0517-0375-05. Date of revision 02/2017. 
 
3. 
Barbieri R. Farewell to Indigo Carmine. OBG Management 2014; 26(9):8-9,12 
 
4. 
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 
 
5. 
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 
 
6. 
Cohen SA, Carberry CL and Smilen SW. American Urogynecologic Society consensus 
statement: cystoscopy at the time of prolapse repair.  Female Pelvic Med Reconstr Surg 
2018;24:258–259 
 
7. 
Gill EJ, Elser DM, Bonidie MJ, Roberts KM, Hurt WG. The routine use of cystoscopy 
with the Burch procedure. Am J Obstet Gynecol. 2001 Aug;185(2):345-8. 
8. 
Gilmour D and Flowerdew G. Rates of urinary tract injury from gynecologic surgery and 
the role of intraoperative cystoscopy. Obstet Gynecol 2006;107:1366–1372 
 
9. 
Grimes CL, Patankar S, Ryntz T et al.  Evaluating ureteral patency in the post-indigo 
carmine era: a randomized controlled trial. Am J Obstet Gynecol. 2017;217:601.e1 – 10 
 
10. 
Harris RL, Cundiff GW, Theofrastous JP, Yoon H, et al. The value of intraoperative 
cystoscopy in urogynecologic and reconstructive pelvic surgery. Am J Obstet Gynecol 
1997; 177:1367-71. 
 
11. 
Ibeanu O, Chesson RR, Echols KT et al. Urinary tract injury during hysterectomy based 
on universal cystoscopy. Obstet Gynecol 2009;113:6–10 
 
12. 
Jabs CF, Drutz HP. The role of intraoperative cystoscopy in prolapse and incontinence 
surgery. Am J Obstet Gynecol 2001;185(6):1368–73 
 
 


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13. 
Jelovsek JE, Chen G, Roberts SL, et al. Incidence of lower urinary tract injury at the time 
of total laparoscopic hysterectomy. J Soc Laparoendoscopic Surg 2007; 11:422-27. 
 
14. 
Jung M, Kiesslich R. Chromoendoscopy and intravital staining techniques, Baillière’s 
Clinical Gastroenterology 1999; 13(1):11-19 
 
15. 
Lacy, W.W., Ugaz, C., Newman, E.V. The use of indigo carmine for dye dilution curves.  
Circulation Research 1955; 111: 570-574 
 
16. 
Lee M, Sharifi R. Methylene blue versus indigo carmine. Urology 1996;47(5):783-4. 
 
17. 
O’Brien WM, Lynch JH. Simplification of double-dye test to diagnose various types of 
vaginal fistulas. Urology 1990;36(5):456 
18. 
Oravisto, K.J., Investigations into the excretion mechanism of indigo carmine in normal 
human kidney. Ann. Chir. Gynaecol. Fenn Suppl. 1957;46(2):1-79 
 
19. 
Pettit PD, Petrou SP. The value of cystoscopy in major vaginal surgery.  Obstet Gynecol. 
1994;84(2):318-20 
 
20. 
Ribeiro S, Reich H, et al. The Value of intra-operative cystoscopy at the time of 
laparoscopic hysterectomy. Human Reproduction 1999; 14(7): 1727-1729 
 
21. 
Speights SE, Moore RD, Miklos JR. Frequency of lower urinary tract injury at 
laparoscopic burch and paravaginal repair. J Am Assoc Gynecol Laparosc. 2000; 7:515-8 
 
22. 
SmPC (Summary of Product Characteristics). Indigo Carmine 40 mg/5 mL solution for 
injection. SERB S.A, Belgium. Marketing Authorisation Number: PL 43956/0001. Date 
of Revision of the Text: 05/06/2015 
 
23. 
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 
 
24. 
Voelcker F, Joseph, E, Funktionelle Nierendiagnostik ohne Ureterenkatheter. Munch. 
Med. Wschr. 1903; 50: 2081-2089 
 
25. 
Weinstein JR and Anderson S. The aging kidney: physiological changes.  Adv Chronic 
Kidney Dis. 2010 July ; 17: 302–307 
 
 
 


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Appendix 1: 
   Administrative Change #1 Summary of Changes 
 
 Version 1.1 Administrative Change 1 
 
1. Addition of reference cited in document to the bibliography 
2. Update of Sponsor contact information 
3. Correction of mis-numbered section (6.6) 
4. Clarification of timing for prohibited Medications/Foods/Supplements 
5. Clarification of packaging for Indigo Carmine in section 6.2 
 


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Change descriptions in italics are descriptive rather than representative                 Page 61 of 68 
 
 
Appendix 2: 
   Amendment # 1 Summary of Changes 
Page  Section # 
Change 
Reason / 
Rationale 
Title 
page, 
page 
3 
and 
Page 
10 
Study Title 
An Open-Label, Randomized, Multicenter Study 
to Evaluate the Safety, Efficacy, 
Pharmacokinetics and Physician Satisfaction of 
two different doses of 3,3'-Dioxo-2,2'-
bisindolylidene-5,5'-disulfonate disodium Indigo 
Carmine Injection 0.8% solution when used as 
an aid in the determination of ureteral patency. 
To include the 
IUPAC name in 
the title 
Title 
page 
Investigational 
Product 
3,3'-Dioxo-2,2'-bisindolylidene-5,5'-disulfonate 
disodium (Indigo Carmine 0.8% Injection, USP) 
To correlate the 
IUPAC name 
with the USP 
name of the 
investigational 
product 
11 
1.1 
Secondary 
Objectives (5) 
5. 
An exploratory comparison will be 
performed to assess done on the difference 
between the IC high dose vs IC low dose.  
Surgeons will be blinded to the dose of IC. 
Clarity and 
readability 
11 
1.1 
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 3,3'-Dioxo-2,2'-bisindolylidene-5,5'-
disulfonate disodium commonly referred to as 
Indigo Carmine (IC) 0.8% Injection, USP 
solution for injection 
To correlate the 
IUPAC name 
with the USP 
name of the 
investigational 
product 
11 
1.1 
Methodology 
 
 
 
 
 
 
Eligible subjects will be randomized in a 1:1 
ratio to receive a dose of either IC high dose (5 
mL) or IC low dose (2.5 mL). 
To specify the 
randomization 
ration 


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Reason / 
Rationale 
12 
1.1 
Methodology 
 
 
 
 
In a subset of subjects from 2 sites 
(approximately about 16 subjects), subjects will 
be consented to participate in the 
pharmacokinetic (PK) portion of the study 
Readability 
12 
1.1 
Study Drug 
Section Title changed:  Study Medication Drug 
Consistency 
throughout 
protocol 
12 
1.1 
Number of 
Subjects 
Up to 116 subjects will be enrolled from 
approximately 10 sites, including approximately 
16 subjects to participate in PK/metabolite 
analysis at 2 sites 
Clarification and 
consistency 
12 
1.1 
Sample Size 
The study plans to randomize 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% (about 20) subjects may 
be enrolled to account for protocol deviations, 
withdrawal of consent, etc., if necessary. 
A subgroup of subjects at 2 sites (total 
Approximate N=16, at least 8 from each IC dose 
level) will be evaluated for 
pharmacokinetics/metabolites 
Clarification and 
consistency 
14 
1.1 
Safety 
Endpoints (3) 
3. 
Changes in clinically significant 
abnormal 12-ECG 
Clarity and 
readability 


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Page  Section # 
Change 
Reason / 
Rationale 
14 
1.1 
PK Parameters 
(1) 
1. 
AUC0-t, AUC0 t(90%subj), AUC0-∞ 
Consistency with 
statistical 
analysis plan 
14 
PK Analysis 
Non-compartmental analysis model will be used 
to calculate PK parameters using the observed 
concentrations and actual sampling times. 
Clarity and 
accuracy 
15 
1.1 
Procedure 
Summary 
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 the first bowel movements within the first 
24 hours post-surgery in this same subgroup. 
Correction and 
consistency 
throughout 
protocol 
18 
1.3 SOA 
Deleted weight assessment during the Follow-up 
visit 
Post-surgery 
weight not 
required for 
study purposes  
18 
1.3 SOA 
Footnote # 7 
For a total of approximately 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. 
Clarification 
18 
1.3 SOA 
“IVRS/IWRS contact” row renamed to 
“randomization” and “X” removed from all 
columns except for the 
“Randomization/Treatment Day 1” column 
An IVRS/IWRS 
system will not 
be used for this 
study.  
Randomization 
will be 
completed via the 
EDC system 
18 
1.3 SOA 
“Day 2 PK” 
column 
Column deleted 
Based on the PK 
sampling 
schedule a 
separate Day 2 
visit is not 
expected  


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Reason / 
Rationale 
21 
3.1.2 
Secondary 
objective # 4 
4. 
To determine the IC pharmacokinetic 
profile from in a subset of subjects 
approximately 16 subjects from 2 investigational 
sites. 
Clarification 
21 
3.1.2 
Secondary 
objective # 5 
5. 
An exploratory comparison will be done 
on performed to assess the difference between 
the IC high dose vs IC low dose. 
Clarity and 
readability 
26 
5.3 Screen 
Failures 
Minimal information includes demography, 
screen failure details and, eligibility criteria. and 
any serious adverse event (SAE). 
Serious Adverse 
Events will not 
be captured for 
subjects who are 
not dosed with 
study drug. 
28 
6.3 Measures to 
minimum Bias 
IVRS/IWRS 
Randomization 
All subjects will be centrally 
assigned to randomized study 
drug using an Interactive 
Voice/Web Response System 
(IVRS/IWRS).the 
randomization feature of the 
electronic data capture (EDC) 
system.  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. 
An IVRS/IWRS 
system will not 
be used for 
randomization 
29 
6.4 Study Drug 
Compliance 
The randomized dose of IC treatment will be 
provided by IVRS/IWRS the EDC system to the 
designated unblinded site staff 
An IVRS/IWRS 
system will not 
be used for 
randomization 
30 
6.6 
Prohibited medications, foods, and/or supplements 
would be are anything that could discolor urine. 
 


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Change 
Reason / 
Rationale 
33 
8 
Screening 
o 
Once consented, the subject will be entered 
into IVRS/IWRS the EDC system.  Randomization will 
be a 1:1 ratio to be assigned to low dose (2.5 mL IC) 
or high dose (5 mL IC). 
An IVRS/IWRS 
system will not 
be used for 
randomization 
33 
8 
Randomization/ 
Treatment 
o 
After a subject is confirmed to be eligible, 
the subject will be randomized via IVRS/IWRS the 
EDC system. 
An IVRS/IWRS 
system will not 
be used for 
randomization 
33 
8 
Dosing step by 
step: 1 
by identifying the efflux of urine from the ureteral 
orifices for up to 10 minutes for each injection post 
injection. 
Clarity 
33 
8 
Dosing step by 
step: 1c 
c. 
The surgeon will document the UOVS score 
following the use of saline in the assessment of the 
patency of the ureters. 
Addition of step 
for clarity of 
procedure 
33 
8 
Dosing step by 
step: 5 
Urine and stool will be collected at specified time 
points in this same subgroup. 
Added for 
completeness 
36 
8.2.1 
 
These will be repeated at the follow-up visit except 
for height and weight. 
Weight is not 
required during 
the follow-up 
visit for study 
purposes 
37 
8.2.3 
Initial ECGs will be assessed by an investigator, 
addition to the measurements, as normal or 
abnormal finding. 
Typographical 
correction 
38 
8.2.5 
Section Title 
Follow-up for Subjects in whom Ureteral Patency 
was Not Visualized or Poorly Visualized After IC 
Treatment 
Clarity 
38 
8.25 
Subjects in whom ureteral patency was not 
visualized or was poorly visualized after IC 
treatment will be treated with appropriate follow-
up as per the discretion of the surgeon. 
Clarity 


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Reason / 
Rationale 
38 
8.3.1 
 
All AEs and SAEs will be collected from the start of 
the saline injection until the follow-up visit. 
to specify the 
timing of AE 
collection 
39 
8.3.5 
Details of all pregnancies in female subjects or 
female partners of male subjects 
To include 
pregnancy 
follow-up for 
pregnancies that 
occur during the 
study in female 
partners of male 
subjects 
40 
8.4 
PK samples of plasma, urine, and stool will be 
collected from approximately 16 subjects 
Clarification 
40 
8.4.1 
 
• 
Approximately 2 mL of plasma (4 ml of 
whole blood) will be collected for each 
measurement of plasma concentrations 
Clarity 
40 
8.4.3 
• 
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 
Correction and 
consistency 
throughout 
protocol 
40 
8.4.3 
The pharmacokinetic (PK) analysis will include at 
least the following parameters:  
1. 
AUC0-t, AUC0 t(90%subj), AUC0-∞ 
Consistency with 
statistical 
analysis plan 
41 
8.4.3 
Non-compartmental Analysis model will be used to 
calculate PK parameters using the observed 
concentrations and actual sampling times. 
Clarity 
42 
9.2 
The following empirical distribution table (Table 1) is 
created to illustrate this effect size; sample size was 
is determined using the two-group chi-square test 
comparing the difference in proportions in 3 
categories (Table 2).     
Accuracy and 
readability 
43 
9.2.2 
A subgroup of subjects at 2 sites (total approximate 
N=16, 8 from each IC dose level) will be evaluated 
for pharmacokinetics/metabolites. 
Clarification 


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Rationale 
43 
9.3 
Per Protocol 
Analysis Set 
This Analysis Set will serve as a confirmation analysis 
set for efficacy analysis.  who   
Correction of 
typographical 
error 
43 
9.3 
Safety Analysis 
Set 
Subjects will be analyzed according to the drug they 
actually received should the randomized IC dose is 
be different from the actual received IC dose. 
Correction of 
typographical 
error 
45 
9.4.2 
An exploratory comparison will be performed to 
assess 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. 
Clarity and 
readability 
45 
9.4.3 
In this approach a A new covariate indicating 
evaluator (surgeon vs blinded central reviewer) will 
be added to the GEE model. 
Readability and 
clarity 
45 
9.44 
Potential covariates (such as randomization strata 
and site will also be evaluated) if data warrants. 
Accuracy as 
there are no 
randomization 
strata planned 
46 
9.4.6 
The effect of procedure on the response 
(randomization stratum) will be assessed via 
covariate analysis.  An appropriate model will be 
chosen for specific parameter. 
Accuracy as 
there are no 
randomization 
strata planned 
46 
9.4.8 
PK parameters will be estimated using Non-
compartmental analysis model based on the 
observed concentration; 
Clarity 
56 
10.2.3 
Follow-up of 
AEs and SAEs 
Bullet #4 
• 
The investigator will submit any new or 
updated SAE data to PV within 24 hours of receipt of 
the information via the SAE Reporting Form. 
 


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Page  Section # 
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Reason / 
Rationale 
56 
10.2.4 
SAE Reporting 
to PV via an 
Electronic Data 
Collection Tool 
Section number and Entire table deleted 
Reporting of 
SAEs through an 
electronic data 
collection tool is 
not applicable for 
this study 
56 
10.2.3 
SAE Reporting 
to PV via Paper 
SAE Reporting 
Form 
SAE Reporting to PV via Paper SAE Reporting Form 
CRF 
SAE Reporting 
will be via a 
reporting form 
not a paper CRF. 
56 
10.2.3 
Bullet 1  
• 
The primary mechanism for reporting an 
SAE to PV will be via email of a Facsimile 
transmission or scanned copy of the completed SAE 
paper CRF reporting form to PV.  If email is not 
available, facsimile transmission is acceptable are 
the preferred method to transmit this information 
to PV. 
The primary 
method of SAE 
Reporting will be 
via a paper SAE 
reporting form 
not a paper CRF. 
59 
11. 
 
20. 
Ribeiro S, Reich H, et al. The Value of intra-
operative cystoscopy at the time of laparoscopic 
hysterectomy. Human Reproduction 1999; 14(7): 
1727-1729 
Reference added 
All 
N/A 
Corrections of minor typographical and spelling 
errors that had no effect on meaning were also made 
throughout the document  
N/A 
 

Prot_004.pdf: 
Clinical Study Protocol 
An Open-Label, Randomized, Multicenter Study to Evaluate the 
Safety, Efficacy, Pharmacokinetics and Physician Satisfaction of Two 
Different Doses of 3,3'-Dioxo-2,2'-bisindolylidene-5,5'-disulfonate 
disodium 0.8% Solution When Used as an Aid in the Determination of 
Ureteral Patency. 
PVP-19IC01 
Version 3.0 
Amendment 2 
Development Phase: 
3 
Investigational Product: 
3,3'-Dioxo-2,2'-bisindolylidene-5,5'-disulfonate 
disodium (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: 
01 July 2019 
Administrative Change 1 Date: 
18 October 2019 
Amendment 1 Date: 
10 December 2019 
Amendment 2 Date: 
02 September 2020 
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 R2 GCP) and regulatory requirements as 
applicable. 
NCT#: 04228445


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CONFIDENTIAL INFORMATION 
This document is the sole property of Provepharm Inc. This document and any and all 
information contained herein have 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. 




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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 
EDC 
Electronic Data Capture 
eCRF 
Electronic Case Report Form 
FDA 
Food and Drug Administration 
FPFV 
First Patient First Visit 
GCP 
Good Clinical Practices 
GEE 
Generalized Estimating Equation 
GFR 
Glomerular Filtration Rate 
GLP 
Good Laboratory Practices 


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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 
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 
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 


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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 
VS 
Vital Signs 
WBC 
White Blood Cell 
WOCBP 
Woman of Childbearing Potential 


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Table of Contents 
Sponsor Signature Page .................................................................................................................3 
ABBREVIATIONS ........................................................................................................................4 
1 
PROTOCOL SUMMARY ...........................................................................................10 
1.1. 
Protocol Synopsis ...........................................................................................................10 
1.2. 
Study Schema .................................................................................................................18 
1.3. 
Schedule of Assessments (SoA) .....................................................................................19 
2. 
INTRODUCTION ........................................................................................................20 
2.1. 
Study Rationale ...............................................................................................................20 
2.2. 
Background .....................................................................................................................21 
3. 
OBJECTIVES AND ENDPOINTS .............................................................................22 
3.1. 
Objectives .......................................................................................................................22 
3.1.1. 
Primary Objective ...........................................................................................................22 
3.1.2. 
Secondary Objectives ......................................................................................................22 
3.2. 
Endpoints ........................................................................................................................23 
3.2.1. 
Efficacy Endpoints ..........................................................................................................23 
3.2.2. 
Safety Endpoints .............................................................................................................23 
4. 
STUDY DESIGN ..........................................................................................................25 
4.1. 
Overall Design ................................................................................................................25 
4.2. 
Scientific Rationale for Study Design ............................................................................25 
4.3. 
Scientific Rationale for Development of a New Conspicuity 
Score ...............................................................................................................................26 
4.4. 
Justification for Dose ......................................................................................................26 
4.5. 
End of Study Definition ..................................................................................................26 
5. 
STUDY POPULATION ...............................................................................................27 
5.1. 
Inclusion Criteria ............................................................................................................27 
5.2. 
Exclusion Criteria ...........................................................................................................27 
5.3. 
Screen Failures ................................................................................................................28 
6. 
STUDY DRUG ..............................................................................................................29 
6.1. 
Study Drug(s) Administered ...........................................................................................29 
6.2. 
Preparation/Handling/Storage/Accountability ................................................................30 
6.3. 
Measures to Minimize Bias: Randomization and Blinding ............................................30 
6.4. 
Study Drug Compliance..................................................................................................31 
6.5  
Concomitant Therapy .....................................................................................................31 
6.6 
Prohibited Concomitant Medications/Foods/Supplements .............................................32 
7. 
DISCONTINUATION OF STUDY DRUG AND SUBJECT 
DISCONTINUATION/WITHDRAWAL ...................................................................33 
7.1. 
Subject Discontinuation/Withdrawal from the Study .....................................................33 
7.2. 
Lost to Follow up ............................................................................................................33 
8. 
STUDY ASSESSMENTS AND PROCEDURES .......................................................34 
8.1. 
Efficacy Assessments .....................................................................................................36 
8.1.1. 
Conspicuity Assessment .................................................................................................36 
8.1.2. 
Physician Satisfaction Agreement Scale (PSAS) ...........................................................36 
8.1.3. 
Videography Specifics and Central Review Process ......................................................36 
8.2. 
Safety Assessments .........................................................................................................37 
8.2.1. 
Physical Examinations ....................................................................................................37 


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8.2.2. 
Vital Signs .......................................................................................................................37 
8.2.3. 
Electrocardiograms .........................................................................................................38 
8.2.4. 
Clinical Safety Laboratory Assessments ........................................................................38 
8.3. 
Adverse Events and Serious Adverse Events .................................................................39 
8.3.1. 
Time Period and Frequency for Collecting AE and SAE 
Information .....................................................................................................................39 
8.3.2. 
Method of Detecting AEs and SAEs ..............................................................................39 
8.3.3. 
Follow-up of AEs and SAEs ...........................................................................................40 
8.3.4. 
Regulatory Reporting Requirements for SAEs ...............................................................40 
8.3.5. 
Pregnancy ........................................................................................................................40 
8.4. 
Pharmacokinetics ............................................................................................................40 
8.4.1. 
Plasma samples ...............................................................................................................41 
9. 
STATISTICAL CONSIDERATIONS ........................................................................42 
9.1. 
Statistical Hypotheses .....................................................................................................42 
9.2. 
Sample Size Determination ............................................................................................42 
9.2.1. 
Distribution of Conspicuity Score and IC Treatment Effect Size 
[1, 2] ................................................................................................................................42 
9.3. 
Populations for Analyses ................................................................................................43 
9.4. 
Statistical Analyses .........................................................................................................44 
9.4.1. 
Interim Analysis ..............................................................................................................44 
9.4.2. 
Type 1 Error Control for the Primary Efficacy Endpoint ...............................................46 
9.4.3. 
Treatment Effect on Conspicuity Score ..........................................................................46 
9.4.4. 
Time to Visualization ......................................................................................................47 
9.4.5. 
Assessment of PSAS with IC Treatment ........................................................................48 
9.4.6. 
Assessment of Covariates ...............................................................................................48 
9.4.7. 
Safety Analysis ...............................................................................................................48 
9.4.8. 
PK Analysis ....................................................................................................................48 
10. 
SUPPORTING DOCUMENTATION AND OPERATIONAL 
CONSIDERATIONS ....................................................................................................49 
10.1. 
Regulatory, Ethical, and Study Oversight Considerations .............................................49 
10.1.1. Regulatory and Ethical Considerations ...........................................................................49 
10.1.2. Financial Disclosure ........................................................................................................49 
10.1.3. Informed Consent Process ..............................................................................................49 
10.1.4. Institutional Review Board/Independent Ethics Committee (IRB) ................................50 
10.1.5. Data Protection................................................................................................................51 
10.1.6. Dissemination of Clinical Study Data .............................................................................51 
10.1.7. Data Quality Assurance ..................................................................................................52 
10.1.8. Source Documents ..........................................................................................................52 
10.1.9. Study and Site Start and Closure ....................................................................................53 
10.1.10. Publication Policy ...........................................................................................................53 
10.2. 
Adverse Events: Definitions and Procedures for Recording, 
Evaluating, Follow-up, and Reporting ............................................................................54 
10.2.1. Definition of AE .............................................................................................................54 
10.2.2. Definition of SAE ...........................................................................................................55 
10.2.3. Recording and Follow-Up of AE and/or SAE ................................................................56 
11. 
REFERENCES .............................................................................................................59 


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Appendix 1: 
   Administrative Change #1 Summary of Changes ......................................61 
Appendix 2: 
   Amendment # 1 Summary of Changes ........................................................62 
Appendix 3: 
   Amendment # 2 Summary of Changes ........................................................71 
 


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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 3,3'-Dioxo-2,2'-bisindolylidene-5,5'-disulfonate disodium 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 a visualization aid in the determination of ureter 
patency. Visualization will be measured by a conspicuity scale designed to 
provide an objective tool applicable to the visualization of the urine jet 
stream when determining ureter patency. 
The 5-point conspicuity score is defined as follows: 
1 = No jet observed 
2 = Weak jet, little color contrast 
3 = Color contrast or significant jet flow 
4 = Strong jet flow with good color contrast 
5 = Strong jet flow with striking contrast in color 
Secondary Objectives 
1. To evaluate the safety profile of IC when used as an aid in the 
determination of ureteral patency.  
2. 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).    
3. To determine proportion of subjects meeting the responder 
definition in conspicuity score following IC treatment based on the 
blinded central reviewers’ assessment.  A subject is a responder 
when there is >=1 point improvement in the conspicuity scores 
following the IC vs saline treatment (IC – Saline >=1) and the 








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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 
Urine jet conspicuity score provided by the blinded central independent 
image review process.   
Secondary Efficacy Endpoints 
1. Physician’s overall satisfaction with the IC treatment as an aid for 
the assessment of ureter patency 
2. Proportion of responders. A subject is considered a responder to IC 
if there is >=1 point improvement in the conspicuity score following 
the IC treatment vs the saline treatment and the conspicuity score for 
the IC treatment must be (3, 4, or 5).  The responder criteria will be 
assessed separately for each ureter (left or right) based on the 
blinded central reviewer’s assessment 
3. Time (minutes) to visualization (TTV) of blue color in the urine jet 
flow following administration of IC treatment.  
Other Endpoints 
1. Urine jet conspicuity score assessed by the surgeon. 
2. Concordance of the blinded central review urine jet conspicuity 
scores and the surgeon’s urine jet conspicuity scores. 
Safety 
Endpoints 
 Safety Endpoints include 
1. Incidence of treatment emergent adverse events 
2. Changes in safety laboratory tests and vital signs 
3. Changes in 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.  






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effect at the interim analysis; the final decision will be dependent on the 
size of the estimated conditional power.   
Statistical 
Analysis 
Methodology 
The statistical analysis for this study will be carried out using SAS® 9.4 or 
later and described in a detailed statistical analysis plan (SAP), which was 
finalized prior to first patient first visit and will be amended to reflect 
changes in Protocol Amendment No. 2. Any deviations from the planned 
analysis as described in the SAP will be justified and recorded in the final 
clinical study report (CSR). 




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1.3. 
Schedule of Assessments (SoA) 
 
Screening 
period 
Day -30 to -11 
Randomization/ 
Treatment 
Day 1 
Follow-up 
Visit Day 7 to 
Day 32 or 
Early 
Termination2 
Telephone 
follow-up if 
Follow-up 
visit 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 
 
 
 
Vital Signs 
X 
X3 
X 
 
12 Lead ECG 
X 
 
X 
 
Randomization 
 
X 
 
 
Concomitant Medications 
X 
X 
X 
X 
AE/SAE 
 
X 
X 
X 
Blood Safety Labs4 
X 
 
X5 
 
PK Blood6, Urine7, and Stool8 collection 
 
X 
 
 
Bowel Prep9 (PK group) 
X (Day -1) 
 
 
 
Surgery 
 
X 
 
 
Surgeon satisfaction with IC treatment 
 
X 
 
 
0.9% saline injection 
 
X 
 
 
0.8% IC injection 
 
X 
 
 
Video filming of ureteral jet flow with 
saline and with IC  
 
X 
 
 
Surgeon conspicuity score of urine Jet flow 
 
X10 
 
 
Urine Pregnancy Test11 
 
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 approximately 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 approximately 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. 
The first post-op stool will be collected and sent for analysis for IC and/or its breakdown products. 
9. 
Bowel prep will be required preoperatively (Day -1) for Subjects in the PK/Breakdown product collection patients. 
10. The Surgeon should review the videos post-surgery to perform their conspicuity scoring of the urine jet flows.  Scoring should not be done during 
surgery 
11. 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 (Voelcker 
1903) 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 (Chi 
2016, Cohen 2018).  
 
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 (Ibeanu 2009).  
 
 
 


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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 (Grimes 2017). In the US, approximately 600,000 
hysterectomies, 226,000 prolapse repairs, and 135,000 stress incontinence operations are performed 
annually (Vakili 2005; 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 5-point conspicuity score:  
1 = No jet observed 
2 = Weak jet, little color contrast 
3 = Color contrast or significant jet flow 
4 = Strong jet flow with good color contrast 
5 = Strong jet flow with striking contrast in color 
 
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 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 surgeon’s evaluation is considered satisfactory if the rating is either a 1 (strongly 
agree) or 2 (agree).    
3. To determine proportion of subjects meeting the responder definition in conspicuity score 
following IC treatment based on the blinded central reviewers’ assessment.  A subject is a 
responder when there is >=1 point improvement in the conspicuity scores following the IC 
vs saline treatment (IC – Saline >=1) and the conspicuity score following the IC treatment is 
(3, 4, or 5). The responder criteria will be assessed separately for each ureter for each 
subject. 
4. To describe the time to visualization (TTV) of blue color in the ureteral jets flow following 
IC treatment when used as a visualization aid during urological and gynecological surgical 
procedures  


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5. To determine the IC pharmacokinetic profile from approximately 16 subjects from 2 
investigational sites. 
6. An exploratory comparison will be performed to assess the difference between the IC high 
dose vs IC low dose. Surgeons will be blinded to the dose of IC. 
 
3.2. Endpoints 
3.2.1. Efficacy Endpoints 
The primary efficacy endpoint is the blinded central reader’s visualization conspicuity score, a 
5-point ordinal scale score:  
1 = No jet observed 
2 = Weak jet, little color contrast 
3 = Color contrast or significant jet flow 
4 = Strong jet flow with good color contrast 
5 = Strong jet flow with striking contrast in color 
The secondary efficacy endpoints include: 
1. Proportion of physicians who agree that compared to saline, IC treatment improves 
visualization as an aid for the assessment of ureteral patency  
2. Proportion of responders.  A subject is a responder when there is >=1 point improvement in 
the conspicuity scores following the IC vs saline treatment (IC – Saline >=1)  and the 
conspicuity score following the IC treatment is (3, 4, or 5).  The responder criteria will be 
assessed separately for each ureter for each subject based on the blinded central reviewer’s 
conspicuity score. 
3. Time (minutes) to visualization (TTV) of blue color in the ureteral jets flow following IC 
treatment  
 
Other efficacy endpoints will include: 
1. Conspicuity score provided by surgeon  
2. Concordance in conspicuity 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 


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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. 
Scientific Rationale for Development of a New Conspicuity Score 
 
 
 
 
 
  
 
 
 
 
 
 
 
4.4. 
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). 
 
4.5. 
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 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 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. 
E09. Subjects who are pregnant or breast-feeding 
 


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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 and eligibility criteria. 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.  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 
 
Randomization 
All subjects will be centrally assigned to randomized study drug using the randomization feature of the 
electronic data capture (EDC) system.  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. 
Blinded 
Conspicuity 
score 
Both the central review and surgeon conspicuity score results will be blinded form all members of the 
study team except for unblinded data management and statistical personnel directly involved with 
collecting and analyzing the data for the interim analysis.  


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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 eCRF.  The randomized dose of IC 
treatment will be provided by the randomization system 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. 
 
 


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6.6 
Prohibited Concomitant Medications/Foods/Supplements 
Prohibited medications, foods, and/or supplements are anything that could discolor urine. The length 
of time required from last dose to study randomization is at the discretion of the investigator and 
should be based on the known duration of the effect on urine coloration and documented in the 
source documentation. Examples of prohibited medications are: 
 
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 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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8.1. 
Efficacy Assessments 
8.1.1. 
Conspicuity Assessment 
The ureteral orifices and the jet flow will be captured by video for 10 minutes post-completion of 
each injection for conspicuity assessment.  To capture this information the following will be 
recorded: 
• 
Type of surgical procedure/visualization  
o Cystoscopic 
o Robotic 
o Open  
• 
Time each injection started 
• 
Time each injection completed 
• 
Time to visualization of blue urine following indigo carmine administration 
• 
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. 
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 
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 and Central Review Process 
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 


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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 for 10 minutes. A 
 
 
 
  
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 2 blinded central reviewers for conspicuity assessment using the same 
assessment tool used by the surgeon.  The consistency check between the two central reviewers will 
be performed; the two reviewers will be considered to be consistent if their scores for a given 
ureter/video is within (+/-) 1 point; in this case, the average score of the two reviewers will be the 
final score for efficacy analysis.  Otherwise, a third reviewer will serve as a judicator.  The judicator 
will review the questioned video and the score from the judicator will be the final score for the 
efficacy analysis.   
The surgeon will also review the videos and score the jet flows using the same conspicuity scale.  
The surgeon’s score should be based on a review of the videos and does not have to occur on the day 
of surgery. The concordance in the conspicuity scores between the surgeon’s assessment and the 
central reader process will be evaluated 
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 and weight.  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). 


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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. 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, 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. 
• 
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. 


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• 
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 After IC Treatment 
Subjects in whom ureteral patency was not visualized or was poorly visualized after IC treatment 
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 AEs and 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. 
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. 


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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 or female partners of male 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. 
 
8.4. 
Pharmacokinetics 
PK samples of plasma, urine, and stool will be collected from approximately 16 subjects at 2 
participating sites who have agreed to participate and have been trained in the specific requirements 
for the study. 


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8.4.1. 
Plasma samples  
• 
Approximately 2 mL of plasma (4 ml of whole blood) 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. 
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-∞  
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 model 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 assay methods.  The Bioanalytical Report and Pharmacokinetic Analysis 
Report will be provided as appendices to the final study report. 




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sample size for this study will stay the same (approximately 116) as planned before the protocol 
amendment. 
A subgroup of subjects at 2 sites (approximate 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) and who have a video at 
approximately 10 minutes in length following each treatment.  This 
will be the primary population for efficacy assessments.  
Note: the first 21 patients that were enrolled prior to the 
development of conspicuity score will be EXCLUDED from the 
efficacy population.   
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.  
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 be different from the actual 
received IC dose. 
Note: the first 21 patients enrolled prior to the development of the 
conspicuity score will be INCLUDED in the safety population.   
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 was finalized prior to First Patient First Visit (FPFV) of this study.  The 
SAP amendment will be finalized along with Protocol Amendment No. 2 finalization to reflect the 
changes in Protocol Amendment No. 2, 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. Interim Analysis 
A formal interim analysis is planned to confirm the observed treatment effect size on the conspicuity 
score from the test dataset.  The study will have a total of approximately 95 patients remained to be 
enrolled under the protocol Amendment No. 2.  This formal interim analysis will be held when 50 of 
the 95 remaining subjects are randomized and treated.  The interim analysis will only analyze the 
conspicuity score from the 50 patients provided by the central reviewers.  
Since randomization to the IC dose level will not be unblinded during the interim analysis, the 
overall IC treatment effect will be analyzed using the proportional odds model; the model will 
include the main effect of treatment (IC vs Saline) and ureter (left vs right) and control for the 
repeated measures within a subject. This model is identified as the reduced model in the primary 
efficacy analysis of the primary endpoint in Section 9.4.3.1.   
Conditional Power (CP) will be calculated based on estimated odds ratio and standard error (in log 
scale) at interim analysis.  The prediction of the conditional power for a statistically significant result 
from the final analysis (at the two-sided 0.05 level) given the observed difference between 
treatments at a formal interim analysis can be based on the estimated treatment effect; 2-sided 95% 
confidence intervals of the CP will also be calculated.  Interim analysis results will be then classified 
to three zones (unfavorable, promising, favorable) based on the estimated CP and decision to be 
made in each zone is specified as follows. 
 
 


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Table 1:  Interim Analysis Conditional Power and Decision Principle        
Conditional 
Power 
Decision 
Rationale 
 CP <0.20 
Stop the study because the 
evidence for unfavorable efficacy 
is sufficiently convincing that no 
additional efficacy data is 
necessary.  
An Unfavorable zone: the conditional power 
(CP) is fairly low, suggesting that the final 
result is not likely to achieve significance, 
hence, the study has fairly low probability to 
reject the null hypothesis.  This zone is defined 
to be CP < 40% 
0.2 <=CP 
<0.4 
No sample size re-estimate because 
of the evidence for unfavorable 
efficacy. Complete the study per 
current protocol.  
This is part of the Unfavorable zone. Although 
the evidence of efficacy is low, the study will 
be completed as planned without an increase in 
sample size.    
0.4 <=CP 
<=0.8 
Re-estimate sample size. Study 
continues to complete the 
enrollment per re-estimated sample 
size 
A Promising zone: the conditional power is in 
between 40% to 80%, suggesting that the 
interim analysis result is promising, but the 
study has to have an increased sample size to 
have at least 80% power to reject the null 
hypothesis 
CP > 0.8 
No sample size re-estimate. 
Complete the study per current 
protocol. 
A Favorable zone: the conditional power is 
fairly high, suggesting that the interim result is 
as good or better than the expectation, the 
study has fairly high probability to reject the 
null hypothesis as is.  This zone is defined to 
be CP > 80%. 
 
To protect the data integrity, an independent statistician/programmer who is not involved with the 
study will be responsible to perform the analysis.  A Data Monitoring Committee (DMC) will be 
formed to review the interim analysis results.  All members of the DMC will not be involved with 
the conduct of the study. To perform the interim analysis, the independent statistician will receive 
the final conspicuity score assessed by the central reviewers and the randomization code associated 
with each video/subject.  It is worthy to point out that randomization is only pertained to the 
treatment (IC vs Saline) and the dose level of IC treatment will not be revealed at interim analysis.  
The DMC may share with the study team (sponsor, investigator, CRO) the aggregated results/data 
(e.g., summary tables) but the individual subject data will not be shared with the study team. 
 
 


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9.4.2. 
Type 1 Error Control for the Primary Efficacy Endpoint 
The study has a planned interim analysis when the enrollment is at approximately 50 subjects.  The 
objective of this interim analysis is to assess the treatment effect size on the conspicuity score based 
on the blinded central reviewer.  To preserve the overall Type I error for the study, an alpha of 
0.0001 will be used for this interim analysis and alpha of 0.0499 will be used for the final analysis. 
For the final analysis, there are two null hypotheses for the primary efficacy endpoint conspicuity 
score. 
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 0.0499, when both nominal p-values are 
less or equal to 0.0499, 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 (the 
one with the higher value) of the two nominal p-values is greater than or equal to 0.05 but the second 
nominal p-value (the one with the lower 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 conspicuity score only. 
 
9.4.3. 
Treatment Effect on Conspicuity Score  
9.4.3.1. 
Conspicuity Score per Blinded Central Reviewers  
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 conspicuity scores.  The conspicuity score 
provided by the blinded central reviewer will be the primary efficacy endpoint whereas the score 
provided by the surgeon will serve only as an estimate of concordance.   
The treatment effect on the conspicuity score will be evaluated using a Generalized Estimating 
Equation (GEE) for Repeated Measures to control for the intra-subject correlation.  A proportional 
odds model will be fitted to estimate the treatment effect measured by odds that the conspicuity 
score following saline treatment is less than that following the IC treatment. 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=5) 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 


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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.    
An exploratory comparison will be performed to assess 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.    
 
9.4.3.2. 
Responders to IC Treatment per Blinded Central Reviewer 
A responder analysis will be performed to assess the ‘added benefit’ of IC over the saline within a 
subject.  That is, since each subject will serve as his/her own control, the difference in conspicuity 
score following the IC treatment and Saline will be used to define a responder. 
A subject is considered a responder if there is >=1 point improvement in the conspicuity score 
following the IC treatment vs Saline treatment and the conspicuity score following the IC treatment 
is >= 3.  This responder criteria will be assessed twice for each subject: once for the left side ureter 
and once for the right side of the ureter based on the blinded central reviewers assessment.   
Number (%) of subjects meeting the responder criteria will be tabulated by IC dose and each ureter 
(left only, right only, and left and right sides).  A 2-sided 95% confidence intervals will also be 
estimated using the normal approximation.  
 
9.4.3.3. 
Concordance in Conspicuity Score Assessment 
Concordance in conspicuity score will be assessed following 3 steps.  
First, the conspicuity score provided by the surgeons will be analyzed using the same GEE approach 
to evaluate treatment effect on the conspicuity described in Section 9.4.3.1.  Second, to assess the 
consistency between the surgeon and central rater by covariate analysis approach, in which the effect 
of raters (surgeon or blinded central rater) on the response will be evaluated in the GEE model. The 
third step is to derive the difference in conspicuity score between the surgeon and the central rater.  
This difference will have a value ranging (-4 to +4) for each of the 4 pairs of readings per subject, 
with 0 indicating a perfected agreement.  If a pair of ratings between the central readers and the 
surgeon is within (+/-) 1 point (i.e., the difference is ranging (-1 to +1), inclusive), the pair of ratings 
is marked as ‘agree’, otherwise, the pair of ratings is ‘not agree’.  The association of this 
concordance response variable (agree vs not agree) with treatment and ureter will also be evaluated 
using the GEE model based on the concept of logistic regression for dichotomized response. 
9.4.4. 
Time to Visualization  
Time (minutes) to visualization (TTV) will be evaluated for IC treatment 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 stratified log-rank test for the homogeneity of the survival 


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curves.  Cox Proportional hazards model approach may be used to estimate hazards ratios if data 
warrant as an exploratory analysis to assess differences between the low dose and high dose. 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 site) will be 
evaluated if data warrants.  
IC 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.   
      
9.4.6. 
Assessment of Covariates 
The effect of procedure on the response will be assessed via covariate analysis.  An appropriate 
model will be chosen for specific parameter.   In addition, if data warrant, demographic variables 
(age group, sex, and race) will be assessed as a potential covariance.  If data warrant, subgroup 
analysis per surgery type, or demographic variables may be performed.   
 
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 model 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. 
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. 


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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 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. 
The results of this study may be published or presented at scientific meetings. If this is foreseen, the 
investigator agrees to submit all manuscripts or abstracts to the sponsor before submission. This 
allows the sponsor to protect proprietary information and to provide comments. 












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11. 
REFERENCES 
 
1. 
AAGL Practice Committee.  AAGL Practice Report: Practice guidelines for intraoperative 
cystoscopy in laparoscopic hysterectomy. J Minim Invasive Gynecol 2012;19:407-411 
 
2. 
American Regent Package Insert. Indigo Carmine Injection American Regent, Inc. 
NDC:0517-0375-05. Date of revision 02/2017. 
 
3. 
Barbieri R. Farewell to Indigo Carmine. OBG Management 2014; 26(9):8-9,12 
 
4. 
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 
 
5. 
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 
 
6. 
Cohen SA, Carberry CL and Smilen SW. American Urogynecologic Society consensus 
statement: cystoscopy at the time of prolapse repair.  Female Pelvic Med Reconstr Surg 
2018;24:258–259 
 
7. 
Gill EJ, Elser DM, Bonidie MJ, Roberts KM, Hurt WG. The routine use of cystoscopy with 
the Burch procedure. Am J Obstet Gynecol. 2001 Aug;185(2):345-8. 
8. 
Gilmour D and Flowerdew G. Rates of urinary tract injury from gynecologic surgery and the 
role of intraoperative cystoscopy. Obstet Gynecol 2006;107:1366–1372 
 
9. 
Grimes CL, Patankar S, Ryntz T et al.  Evaluating ureteral patency in the post-indigo carmine 
era: a randomized controlled trial. Am J Obstet Gynecol. 2017;217:601.e1 – 10 
 
10. 
Harris RL, Cundiff GW, Theofrastous JP, Yoon H, et al. The value of intraoperative 
cystoscopy in urogynecologic and reconstructive pelvic surgery. Am J Obstet Gynecol 1997; 
177:1367-71. 
 
11. 
Ibeanu O, Chesson RR, Echols KT et al. Urinary tract injury during hysterectomy based on 
universal cystoscopy. Obstet Gynecol 2009;113:6–10 
 
12. 
Jabs CF, Drutz HP. The role of intraoperative cystoscopy in prolapse and incontinence 
surgery. Am J Obstet Gynecol 2001;185(6):1368–73 
 
 


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Indigo Carmine 0.8%  
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13. 
Jelovsek JE, Chen G, Roberts SL, et al. Incidence of lower urinary tract injury at the time of 
total laparoscopic hysterectomy. J Soc Laparoendoscopic Surg 2007; 11:422-27. 
 
14. 
Jung M, Kiesslich R. Chromoendoscopy and intravital staining techniques, Baillière’s 
Clinical Gastroenterology 1999; 13(1):11-19 
 
15. 
Lacy, W.W., Ugaz, C., Newman, E.V. The use of indigo carmine for dye dilution curves.  
Circulation Research 1955; 111: 570-574 
 
16. 
Lee M, Sharifi R. Methylene blue versus indigo carmine. Urology 1996;47(5):783-4. 
 
17. 
O’Brien WM, Lynch JH. Simplification of double-dye test to diagnose various types of 
vaginal fistulas. Urology 1990;36(5):456 
18. 
Oravisto, K.J., Investigations into the excretion mechanism of indigo carmine in normal 
human kidney. Ann. Chir. Gynaecol. Fenn Suppl. 1957;46(2):1-79 
 
19. 
Pettit PD, Petrou SP. The value of cystoscopy in major vaginal surgery.  Obstet Gynecol. 
1994;84(2):318-20 
 
20. 
Ribeiro S, Reich H, et al. The Value of intra-operative cystoscopy at the time of laparoscopic 
hysterectomy. Human Reproduction 1999; 14(7): 1727-1729 
 
21. 
Speights SE, Moore RD, Miklos JR. Frequency of lower urinary tract injury at laparoscopic 
burch and paravaginal repair. J Am Assoc Gynecol Laparosc. 2000; 7:515-8 
 
22. 
SmPC (Summary of Product Characteristics). Indigo Carmine 40 mg/5 mL solution for 
injection. SERB S.A, Belgium. Marketing Authorisation Number: PL 43956/0001. Date of 
Revision of the Text: 05/06/2015 
 
23. 
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 
 
24. 
Voelcker F, Joseph, E, Funktionelle Nierendiagnostik ohne Ureterenkatheter. Munch. Med. 
Wschr. 1903; 50: 2081-2089 
 
25. 
Weinstein JR and Anderson S. The aging kidney: physiological changes.  Adv Chronic 
Kidney Dis. 2010 July ; 17: 302–307 
 
 
 


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Appendix 1: 
   Administrative Change #1 Summary of Changes 
 
 Version 1.1 Administrative Change 1 
 
1. Addition of reference cited in document to the bibliography 
2. Update of Sponsor contact information 
3. Correction of mis-numbered section (6.6) 
4. Clarification of timing for prohibited Medications/Foods/Supplements 
5. Clarification of packaging for Indigo Carmine in section 6.2 
 


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Appendix 2: 
   Amendment # 1 Summary of Changes 
Page  Section # 
Change 
Reason / 
Rationale 
Title 
page, 
page 
3 
and 
Page 
10 
Study Title 
An Open-Label, Randomized, Multicenter Study 
to Evaluate the Safety, Efficacy, 
Pharmacokinetics and Physician Satisfaction of 
two different doses of 3,3'-Dioxo-2,2'-
bisindolylidene-5,5'-disulfonate disodium Indigo 
Carmine Injection 0.8% solution when used as 
an aid in the determination of ureteral patency. 
To include the 
IUPAC name in 
the title 
Title 
page 
Investigational 
Product 
3,3'-Dioxo-2,2'-bisindolylidene-5,5'-disulfonate 
disodium (Indigo Carmine 0.8% Injection, USP) 
To correlate the 
IUPAC name 
with the USP 
name of the 
investigational 
product 
11 
1.1 
Secondary 
Objectives (5) 
5. 
An exploratory comparison will be 
performed to assess done on the difference 
between the IC high dose vs IC low dose.  
Surgeons will be blinded to the dose of IC. 
Clarity and 
readability 
11 
1.1 
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 3,3'-Dioxo-2,2'-bisindolylidene-5,5'-
disulfonate disodium commonly referred to as 
Indigo Carmine (IC) 0.8% Injection, USP 
solution for injection 
To correlate the 
IUPAC name 
with the USP 
name of the 
investigational 
product 


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Page  Section # 
Change 
Reason / 
Rationale 
11 
1.1 
Methodology 
 
 
 
 
 
 
Eligible subjects will be randomized in a 1:1 
ratio to receive a dose of either IC high dose (5 
mL) or IC low dose (2.5 mL). 
To specify the 
randomization 
ration 
12 
1.1 
Methodology 
 
 
 
 
In a subset of subjects from 2 sites 
(approximately about 16 subjects), subjects will 
be consented to participate in the 
pharmacokinetic (PK) portion of the study 
Readability 
12 
1.1 
Study Drug 
Section Title changed:  Study Medication Drug 
Consistency 
throughout 
protocol 
12 
1.1 
Number of 
Subjects 
Up to 116 subjects will be enrolled from 
approximately 10 sites, including approximately 
16 subjects to participate in PK/metabolite 
analysis at 2 sites 
Clarification and 
consistency 


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Page  Section # 
Change 
Reason / 
Rationale 
12 
1.1 
Sample Size 
The study plans to randomize 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% (about 20) subjects may 
be enrolled to account for protocol deviations, 
withdrawal of consent, etc., if necessary. 
A subgroup of subjects at 2 sites (total 
Approximate N=16, at least 8 from each IC dose 
level) will be evaluated for 
pharmacokinetics/metabolites 
Clarification and 
consistency 
14 
1.1 
Safety 
Endpoints (3) 
3. 
Changes in clinically significant 
abnormal 12-ECG 
Clarity and 
readability 
14 
1.1 
PK Parameters 
(1) 
1. 
AUC0-t, AUC0 t(90%subj), AUC0-∞ 
Consistency with 
statistical 
analysis plan 
14 
PK Analysis 
Non-compartmental analysis model will be used 
to calculate PK parameters using the observed 
concentrations and actual sampling times. 
Clarity and 
accuracy 
15 
1.1 
Procedure 
Summary 
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 the first bowel movements within the first 
24 hours post-surgery in this same subgroup. 
Correction and 
consistency 
throughout 
protocol 
18 
1.3 SOA 
Deleted weight assessment during the Follow-up 
visit 
Post-surgery 
weight not 
required for 
study purposes  


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Page  Section # 
Change 
Reason / 
Rationale 
18 
1.3 SOA 
Footnote # 7 
For a total of approximately 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. 
Clarification 
18 
1.3 SOA 
“IVRS/IWRS contact” row renamed to 
“randomization” and “X” removed from all 
columns except for the 
“Randomization/Treatment Day 1” column 
An IVRS/IWRS 
system will not 
be used for this 
study.  
Randomization 
will be 
completed via the 
EDC system 
18 
1.3 SOA 
“Day 2 PK” 
column 
Column deleted 
Based on the PK 
sampling 
schedule a 
separate Day 2 
visit is not 
expected  
21 
3.1.2 
Secondary 
objective # 4 
4. 
To determine the IC pharmacokinetic 
profile from in a subset of subjects 
approximately 16 subjects from 2 investigational 
sites. 
Clarification 
21 
3.1.2 
Secondary 
objective # 5 
5. 
An exploratory comparison will be done 
on performed to assess the difference between 
the IC high dose vs IC low dose. 
Clarity and 
readability 
26 
5.3 Screen 
Failures 
Minimal information includes demography, 
screen failure details and, eligibility criteria. and 
any serious adverse event (SAE). 
Serious Adverse 
Events will not 
be captured for 
subjects who are 
not dosed with 
study drug. 


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Page  Section # 
Change 
Reason / 
Rationale 
28 
6.3 Measures to 
minimum Bias 
IVRS/IWRS 
Randomization 
All subjects will be 
centrally assigned to 
randomized study drug 
using an Interactive 
Voice/Web Response 
System (IVRS/IWRS).the 
randomization feature of 
the electronic data capture 
(EDC) system.  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. 
An IVRS/IWRS 
system will not 
be used for 
randomization 
29 
6.4 Study Drug 
Compliance 
The randomized dose of IC treatment will be 
provided by IVRS/IWRS the EDC system to the 
designated unblinded site staff 
An IVRS/IWRS 
system will not 
be used for 
randomization 
30 
6.6 
Prohibited medications, foods, and/or 
supplements would be are anything that could 
discolor urine. 
 
33 
8 
Screening 
o 
Once consented, the subject will be 
entered into IVRS/IWRS the EDC system.  
Randomization will be a 1:1 ratio to be assigned 
to low dose (2.5 mL IC) or high dose (5 mL IC). 
An IVRS/IWRS 
system will not 
be used for 
randomization 
33 
8 
Randomization/ 
Treatment 
o 
After a subject is confirmed to be 
eligible, the subject will be randomized via 
IVRS/IWRS the EDC system. 
An IVRS/IWRS 
system will not 
be used for 
randomization 
33 
8 
Dosing step by 
step: 1 
by identifying the efflux of urine from the 
ureteral orifices for up to 10 minutes for each 
injection post injection. 
Clarity 


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Page  Section # 
Change 
Reason / 
Rationale 
33 
8 
Dosing step by 
step: 1c 
c. 
The surgeon will document the 
CONSPICUITY score following the use of 
saline in the assessment of the patency of the 
ureters. 
Addition of step 
for clarity of 
procedure 
33 
8 
Dosing step by 
step: 5 
Urine and stool will be collected at specified 
time points in this same subgroup. 
Added for 
completeness 
36 
8.2.1 
 
These will be repeated at the follow-up visit 
except for height and weight. 
Weight is not 
required during 
the follow-up 
visit for study 
purposes 
37 
8.2.3 
Initial ECGs will be assessed by an investigator, 
addition to the measurements, as normal or 
abnormal finding. 
Typographical 
correction 
38 
8.2.5 
Section Title 
Follow-up for Subjects in whom Ureteral 
Patency was Not Visualized or Poorly 
Visualized After IC Treatment 
Clarity 
38 
8.25 
Subjects in whom ureteral patency was not 
visualized or was poorly visualized after IC 
treatment will be treated with appropriate 
follow-up as per the discretion of the surgeon. 
Clarity 
38 
8.3.1 
 
All AEs and SAEs will be collected from the 
start of the saline injection until the follow-up 
visit. 
to specify the 
timing of AE 
collection 
39 
8.3.5 
Details of all pregnancies in female subjects or 
female partners of male subjects 
To include 
pregnancy 
follow-up for 
pregnancies that 
occur during the 
study in female 
partners of male 
subjects 
40 
8.4 
PK samples of plasma, urine, and stool will be 
collected from approximately 16 subjects 
Clarification 


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40 
8.4.1 
 
• 
Approximately 2 mL of plasma (4 ml of 
whole blood) will be collected for each 
measurement of plasma concentrations 
Clarity 
40 
8.4.3 
• 
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 
Correction and 
consistency 
throughout 
protocol 
40 
8.4.3 
The pharmacokinetic (PK) analysis will include 
at least the following parameters:  
1. 
AUC0-t, AUC0 t(90%subj), AUC0-∞ 
Consistency with 
statistical 
analysis plan 
41 
8.4.3 
Non-compartmental Analysis model will be used 
to calculate PK parameters using the observed 
concentrations and actual sampling times. 
Clarity 
42 
9.2 
The following empirical distribution table (Table 
1) is created to illustrate this effect size; sample 
size was is determined using the two-group chi-
square test comparing the difference in 
proportions in 3 categories (Table 2).     
Accuracy and 
readability 
43 
9.2.2 
A subgroup of subjects at 2 sites (total 
approximate N=16, 8 from each IC dose level) 
will be evaluated for 
pharmacokinetics/metabolites. 
Clarification 
43 
9.3 
Per Protocol 
Analysis Set 
This Analysis Set will serve as a confirmation 
analysis set for efficacy analysis.  who   
Correction of 
typographical 
error 
43 
9.3 
Safety Analysis 
Set 
Subjects will be analyzed according to the drug 
they actually received should the randomized IC 
dose is be different from the actual received IC 
dose. 
Correction of 
typographical 
error 
45 
9.4.2 
An exploratory comparison will be performed to 
assess 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. 
Clarity and 
readability 


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9.4.3 
In this approach a A new covariate indicating 
evaluator (surgeon vs blinded central reviewer) 
will be added to the GEE model. 
Readability and 
clarity 
45 
9.44 
Potential covariates (such as randomization 
strata and site will also be evaluated) if data 
warrants. 
Accuracy as 
there are no 
randomization 
strata planned 
46 
9.4.6 
The effect of procedure on the response 
(randomization stratum) will be assessed via 
covariate analysis.  An appropriate model will be 
chosen for specific parameter. 
Accuracy as 
there are no 
randomization 
strata planned 
46 
9.4.8 
PK parameters will be estimated using Non-
compartmental analysis model based on the 
observed concentration; 
Clarity 
56 
10.2.3 
Follow-up of 
AEs and SAEs 
Bullet #4 
• 
The investigator will submit any new or 
updated SAE data to PV within 24 hours of 
receipt of the information via the SAE Reporting 
Form. 
 
56 
10.2.4 
SAE Reporting 
to PV via an 
Electronic Data 
Collection Tool 
Section number and Entire table deleted 
Reporting of 
SAEs through an 
electronic data 
collection tool is 
not applicable for 
this study 
56 
10.2.3 
SAE Reporting 
to PV via Paper 
SAE Reporting 
Form 
SAE Reporting to PV via Paper SAE Reporting 
Form CRF 
SAE Reporting 
will be via a 
reporting form 
not a paper CRF. 


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10.2.3 
Bullet 1  
• 
The primary mechanism for reporting an 
SAE to PV will be via email of a Facsimile 
transmission or scanned copy of the completed 
SAE paper CRF reporting form to PV.  If email 
is not available, facsimile transmission is 
acceptable are the preferred method to transmit 
this information to PV. 
The primary 
method of SAE 
Reporting will be 
via a paper SAE 
reporting form 
not a paper CRF. 
59 
11. 
 
20. 
Ribeiro S, Reich H, et al. The Value of 
intra-operative cystoscopy at the time of 
laparoscopic hysterectomy. Human 
Reproduction 1999; 14(7): 1727-1729 
Reference added 
All 
N/A 
Corrections of minor typographical and spelling 
errors that had no effect on meaning were also 
made throughout the document  
N/A 


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Appendix 3: 
   Amendment # 2 Summary of Changes 
 
Page  
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Reason / 
Rationale 
10 -11 
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 a visualization aid in the 
determination of ureteral patency. 
Visualization will be measured by a 3
point Ureteral Orifice Visualization Scale 
(UOVS): conspicuity scale designed to 
provide an objective tool applicable to the 
visualization of the urine jet stream when 
determining ureter patency. 
1 =  non visualization 
2 = inadequate/equivocal 
visualization 
3 = adequate/unequivocal 
visualization of the urine jet 
stream after administration of 
test agent 
The 5-point conspicuity score is defined 
as follows. 
1 = No jet observed 
2 = Weak jet, little color contrast 
3 = Color contrast or significant 
jet flow 
4 = Strong jet flow with good 
color contrast 
5 = Strong jet flow with striking 
contrast in color 
Secondary Objectives 
1. To evaluate the safety profile of 
IC when used as an aid in the 
determination of ureteral patency.  
To implement 
the conspicuity 
score to 
evaluate the 
potential 
visualization 
advantage 
provided by 
indigo carmine 


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2. To describe the time to 
visualization (TTV) of ureteral 
jets during urological and 
gynecological surgical procedures 
2. 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).    
3. To determine proportion of 
subjects meeting the responder 
definition in conspicuity score 
following IC treatment based on 
the blinded central reviewer’s 
assessment.  A subject is a 
responder when there is >=1 point 
improvement in the conspicuity 
scores following the IC vs saline 
treatment (IC – Saline >=1) and 
the conspicuity score following 
the IC treatment is (3, 4, or 5). 
The responder criteria will be 


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assessed separately for each ureter 
for each subject. 
4. To describe the time to 
visualization (TTV) of blue color 
in the urine jet flow following IC 
treatment when used as a 
visualization aid during urological 
and gynecological surgical 
procedures  
5. To determine the IC 
pharmacokinetic profile in a 
subset of subjects from 2 
investigational sites. 
6. An exploratory comparison will 
be performed to assess the 
difference between the IC high 
dose vs IC low dose.  Surgeons 
will be blinded to the dose of IC. 


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11 
Methodology 
This is an open-label, randomized, 
multicenter study to evaluate the efficacy, 
safety, and pharmacokinetics of two 
doses dose levels (2.5 mL and 5.0 mL) of 
3,3'-Dioxo-2,2'-bisindolylidene-5,5'-
disulfonate disodium commonly referred 
to as 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.approximately 116 subjects from 
10 – 20 study centers in the United 
States. When the 5 point conspicuity 
score was developed, the study had 
enrolled and treated 21 subjects. These 
initial 21 subjects will be included in all 
safety evaluations but they will not be 
evaluated for efficacy. The total sample 
size for the study will stay the same, so 
approximately 95 patients will be 
enrolled following Protocol Amendment 
No. 2.   
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, review of 
inclusion and exclusion criteria will be 
performed, the collection of concomitant 
medications, medical history, physical 
examination, baseline laboratory testing, 
pregnancy testing, 12-lead ECG, and vital 
sign measurements will be completed 
during the screening visit. 
Clarification of 
study sample 
size 


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11-12 
Methodology 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
.  
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 
To clarify the 
dosing and 
videography 
procedures 
associated with 
the urine jet 
conspicuity 
scale 


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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. 
After the procedure the surgeon will also 
review the videos of his/her patients to 
provide the 5-point conspicuity score for 
left and right ureter after each treatment. 
The surgeon’s conspicuity score will be 
utilized for conspicuity concordance 
analysis.  The surgeon evaluation does 
not have to occur on the day of surgery. 
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 review process 
based on the urine jet conspicuity scale.  
Each ureter jet flow will be scored for 
conspicuity independent of the other 
ureter jet flow, hence, each subject will 
have 4 assessments for urine jet flow 
conspicuity (two following saline 
administration and two following IC 
administration).  The primary endpoint 
analysis will be based on the central 
review score. 
There will be 2 central reviewers for each 
video and consistency between the two 
central reviewers will be checked.  The 
two reviewers will be considered to be 
consistent if their scores for a given 
ureter /video is within (+/-) 1 point. In 
this case, the average score from the two 
reviewers will be the final score for 
efficacy analysis. Otherwise, a judicator 
will review the same video and the scores 


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from this judicator will be the final score 
for efficacy analysis. 
For consistency and ease of comparison 
between the central reviewer and surgeon 
conspicuity scores, ureter jet flows and 
conspicuity scores will be referred to as 
left and right based on the perceived 
orientation on the videos irrespective of 
the actual orientation of the patient. 
13 
Number of 
Subjects 
Up to Approximately 116 subjects will be 
enrolled from approximately 10 - 20 sites 
To allow for 
additional sites  
13 
Sample Size 
The study plans to randomize 96 
approximately 116 subjects; 458 subjects 
to 2.5 mL IC and 4858 subjects to 5 mL 
IC. This sample size calculation was 
determined based on two group Chi
square empirical efficacy assumption plus 
10% drop-out.   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
   
The sample size does not account for 
dropouts, protocol deviations, withdrawal 
To clarify the 
sample size 
and provide 
provisions for 
an interim 
analysis 


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of consent, etc. Up to an additional 20% 
(about 20) subjects may be enrolled to 
account for protocol deviations, 
withdrawal of consent, etc., if necessary. 
A formal interim analysis is planned at 
the time when approximately 50 subjects 
of the remaining sample size are 
randomized and treated under this 
protocol amendment.  The objective of 
the interim analysis is to evaluate the 
sample size assumption following the 
conditional power approach.   
14 
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  
Urine jet conspicuity score provided by 
the blinded central independent image 
review process.   
Secondary Efficacy Endpoints 
1 = Time (minutes) to 
visualization (TTV) of the 
ureteral jets after each study 
drug administration 
1. Physician’s overall 
satisfaction with the IC 
treatment as an aid for the 
assessment of ureter patency 
Specify 
endpoints to be 
evaluated 


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2. Proportion of responders. A 
subject is considered a 
responder to IC if there is >=1 
point improvement in the 
conspicuity score following 
the IC treatment vs the saline 
treatment and the conspicuity 
score for the IC treatment 
must be (3, 4, or 5).  The 
responder criteria will be 
assessed separately for each 
ureter (left or right) based on 
the blinded central reviewer’s 
assessment.  
3. Time (minutes) to 
visualization (TTV) of blue 
color in the urine jet flow 
following administration of IC 
treatment.  
Other Endpoints 
1. UOVS scores Urine jet 
conspicuity score assessed by 
the blinded central reviewer 
surgeon. 
2. Concordance of the surgeons’ 
UOVS blinded central review 
urine jet conspicuity scores 
and the blinded central 
reviewer’s UOVS score 
surgeon’s urine jet 
conspicuity scores. 
15 
Videography 
 
 
 
 
 
To clarify the 
vasography 
procedure 
required 


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ntil 
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.for the duration of the 10-minute 
recording time.   
15-16 
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 in conjunction with the ureteral 
patency check performed as standard 
of care for the surgery being 
performed. 
3. 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
To clarify and 
specify the 
procedure for 
drug 
administration 
and 
videography 


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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 
After 10 minutes has elapsed the 
videography will be stopped, and the 
video file saved separately from the 
post saline video file. 
a. 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. 
5. The surgeon will document the UOVS 
score assessing the patency of the 
ureters following the saline injection. 
6. After the normal saline observation 
period of up to 10 minutes, a blinded 
dose of IC (2.5 or 5 mL, based on 


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randomization) will be administered 
over 1 minute intravenously by the 
non surgeon unblinded administrator, 
noting the exact time of injection.   
7. 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. 
5. Once identified, the surgeon will note 
the time of identification of efflux of 
the IV first blue urine following IC 
injection  
6. During the videography the surgeon 
should assess the patency of the 
ureters by identifying the efflux of 
urine from the ureteral orifices, 
however each video should be 
recorded for a full 10 minutes 
regardless of if and when the ureters 
are deemed patent. 
7. The surgeon will document his/her 
overall satisfaction by completing 
overall Physician Satisfaction 
Assessment Scale (PSAS) for each 
subject. 
8. 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 the first bowel 
movement post-surgery in this same 
subgroup. 
9. Subjects will be followed for 30 days 
(± 2 days) for adverse events. 


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10.The videos will be sent to a central 
imaging group who will pool and 
blind the videos. The videos will then 
be assessed and scored by a blinded 
central reviewer review process for 
ureteral patency conspicuity of the 
urine jet flow based on the urine jet 
flow conspicuity scale.  The surgeon 
will also review the videos and score 
the jet flows using the same UOVS 
conspicuity scale.  The surgeon’s 
score should be based on a review of 
the videos and does not have to occur 
on the day of surgery. The 
concordance in the UOVS conspicuity 
scores between the surgeon’s 
assessment and the central blinded 
reader process will be evaluated.   
16 
Interim analysis 
A Data Monitoring Committee (DMC) 
will be formed with the purpose of 
conducting an interim analysis after 
approximately 50 subjects have been 
enrolled and their videos have been 
reviewed by the central readers. These 50 
subjects do not include the initial 21 
subjects who were enrolled and treated 
before the 5-point conspicuity score was 
developed. The purpose of the DMC 
review will be to reevaluate the planned 
sample size of the study based on the 
observed treatment effect.  The DMC 
may also recommend that the study be 
stopped for futility if appropriate.  Study 
enrollment will not be held while the 
DMC is performing the data review.  The 
interim analysis will estimate the 
conditional power using the observed 
treatment effect at the interim analysis; 
the final decision will be dependent on 
Section added 
to specify an 
interim 
analysis by a 
DMC 


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the size of the estimated conditional 
power.   
16-17 
Statistical 
Analysis 
Methodology 
The statistical analysis for this study will 
be carried out using SAS® 9.34 or later 
and described in a detailed statistical 
analysis plan (SAP), which will be was 
finalized prior to first patient first visit 
and will be amended to reflect changes in 
Protocol Amendment No. 2. 
Clarification 
19 
Schedule of 
Assessments 
The SOA was updated as required to 
match procedure changes specified 
throughout the protocol amendment 
Consistency 
throughout 
protocol 
23 
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 35-point Ureteral Orifice 
Visualization Scale (UOVS):conspicuity 
score:  
1 = non visualization 
2 = inadequate/equivocal 
visualization  
3 = adequate/unequivocal 
visualization of the urine jet 
stream after administration of 
test agent 
 
1 = No jet observed 
2 = Weak jet, little color contrast 
3 = Color contrast or significant 
jet flow 
4 = Strong jet flow with good 
color contrast 
5 = Strong jet flow with striking 
contrast in color 
To implement 
the conspicuity 
score to 
evaluate the 
potential 
visualization 
advantage 
provided by 
indigo carmine 


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23 
3.1.2 Secondary 
Objectives 
2. To describe the time to visualization 
(TTV) of ureteral jets during urological 
and gynecological surgical procedures. 
3.2. 
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).    
3. To determine proportion of subjects 
meeting the responder definition in 
conspicuity score following IC 
treatment based on the blinded central 
reviewer’s assessment.  A subject is a 
responder when there is >=1 point 
improvement in the conspicuity scores 
following the IC vs saline treatment 
(IC – Saline >=1) and the conspicuity 
score following the IC treatment is (3, 
4, or 5). The responder criteria will be 
assessed separately for each ureter for 
each subject. 
4. To describe the time to visualization 
(TTV) of blue color in the ureteral jets 
To modify the 
secondary 
objectives of 
the trial based 
on the revised 
conspicuity 
scale and 
revised 
procedures. 


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flow following IC treatment when 
used as a visualization aid during 
urological and gynecological surgical 
procedures 
24 
3.2.1 Efficacy 
Endpoints 
The primary efficacy endpoint is the 
surgeon’s assessment of ureteral 
patency by UOVS: blinded central 
reader’s visualization conspicuity 
score, a 5-point ordinal scale score:  
1 =Not visualized  I cannot see the 
ureteral No jet observed 
2 =Weak jet, little color contrast 
1 =3 = Color contrast or significant jet 
flow 
1 =Inadequately visualized or 
equivocal  I am less than 
completely confident that the ureter 
is patent  
2 =Adequately visualized or 
unequivocal  I am completely 
confident that the ureter is patent  
4 =Strong jet flow with good color 
contrast 
5 =Strong jet flow with striking contrast 
in color 
The secondary efficacy endpoints 
include: 
1. Time (minutes) to visualization (TTV) 
of the ureteral jets after each study 
drug administration 
1. Proportion of physicians who agree 
that compared to saline, IC treatment 
improves visualization as an aid for the 
assessment of ureteral patency  
To modify the 
efficacy 
endpoints of 
the trial based 
on the revised 
conspicuity 
scale and 
revised 
procedures. 


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2. Proportion of responders.  A subject is 
a responder when there is >=1 point 
improvement in the conspicuity scores 
following the IC vs saline treatment 
(IC – Saline >=1)  and the conspicuity 
score following the IC treatment is (3, 
4, or 5).  The responder criteria will be 
assessed separately for each ureter for 
each subject based on the blinded 
central reviewer’s conspicuity score. 
3. Time (minutes) to visualization (TTV) 
of blue color in the ureteral jets flow 
following IC treatment  
 
Other efficacy endpoints will include: 
1. UOVS scores assessed by the blinded 
central reviewer 
1. Conspicuity score provided by surgeon  
2. Concordance of the UOVS in 
conspicuity scores between the 
surgeons’ assessments and the blinded 
central reviewer assessments. 
 
27 
4.3  
Scientific 
Rationale for 
Development of a 
New Conspicuity 
Score 
In a recent meeting with the FDA, the 
consideration of a new objective 
visualization elements of the scale such 
as conspicuity of ureteral outflow was 
discussed. The objective of development 
of this new conspicuity score is to be able 
to capture added benefit that IC may 
provide when used as an aid in the 
determination of ureteral patency.  Being 
able to visualize the contrasting blue 
color in the ureteral jet is a critical 
addition that IC treatment provides over 
the saline.  Hence, color contrast is an 
Section added 


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important factor for higher scores on the 
conspicuity scale  
The new 5-point conspicuity score tool 
was then developed using the videos 
collected from the first 18 patients 
enrolled into the study.  To test the tool 
the videos following the IC and saline 
treatment for each ureter for the first 18 
patients enrolled were provided to 4 
reviewers who were blinded to the 
treatment of the videos.  These reviewers 
scored the videos using the new scoring 
tool.  Inter reader consistency and 
treatment effect were evaluated and 
found to be satisfactory. Intra reader 
consistency testing is ongoing as it is 
being performed in conjunction with the 
central review process development and 
testing.  
 
Previous Sections 4.3 and 4.4 have been 
re-numbered as 4.4 and 4.5 to 
accommodate the addition of this 
section 
31 
6.3 Measures to 
Minimize Bias: 
Randomization 
and Blinding 
Blinded 
Conspicuity 
score 
Both the central review 
and surgeon conspicuity 
score results will be 
blinded form all 
members of the study 
team except for 
unblinded data 
management and 
statistical personnel 
directly involved with 
collecting and 
analyzing the data for 
the interim analysis.  
To specify who 
will have 
access to 
primary 
endpoint data 
32 
6.4. Study Drug 
Compliance 
The date and time of each dose (a dose of 
saline followed by a dose of IC 
treatment) administered in the clinic will 
Accuracy and 
clarity 


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be recorded in the source documents and 
recorded in the eCRF.  The randomized 
dose of IC treatment will be provided by 
the EDC randomization system to the 
designated unblinded site staff (i.e. 
pharmacist, coordinator, anesthesiologist, 
and or operating room/procedure nurse). 
35 
8.Study 
Assessments and 
Procedures 
(Screening) 
o 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. 
Above paragraphed moved for clarity 
Clarity and 
accuracy 
35 
8. Study 
Assessments and 
Procedures 
(Randomization / 
treatment) 
o 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. 
o After a subject is confirmed to be 
eligible, the subject will be 
randomized via the EDC online 
randomization system. 
Randomization will be a 1:1 ratio 
to be assigned to low dose (2.5 
mL IC) or high dose (5 mL IC).    
o Study procedures and their timing 
are summarized in the SoA. 
Protocol waivers or exemptions 
are not allowed. 
Clarity and 
accuracy 




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1.3.
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 or until 
adequate/unequivocal 
visualization is observed.  Then 
the camera may remain focused 
on the other ureteral orifice until 
adequate/unequivocal 
visualization is seen or the 


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completion of the 10 minute 
observation period has lapsed 
After 10 minutes has elapsed the 
videography will be stopped, and 
the video file saved separately 
from the post saline video file. 
a. If visualization of Once identified, 
the ureter is adequate/unequivocal 
and surgeon will note the time is 
less than 10 minutes, then the 
time of identification of 
visualization is to be noted for 
each ureter and the video may be 
stopped.  
b. The surgeon will document the 
UOVS score efflux of the first 
blue urine following the use of 
saline in the assessment of the 
patency of the ureters.  
 
3.5. Once identified, the surgeon will 
note the time of identification of 
efflux of the first blue urine 
following IC injection 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.   
4.6. Again,During the videography the 
surgeon will should assess the 
patency of the ureters by 
identifying the efflux of blue urine 
from each the ureteral orifice 
orifices, however each video 
should be recorded for up to a full 
10 minutes.  If / regardless of if 
and when visualization of each 
ureter is adequate/unequivocal 


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then the time of visualization is to 
be noted for each ureter and the 
procedure may continue.the 
ureters are deemed patent.  
2. The surgeon will document the 
UOVS score with the use of IC in 
the assessment of the patency of 
the ureters.  
3. 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. Urine and stool will be 
collected at specified time points 
in this same subgroup. 
5.7. The surgeon will document 
his/her overall satisfaction by 
completing oneoverall Physician 
Satisfaction Assessment for each 
subject. 
8. 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 the first 
bowel movement post-surgery in 
this same subgroup. 
6.9. Subjects will be followed for 30 
days (± 2 days) for adverse 
events. 
10. The videos will be sent to a 
central imaging group who will 
pool and blind the videos. The 
videos will then be assessed and 
scored by a blinded central review 
process for conspicuity of the 
urine jet flow based on the urine 
jet flow conspicuity scale.  The 


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surgeon will also review the 
videos and score the jet flows 
using the same conspicuity scale.  
The surgeon’s score should be 
based on a review of the videos 
and does not have to occur on the 
day of surgery. The concordance 
in the conspicuity scores between 
the surgeon’s assessment and the 
central reader process will be 
evaluated.   
37 
8.1.1. Ureter 
Conspicuity 
Assessment 
 
 
• 
Type of surgical 
procedure/visualization  
o Cystoscopic 
o Robotic 
o Open  
• 
Time each injection started 
• 
Time each injection completed 
• 
Time of to visualization of the 
ureter jet flow at the ureteral 
orifice blue urine following indigo 
carmine administration. 
• 
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 
To update 
efficacy 
endpoint 
procedures to 
allow for 
implementation 
of the urine jet 
conspicuity 
scale as the 
primary 
endpoint 


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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 
37-38 
8.1.3. 
Videography 
Specifics and 
Central Review 
Process 
 
 
 
 
 
 
 
 
 
 
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 
To update 
videography 
procedures to 
allow for 
implementation 
of the urine jet 
conspicuity 
scale as the 
primary 
endpoint 


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period is complete.for the entire 10 
minutes of the recording.  
 
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. 2 blinded central reviewers 
for conspicuity assessment using the 
same assessment tool used by the 
surgeon.  The consistency check between 
the two central reviewers will be 
performed; the two reviewers will be 
considered to be consistent if their scores 
for a given ureter/video is within (+/-) 1 
point; in this case, the average score of 
the two reviewers will be the final score 
for efficacy analysis.  Otherwise, a third 
reviewer will serve as a judicator.  The 
judicator will review the questioned 
video and the score from the judicator 
will be the final score for the efficacy 
analysis.   
 
The surgeon will also review the videos 
and score the jet flows using the same 
conspicuity scale.  The surgeon’s score 
should be based on a review of the videos 
and does not have to occur on the day of 
surgery. The concordance in the 
conspicuity scores between the surgeon’s 
assessment and the central reader process 
will be evaluated 
43 
9.1. Statistical 
Hypotheses 
It is expected that the IC treatment will 
improve the ureter visualization with 
effect size greater or equal compared to 
To update 
hypothesis 
based on 


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0.10 comparing to the normal saline 
using the 35-point UOVS measurement 
conspicuity score. 
implementation 
of the urine jet 
conspicuity 
scale as the 
primary 
endpoint 
43 
9.2. Sample Size 
Determination 
The following empirical distribution table 
(Table 1) is created to illustrate this effect 
size; initial sample size of approximately 
116 total was determined using the two-
group chi-square test comparing the 
difference in proportions in 3 categories 
(Table 2).  using an empirical 
assumption.  This empirical assumption 
was soon to be found false resulting in 
development of this new 5 point 
conspicuity score.  
 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
To justify the 
sample size 
using the 
revised 
primary 
endpoint 
measure 


Pr o ve p h ar m I nc.  
 
I n di g o C ar mi ne 0. 8 %  
Pr ot oc ol P V P -1 9I C 0 1 A me n d m e nt # 2  
C O N FI D E N TI A L  
Versi o n 3. 0, d ate d 0 2 Se pte m ber 2 0 1 9  
 
C h a n ge descri pti o ns i n it alics are descri ptive r at her t h a n re prese nt ative              Pa ge 9 8  of 1 0 9  
 
 
P a ge  
Secti o n #  
C h a n ge  
Re as o n / 
R ati o n ale 
 
 
 
 
 
 
9. 2. 1.  Ta ble 1  
E m pirical 
Distri b uti o n of Pr o p orti o n of S u bjects i n 
Eac h U O V S  Cate g ories a n d Effect Size 
bet wee n t he I C D ose Gr o u p vs Sali ne     
 
 
 
  
 
% s u bjects b y Ureteral 
Orifice Vis ualizati o n Scale 
[ 1] 
 
1 
2 
3 
Effect 
Size ∆² 
Sali ne 
( π1) 
0. 3 0 
0. 3 0 
0. 4 0 
 
I C 
L o w 
D ose 
( π2) 
0. 1 0 
0. 2 0 
0. 7 0 
0. 1 0 0 9 
[ 2] 
I C 
Hi g h 
D ose 
( π2) 
0. 0 5 
0. 1 0 
0. 8 5 
0. 2 2 0 3 
[ 3] 
[ 1] Sc ore 1 = N ot vis ualize d; 2 = I na de q uatel y 
vis ualize d or e q ui v ocal; 3 = A de q uatel y 
vis ualize d or u ne q ui v ocal.  
[ 2] Effect size bet wee n I C L o w d ose a n d Sali ne 
[ 3] Effect size bet wee n I C Hi g h d ose a n d Sali ne  
Effect size, ² = (2j 1j)²/[ 2(2j+ 1j)] 
 
9. 2. 2.  Ta ble 2   
N u m ber of 
S u bjects Per Gr o u p Usi n g T w o gr o u p 






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Therefore, the 
total sample size does not account for 
dropouts,for this study will stay the same 
(approximately 116) as planned before 
the 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. amendment. 
44 
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) and who have 
a video at 
approximately 10 
minutes in length 
following each 
treatment.  This will 
be the primary 
population for 
efficacy 
assessments.  
To clarify how 
subjects 
enrolled under 
the prior 
amendment 
will be 
analyzed 


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Note: the first 21 
patients that were 
enrolled prior to the 
development of 
conspicuity score 
will be 
EXCLUDED from 
the efficacy 
population.   
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.  
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 
be different from 
the actual received 
IC dose. 
Note: the first 21 
patients enrolled 
prior to the 
development of the 
conspicuity score 
will be INCLUDED 


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in the safety 
population.   
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. 
45 
9.4. Statistical 
Analyses 
The statistical analysis plan will be was 
finalized prior to First Patient First Visit 
(FPFV) of this study.  The SAP 
amendment will be finalized along with 
Protocol Amendment No. 2 finalization 
to reflect the changes in Protocol 
Amendment No. 2, 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. 
To specify the 
timing of the 
SAP and 
amendment 
45-46 
9.4.1. Interim 
Analysis 
A formal interim analysis is planned to 
confirm the observed treatment effect 
size on the conspicuity score from the test 
dataset.  The study will have a total of 
approximately 95 patients remained to be 
enrolled under the protocol Amendment 
No. 2.  This formal interim analysis will 
be held when 50 of the 95 remaining 
subjects are randomized and treated.  The 
interim analysis will only analyze the 
conspicuity score from the 50 patients 
provided by the central reviewers.  
Since randomization to the IC dose level 
will not be unblinded during the interim 
analysis, the overall IC treatment effect 
will be analyzed using the proportional 
odds model; the model will include the 
main effect of treatment (IC vs Saline) 
and ureter (left vs right) and control for 
the repeated measures within a subject. 
New Section 
outlining the 
plan for an 
interim 
analysis 


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This model is identified as the reduced 
model in the primary efficacy analysis of 
the primary endpoint in Section 9.4.3.1.   
Conditional Power (CP) will be 
calculated based on estimated odds ratio 
and standard error (in log scale) at 
interim analysis.  The prediction of the 
conditional power for a statistically 
significant result from the final analysis 
(at the two-sided 0.05 level) given the 
observed difference between treatments 
at a formal interim analysis can be based 
on the estimated treatment effect; 2-sided 
95% confidence intervals of the CP will 
also be calculated.  Interim analysis 
results will be then classified to three 
zones (unfavorable, promising, favorable) 
based on the estimated CP and decision 
to be made in each zone is specified as 
follows. 
 
Table 3:  Interim Analysis Conditional 
Power and Decision Principle added to 
protocol to document the interim analysis 
decision principals 
   
To protect the data integrity, an 
independent statistician/programmer who 
is not involved with the study will be 
responsible to perform the analysis.  A 
Data Monitoring Committee (DMC) will 
be formed to review the interim analysis 
results.  All members of the DMC will 
not be involved with the conduct of the 
study. To perform the interim analysis, 
the independent statistician will receive 
the final conspicuity score assessed by 
the central reviewers and the 
randomization code associated with each 
video/subject.  It is worthy to point out 


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that randomization is only pertained to 
the treatment (IC vs Saline) and the dose 
level of IC treatment will not be revealed 
at interim analysis.  The DMC may share 
with the study team (sponsor, 
investigator, CRO) the aggregated 
results/data (e.g., summary tables) but the 
individual subject data will not be shared 
with the study team.      
47 
9.4.2. Type 1 
Error Control for 
the Primary 
Efficacy Endpoint 
The study has a planned interim analysis 
when the enrollment is at approximately 
50 subjects.  The objective of this interim 
analysis is to assess the treatment effect 
size on the conspicuity score based on the 
blinded central reviewer.  To preserve the 
overall Type I error for the study, an 
alpha of 0.0001 will be used for this 
interim analysis and alpha of 0.0499 will 
be used for the final analysis. 
 
For the final analysis, there are two null 
hypotheses for the primary efficacy 
endpoint UOVS conspicuity score. 
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 0.050499, when both nominal 
p-values are less or equal to 0.050499, 
both null hypotheses will be rejected and 
Updated for the 
interim 
analysis and 
new endpoint 
measure 


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one will conclude that both IC dose 
groups are statistically different from the 
saline group in the examined parameter.  
When one (the one with the higher value) 
of the two nominal p-values is greater 
than or equal to 0.05 but the second 
nominal p-value (the one with the lower 
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 
conspicuity score only. 
47 
9.4.3. Treatment 
Effect Based on 
the Operating 
Surgeon’s UOVS 
Scores 
Conspicuity Score  
9.4.3.1. 
Conspicuity Score 
per Blinded 
Central Reviewers 
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.conspicuity scores.  The 
UOVSconspicuity score provided by the 
surgeon blinded central reviewer will be 
the primary efficacy endpoint whereas 
the score provided by the blinded central 
reviewer surgeon will serve only as an 
estimate of concordance.   
The treatment effect on the UOVS 
conspicuity score will be evaluated using 
a Generalized Estimating Equation (GEE) 
for Repeated Measures to control for the 
intra-subject correlation.  A proportional 
odds model will be fitted to estimate the 
treatment effect measured by odds that 
the conspicuity score following saline 
Section added 
based on new 
endpoint 
measure 


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treatment is less than that following the 
IC treatment. 
48 
9.4.3.2. 
Responders to IC 
Treatment per 
Blinded Central 
Reviewer 
A responder analysis will be performed to 
assess the ‘added benefit’ of IC over the 
saline within a subject.  That is, since 
each subject will serve as his/her own 
control, the difference in conspicuity 
score following the IC treatment and 
Saline will be used to define a responder. 
A subject is considered a responder if 
there is >=1 point improvement in the 
conspicuity score following the IC 
treatment vs Saline treatment and the 
conspicuity score following the IC 
treatment is >= 3.  This responder criteria 
will be assessed twice for each subject: 
once for the left side ureter and once for 
the right side of the ureter based on the 
blinded central reviewers assessment.   
Number (%) of subjects meeting the 
responder criteria will be tabulated by IC 
dose and each ureter (left only, right only, 
and left and right sides).  A 2-sided 95% 
confidence intervals will also be 
estimated using the normal 
approximation. 
Section added 
based on new 
endpoint 
measure 
48 
9.4.3.3. 
Concordance in 
Conspicuity Score 
Assessment 
The UOVS Concordance in conspicuity 
score will be assessed following 3 steps.  
First, the conspicuity score provided by 
the blinded central reviewer surgeons will 
be analyzed using the same GEE 
approach.  One way to evaluate treatment 
effect on the conspicuity described in 
Section 9.4.3.1.  Second, to assess the 
consistency between the surgeon and 
central rater is to evaluate by covariate 
analysis approach, in which the effect of 
raters (surgeon or blinded central rater) 
on the response using covariate approach.  
In this approach a new covariate 
Section added 
based on new 
endpoint 
measure 


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indicating evaluator (surgeon vs blinded 
central reviewer) will be added to will be 
evaluated in the GEE model. 
 
Another way to assess the consistency 
between the raters The third step is to 
derive the difference in UOVS 
conspicuity score between the surgeon 
and the central rater.  This difference will 
have a value of ( 2, 1, 0, +1, +2ranging 
(-4 to +4) for each of the 4 pairs of 
readings per subject, with 0 indicating a 
perfected agreement.  If a pair of ratings 
between the central readers and the 
surgeon is within (+/-) 1 point (i.e., the 
difference is ranging (-1 to +1), 
inclusive), the pair of ratings is marked as 
‘agree’, otherwise, the pair of ratings is 
‘not agree’.  The association of the 
difference in score this concordance 
response variable (agree vs not agree) 
with treatment and site ureter will also be 
evaluated using the GEE model.   
Treatment Effect based on the concept of 
logistic regression for dichotomized 
response. 
48-49 
9.4.4. Time to 
Visualization 
Time (minutes) to visualization (TTV) 
will be evaluated for IC treatment 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 stratified log-rank test for 
equal the homogeneity of the survival 
between the groups (IC vs Saline);curves.  
Cox Proportional hazards model 
approach may be used to estimate hazards 
ratios if data warrant as an exploratory 
analysis to assess differences between the 
low dose and high dose. In this analysis 
Section added 
based on new 
endpoint 
measure 


Provepharm Inc. 
 
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Protocol PVP-19IC01 Amendment #2 
CONFIDENTIAL 
Version 3.0, dated 02 September 2019 
 
Change descriptions in italics are descriptive rather than representative             Page 109 of 109 
 
 
Page  
Section # 
Change 
Reason / 
Rationale 
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 site) will 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. 
IC treated subjects with missing data will 
be censored at 10 minutes. 
49 
9.4.6.Assessment 
of Covariates 
The effect of procedure on the response 
will be assessed via covariate analysis.  
An appropriate model will be chosen for 
specific parameter.   In addition, if data 
warrant, demographic variables (age 
group, sex, and race) will be assessed as a 
potential covariance.  If data warrant, 
subgroup analysis per surgery type, or 
demographic variables may be 
performed.   
Section added 
based on new 
endpoint 
measure 
Throughout Various 
Numerous minor corrections or changes 
for readability that did not affect meaning 
were made throughout the document  
Clarity and 
accuracy 
 

Prot_001.pdf: 
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 
Version 1.1 
Administrative Change 1 
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: 
18 October 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. 
NCT# 04228445


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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. 




Provepharm Inc. 
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Protocol PVP-19IC01 
CONFIDENTIAL 
Version 1.1, dated 18, October 2019 
Page 4 of 59 
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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Protocol PVP-19IC01 
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Version 1.1, dated 18, October 2019 
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Table of Contents 
Sponsor Signature Page .................................................................................................................3 
ABBREVIATIONS ........................................................................................................................4 
1 
PROTOCOL SUMMARY ...........................................................................................10 
1.1. 
Protocol Synopsis ...........................................................................................................10 
1.2. 
Study Schema .................................................................................................................17 
1.3. 
Schedule of Assessments (SoA) .....................................................................................18 
2. 
INTRODUCTION ........................................................................................................19 
2.1. 
Study Rationale ...............................................................................................................19 
2.2. 
Background .....................................................................................................................20 
3. 
OBJECTIVES AND ENDPOINTS .............................................................................21 
3.1. 
Objectives .......................................................................................................................21 
3.1.1. 
Primary Objective ...........................................................................................................21 
3.1.2. 
Secondary Objectives ......................................................................................................21 
3.2. 
Endpoints ........................................................................................................................22 
3.2.1. 
Efficacy Endpoints ..........................................................................................................22 
3.2.2. 
Safety Endpoints .............................................................................................................22 
4. 
STUDY DESIGN ..........................................................................................................23 
4.1. 
Overall Design ................................................................................................................23 
4.2. 
Scientific Rationale for Study Design ............................................................................23 
4.3. 
Justification for Dose ......................................................................................................24 
4.4. 
End of Study Definition ..................................................................................................24 
5. 
STUDY POPULATION ...............................................................................................25 
5.1. 
Inclusion Criteria ............................................................................................................25 
5.2. 
Exclusion Criteria ...........................................................................................................25 
5.3. 
Screen Failures ................................................................................................................26 
6. 
STUDY DRUG ..............................................................................................................27 
6.1. 
Study Drug(s) Administered ...........................................................................................27 
6.2. 
Preparation/Handling/Storage/Accountability ................................................................28 
6.3. 
Measures to Minimize Bias: Randomization and Blinding ............................................28 
6.4. 
Study Drug Compliance..................................................................................................29 
6.5  
Concomitant Therapy .....................................................................................................29 
6.6 
Prohibited Concomitant Medications/Foods/Supplements .............................................29 
7. 
DISCONTINUATION OF STUDY DRUG AND SUBJECT 
DISCONTINUATION/WITHDRAWAL ...................................................................30 
7.1. 
Subject Discontinuation/Withdrawal from the Study .....................................................30 
7.2. 
Lost to Follow up ............................................................................................................30 
8. 
STUDY ASSESSMENTS AND PROCEDURES .......................................................32 
8.1. 
Efficacy Assessments .....................................................................................................33 
8.1.1. 
Ureter Assessment ..........................................................................................................33 
8.1.2. 
Physician Satisfaction Agreement Scale (PSAS) ...........................................................34 
8.1.3. 
Videography specifics .....................................................................................................35 


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8.2. 
Safety Assessments .........................................................................................................35 
8.2.1. 
Physical Examinations ....................................................................................................35 
8.2.2. 
Vital Signs .......................................................................................................................35 
8.2.3. 
Electrocardiograms .........................................................................................................36 
8.2.4. 
Clinical Safety Laboratory Assessments ........................................................................36 
8.3. 
Adverse Events and Serious Adverse Events .................................................................37 
8.3.1. 
Time Period and Frequency for Collecting AE and SAE 
Information .....................................................................................................................37 
8.3.2. 
Method of Detecting AEs and SAEs ..............................................................................38 
8.3.3. 
Follow-up of AEs and SAEs ...........................................................................................38 
8.3.4. 
Regulatory Reporting Requirements for SAEs ...............................................................38 
8.3.5. 
Pregnancy ........................................................................................................................38 
8.4. 
Pharmacokinetics ............................................................................................................39 
8.4.1. 
Plasma samples ...............................................................................................................39 
9. 
STATISTICAL CONSIDERATIONS ........................................................................41 
9.1. 
Statistical Hypotheses .....................................................................................................41 
9.2. 
Sample Size Determination ............................................................................................41 
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...............................................................................................................................41 
9.2.2. 
Table 2  Number of Subjects Per Group Using Two-group Chi-
square Test Comparing Proportions in 3 Categories [1, 2] .............................................41 
9.3. 
Populations for Analyses ................................................................................................42 
9.4. 
Statistical Analyses .........................................................................................................43 
9.4.1. 
Type 1 Error Control for the Primary Efficacy Endpoint ...............................................43 
9.4.2. 
Treatment Effect Based on the Operating Surgeon’s UOVS 
Scores ..............................................................................................................................43 
9.4.3. 
Treatment Effect on UOVS Based on Central Rater’s 
Assessment ......................................................................................................................44 
9.4.4. 
Treatment Effect on Time to Visualization ....................................................................44 
9.4.5. 
Assessment of PSAS with IC Treatment ........................................................................44 
9.4.6. 
Assessment of Covariates ...............................................................................................45 
9.4.7. 
Safety Analysis ...............................................................................................................45 
9.4.8. 
PK Analysis ....................................................................................................................45 
10. 
SUPPORTING DOCUMENTATION AND OPERATIONAL 
CONSIDERATIONS ....................................................................................................46 
10.1. 
Regulatory, Ethical, and Study Oversight Considerations .............................................46 
10.1.1. Regulatory and Ethical Considerations ...........................................................................46 
10.1.2. Financial Disclosure ........................................................................................................46 
10.1.3. Informed Consent Process ..............................................................................................47 
10.1.4. Institutional Review Board/Independent Ethics Committee (IRB) ................................47 
10.1.5. Data Protection................................................................................................................48 
10.1.6. Dissemination of Clinical Study Data .............................................................................48 
10.1.7. Data Quality Assurance ..................................................................................................49 
10.1.8. Source Documents ..........................................................................................................49 
10.1.9. Study and Site Start and Closure ....................................................................................50 


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10.1.10. Publication Policy ...........................................................................................................50 
10.2. 
Adverse Events: Definitions and Procedures for Recording, 
Evaluating, Follow-up, and Reporting ............................................................................51 
10.2.1. Definition of AE .............................................................................................................51 
10.2.2. Definition of SAE ...........................................................................................................52 
10.2.3. Recording and Follow-Up of AE and/or SAE ................................................................53 
10.2.4. Reporting of SAEs ..........................................................................................................55 
11. 
REFERENCES .............................................................................................................57 
Appendix 1: 
   Summary of Changes ....................................................................................59 
 


Provepharm Inc. 
 
Indigo Carmine 0.8%  
Protocol PVP-19IC01 
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Version 1.1, dated 18, October 2019 
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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). 




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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 
(Voelcker 1903) 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 (Chi 2016, Cohen 2018).  
 
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 (Ibeanu 2009).  
 
 


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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 (Grimes 2017). In the US, approximately 
600,000 hysterectomies, 226,000 prolapse repairs, and 135,000 stress incontinence operations 
are performed annually (Vakili 2005; 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 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 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. 
E09. Subjects who are pregnant or breast feeding 


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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.  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. 
 
6.6 
Prohibited Concomitant Medications/Foods/Supplements 
Prohibited medications, foods, and/or supplements would be anything that could discolor urine. 
The length of time required from last dose to study randomization is at the discretion of the 
investigator and should be based on the known duration of the effect on urine coloration and 
documented in the source documentation. Examples of prohibited medications are: 
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 
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. 


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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 
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. 


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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 subjects 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. 
• 
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. 


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• 
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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Version 1.1, dated 18 October 2019 
 
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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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Version 1.1, dated 18 October 2019 
 
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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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Protocol PVP-19IC01 
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Version 1.1, dated 18 October 2019 
 
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11. 
REFERENCES 
 
 
1. 
AAGL Practice Committee.  AAGL Practice Report: Practice guidelines for 
intraoperative cystoscopy in laparoscopic hysterectomy. J Minim Invasive Gynecol 
2012;19:407-411 
 
2. 
American Regent Package Insert. Indigo Carmine Injection American Regent, Inc. 
NDC:0517-0375-05. Date of revision 02/2017. 
 
3. 
Barbieri R. Farewell to Indigo Carmine. OBG Management 2014; 26(9):8-9,12 
 
4. 
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 
 
5. 
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 
 
6. 
Cohen SA, Carberry CL and Smilen SW. American Urogynecologic Society consensus 
statement: cystoscopy at the time of prolapse repair.  Female Pelvic Med Reconstr Surg 
2018;24:258–259 
 
7. 
Gill EJ, Elser DM, Bonidie MJ, Roberts KM, Hurt WG. The routine use of cystoscopy 
with the Burch procedure. Am J Obstet Gynecol. 2001 Aug;185(2):345-8. 
8. 
Gilmour D and Flowerdew G. Rates of urinary tract injury from gynecologic surgery and 
the role of intraoperative cystoscopy. Obstet Gynecol 2006;107:1366–1372 
 
9. 
Grimes CL, Patankar S, Ryntz T et al.  Evaluating ureteral patency in the post-indigo 
carmine era: a randomized controlled trial. Am J Obstet Gynecol. 2017;217:601.e1 – 10 
 
10. 
Harris RL, Cundiff GW, Theofrastous JP, Yoon H, et al. The value of intraoperative 
cystoscopy in urogynecologic and reconstructive pelvic surgery. Am J Obstet Gynecol 
1997; 177:1367-71. 
 
11. 
Ibeanu O, Chesson RR, Echols KT et al. Urinary tract injury during hysterectomy based 
on universal cystoscopy. Obstet Gynecol 2009;113:6–10 
 
12. 
Jabs CF, Drutz HP. The role of intraoperative cystoscopy in prolapse and incontinence 
surgery. Am J Obstet Gynecol 2001;185(6):1368–73 
 


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Indigo Carmine 0.8%  
Protocol PVP-19IC01 
CONFIDENTIAL 
Version 1.1, dated 18 October 2019 
 
Page 58 of 59 
 
 
13. 
Jelovsek JE, Chen G, Roberts SL, et al. Incidence of lower urinary tract injury at the time 
of total laparoscopic hysterectomy. J Soc Laparoendoscopic Surg 2007; 11:422-27. 
 
14. 
Jung M, Kiesslich R. Chromoendoscopy and intravital staining techniques, Baillière’s 
Clinical Gastroenterology 1999; 13(1):11-19 
 
15. 
Lacy, W.W., Ugaz, C., Newman, E.V. The use of indigo carmine for dye dilution curves.  
Circulation Research 1955; 111: 570-574 
 
16. 
Lee M, Sharifi R. Methylene blue versus indigo carmine. Urology 1996;47(5):783-4. 
 
17. 
O’Brien WM, Lynch JH. Simplification of double-dye test to diagnose various types of 
vaginal fistulas. Urology 1990;36(5):456 
18. 
Oravisto, K.J., Investigations into the excretion mechanism of indigo carmine in normal 
human kidney. Ann. Chir. Gynaecol. Fenn Suppl. 1957;46(2):1-79 
 
19. 
Pettit PD, Petrou SP. The value of cystoscopy in major vaginal surgery.  Obstet Gynecol. 
1994;84(2):318-20 
 
20. 
Speights SE, Moore RD, Miklos JR. Frequency of lower urinary tract injury at 
laparoscopic burch and paravaginal repair. J Am Assoc Gynecol Laparosc. 2000; 7:515-8 
 
21. 
SmPC (Summary of Product Characteristics). Indigo Carmine 40 mg/5 mL solution for 
injection. SERB S.A, Belgium. Marketing Authorisation Number: PL 43956/0001. Date 
of Revision of the Text: 05/06/2015 
 
22. 
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 
 
23. 
Voelcker F, Joseph, E, Funktionelle Nierendiagnostik ohne Ureterenkatheter. Munch. 
Med. Wschr. 1903; 50: 2081-2089 
 
24. 
Weinstein JR and Anderson S. The aging kidney: physiological changes.  Adv Chronic 
Kidney Dis. 2010 July ; 17: 302–307 
 
 
 


Provepharm Inc. 
 
Indigo Carmine 0.8%  
Protocol PVP-19IC01 
CONFIDENTIAL 
Version 1.1, dated 18 October 2019 
 
Page 59 of 59 
 
Appendix 1: 
   Summary of Changes 
 
 Version 1.1 Administrative Change 1 
 
1. Addition of reference cited in document to the bibliography 
2. Update of Sponsor contact information 
3. Correction of mis-numbered section (6.6) 
4. Clarification of timing for prohibited Medications/Foods/Supplements 
5. Clarification of packaging for Indigo Carmine in section 6.2 
 

Prot_000.pdf: 
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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Protocol PVP-19IC01 
CONFIDENTIAL 
Version 1, dated 1 July 2019 
Page 3 of 60 
 
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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Version 1, dated 1 July 2019 
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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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Version 1, dated 1 July 2019 
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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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Protocol PVP-19IC01 
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Version 1, dated 1 July 2019 
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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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Version 1, dated 1 July 2019 
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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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Version 1, dated 1 July 2019 
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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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Version 1, dated 1 July 2019 
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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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Indigo Carmine 0.8%  
Protocol PVP-19IC01 
CONFIDENTIAL 
Version 1, dated 1 July 2019 
Page 10 of 60 
 
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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