Clinical trial NCT05188261
Conditions: Healthy Volunteers
Keywords: healthy volunteers
Sponsor: Ironwood Pharmaceuticals, Inc.
Start date: 2022-01-18; Completion date: 2022-03-21
Enrollment: 32 participants; Allocation: RANDOMIZED; Intervention model: SEQUENTIAL

Brief summary:
This is a first-in-human study to evaluate the safety and tolerability of single ascending doses of IW-3300. The study drug will be administered rectally as a low-volume (20 mL) enema. Study participants will be randomized in a 3:1 ratio to receive a single dose of IW-3300 or placebo. Up to 5 different doses of IW-3300 will be studied. Safety reviews will be conducted before proceeding to each higher dose.

Detailed description:
This is a Phase 1, single-center, randomized, double-blind, placebo-controlled, single-ascending-dose study assessing the safety, tolerability, and pharmacokinetics (PK) of IW 3300 administered rectally as a low-volume enema in healthy adult volunteers. This first-in-human study will assess participants for safety, tolerability, and PK.

The study includes up to 6 treatments: placebo and up to 5 dose levels of IW-3300 which will be determined after safety reviews of previous cohorts.

This study will enroll a maximum of 40 participants (up to 5 cohorts of 8 participants each). The 8 participants within each cohort will be randomized in a double-blind manner to receive a single dose of IW-3300 (6 participants) or placebo (2 participants), administered rectally as a low-volume \[20 mL\] enema. Each cohort will progress through a Screening Period, Clinic Period, and Follow-up Period. Treatment duration will be 1 day. Participants will remain in the Phase 1 unit for approximately 24 hours after dosing and will be contacted by phone for follow-up approximately 2 weeks after dosing. Total participation will be 22 to 45 days, including the Screening, Clinic, and Follow-up Periods.

Arms: arm 1: Dose 1: within the cohort, 6 participants receive active drug (IW-3300) arm 2: Within the cohort, 2 participants will receive the matching placebo dose arm 3: Dose 2: within the cohort, 6 participants receive active drug (IW-3300) arm 4: Within the cohort, 2 participants will receive the matching placebo dose arm 5: Dose 3: within the cohort, 6 participants receive active drug (IW-3300) arm 6: Within the cohort, 2 participants will receive the matching placebo dose arm 7: Dose 4: within the cohort, 6 participants receive active drug (IW-3300) arm 8: Within the cohort, 2 participants will receive the matching placebo dose
Interventions: intervention 1: IW-3300 intervention 2: Placebo
Intervention descriptions: intervention 1: A single dose of IW-3300 administered rectally (as a low-volume \[20 mL\] enema) following a fast of at least 6 hours. intervention 2: A single dose of placebo administered rectally (as a low-volume \[20 mL\] enema) following a fast of at least 6 hours.

Protocol documents:
Prot_000.pdf: 
Protocol 
C3300-101 
1 
TITLE PAGE
Protocol Title:
A Phase 1 Placebo-controlled Study of the Safety and 
Tolerability of Rectally Administered Single Ascending Doses 
of IW 3300 in Healthy Volunteers
Short Title: 
A Phase 1 Study of Single Ascending Doses of IW 3300 in 
Healthy Volunteers 
Protocol Number:
C3300-101 
Version Number
/Amendment Number: 
Version 4/Amendment 3 
Final Date: 
10 January 2022 
Study Intervention: 
IW 3300 
Study Phase: 
1 
Sponsor: 
Ironwood Pharmaceuticals, Inc. 
100 Summer Street, Suite 2300 
Boston, MA 02110 USA 
Medical Monitor  
Study Contact: 
Medical monitor and study contact information will be 
provided separately.  
Confidentiality Statement 
The contents of this document are confidential and belong to Ironwood Pharmaceuticals, Inc. 
Except as may be otherwise agreed to in writing, by accepting or reviewing these materials, you 
(including any colleagues or subordinates) agree to hold such information in confidence and not 
to disclose it to others (except where required by applicable law), nor to use it for unauthorized 
purposes. In the event of actual or suspected breach of this obligation, Ironwood should be 
promptly notified. 


Protocol 
C3300-101 
2 
PROTOCOL AMENDMENT SUMMARY OF CHANGES TABLE
DOCUMENT HISTORY 
Document 
Date 
Amendment 3 
10 January 2022 
Amendment 2 
24 November 2021
Amendment 1 
22 September 2021 
Original Protocol
06 August 2021
Amendment 3 (10 January 2022) 
Overall Rationale for the Amendment: 
Changes were made at the request of the US FDA, the Institutional Review Board (IRB), and the 
Clinical Research Unit (CRU). 
Section # and Name 
Description of Change 
Brief Rationale 
Substantial/ 
Nonsubstantial 
Section 1.1.5, 
(Synopsis/Overall 
Design), 
Section 1.1.8 
(Synopsis/ 
Intervention Groups 
and Duration), 
Section 2.3.1 (Risk 
Assessment, Row #2: 
Unknown Adverse 
Events [AEs], 
laboratory 
abnormalities, or 
other safety findings 
with a novel study 
drug), Section 4.1 
(Overall Design), 
Section 4.2 
(Scientific Rationale 
for Study Design), 
and Section 6 (Study 
Intervention) 
Sentinel dosing was added. As 
such, in Cohort 1 only, the first 
2 subjects will be randomized in a 
1:1 ratio to 
or placebo 
and dosed at least 4 hours before 
the remaining subjects in the 
cohort. 
The IRB requested 
that sentinel dosing be 
incorporated in 
Cohort 1 only. 
Substantial 


Protocol 
C3300-101 
3 
Section # and Name 
Description of Change 
Brief Rationale 
Substantial/ 
Nonsubstantial 
Section 1.3 (Schedule 
of Activities/ 
Table 1) and 
Section 10.2 
(Appendix 2: Clinical 
Laboratory Tests; 
Table 2) 
The following laboratory tests 
were added at Check-in (Day 
) 
(predose) and Day 2 (Discharge): 
erythrocyte sedimentation rate 
(ESR), C-reactive protein (CRP) 
and hemoccult testing. 
All laboratory tests will now be 
analyzed at a local laboratory with 
the exception of the point of care 
hemoccult tests and the ESR which 
will be analyzed at an outside 
laboratory. 
Because all urine tests will now be 
analyzed at the local laboratory, 
the requirement to enter select 
urine-based screening test results 
into the CRF was deleted from 
Table 2. 
The FDA requested 
these additional 
laboratory 
assessments. 
The CRU now has the 
ability to run several 
laboratory assays 
locally. 
Substantial 
Section 1.3 (Schedule 
of Activities/ 
Table 1) and 
Section 10.2 
(Appendix 2: Clinical 
Laboratory Tests; 
Table 2) 
Alcohol testing at Check-in 
(Day 1) will be done via a urine 
test. References to the alcohol 
screen being done by breath test 
were deleted. 
The CRU now has the 
ability to do this 
screen via a urine test. 
Substantial 
Section 2.3.1 (Risk 
Assessment,  
Row # 1: Potential 
for diarrhea and 
secondary 
dehydration) 
The Bristol Stool Form Scale 
(BSFS) score of 7 (watery stools) 
was added in the evaluation of the 
severity of diarrhea. 
The FDA requested 
that the definition of 
diarrhea that would 
result in temporary 
dose suspension or 
trial discontinuation 
include a criterion for 
stool consistency as 
measured by BSFS. 
Substantial 


Protocol 
C3300-101 
4 
Section # and Name 
Description of Change 
Brief Rationale 
Substantial/ 
Nonsubstantial 
Section 5.1.4 
(Inclusion 
Criteria/Sex) 
Inclusion Criterion #7b was 
removed. 
Criterion #7a was promoted to 
Criterion #7. 
The FDA requested 
this change. Inclusion 
Criterion #7b was 
considered redundant 
as 1) only women of 
nonchildbearing 
potential are eligible 
for enrollment and 
these women would 
not be taking 
hormonal 
contraceptives, and 
2) subjects who are 
taking any 
prescription 
medications (eg, 
hormonal 
contraceptives) are 
excluded from 
enrollment as per 
Exclusion 
Criterion #5. 
Nonsubstantial 
Section 8.2.5 
(Suicidal Ideation 
and Behavior Risk 
Monitoring) 
Section was deleted. 
This protocol section 
was part of 
Ironwood’s standard 
protocol template 
language and did not 
apply to IW 3300 as 
is a 
guanylate cyclase C 
(GC C) agonist that is 
rectally administered 
and acts locally in the 
gastrointestinal tract.  
Nonsubstantial 
Section 8.3.1 (Time 
Period and Frequency 
for Collecting 
Adverse Event [AE] 
and Serious Adverse 
Event [SAE] 
Information) and 
Section 10.3.3.1 (AE 
and SAE Recording)  
The guidance for categorizing and 
recording a subject's medical 
history versus their pretreatment 
adverse events was clarified. 
For subjects who have an SAE and 
fail screening, only their SAE 
information will be recorded in the 
CRF. 
This change was made 
to comport with the 
Sponsor's data 
handling guidelines. 
Nonsubstantial


Protocol 
C3300-101 
5 
Section # and Name 
Description of Change 
Brief Rationale 
Substantial/ 
Nonsubstantial 
Section 10.2 
(Appendix 2: Clinical 
Laboratory Tests; 
Table 2) 
Bullet point #4 was added: 
• The investigator may consider 
repeating a laboratory test for 
any subject who has an 
abnormal laboratory test result 
(eg, positive hemoccult test, out-
of-range chemistry test, 
abnormal urine culture) at 
Discharge (Day 2). The decision 
to repeat a laboratory test 
would be based on the 
investigator's clinical judgment 
after discussion with the 
sponsor's medical monitor and 
depending on the clinical 
context. 
This addition provides 
guidance on repeating 
laboratory tests. 
Nonsubstantial 
Section 10.3.4.2 
(SAE Reporting to 
Ironwood Global 
Patient Safety via 
Paper SAE Report 
Form) 
The email address was updated to: 
ICSRoperations@ironwoodpharma
.com. 
The change reflects 
the update. 
Nonsubstantial 
Section 10.4.2 
(Contraception 
Guidance) 
The reference to hormonal 
contraceptives was removed. 
Because only women 
of nonchildbearing 
potential are eligible 
for enrollment and the 
reference to hormonal 
contraceptives was 
removed. 
Nonsubstantial 
Throughout 
Minor editorial and document 
formatting revisions 
Minor corrections and 
clarifications 
Nonsubstantial 


Protocol 
C3300-101 
6 
TABLE OF CONTENTS
TITLE PAGE ...................................................................................................................................1
PROTOCOL AMENDMENT SUMMARY OF CHANGES TABLE ............................................2
1.
PROTOCOL SUMMARY ..........................................................................................12
1.1.
Synopsis ......................................................................................................................12
1.1.1. 
Protocol Title ..............................................................................................................12
1.1.2. 
Short Title ...................................................................................................................12
1.1.3. 
Rationale .....................................................................................................................12
1.1.4. 
Objectives and Endpoints ...........................................................................................14
1.1.5. 
Overall Design ............................................................................................................14
1.1.6. 
Disclosure Statement ..................................................................................................15
1.1.7. 
Number of Subjects ....................................................................................................15
1.1.8. 
Intervention Groups and Duration ..............................................................................15
1.1.9. 
Data Monitoring Committee: No ................................................................................15
1.2.
Schema ........................................................................................................................16
1.3.
Schedule of Activities .................................................................................................17
2.
INTRODUCTION ......................................................................................................21
2.1.
Study Rationale ...........................................................................................................21
2.2.
Background .................................................................................................................21
2.3.
Benefit/Risk Assessment ............................................................................................23
2.3.1. 
Risk Assessment .........................................................................................................23
2.3.2. 
Benefit Assessment .....................................................................................................24
2.3.3. 
Overall Benefit: Risk Conclusion ...............................................................................25
3.
OBJECTIVES AND ENDPOINTS ............................................................................26
3.1.
Objectives ...................................................................................................................26
3.1.1. 
Primary Objective .......................................................................................................26
3.1.2. 
Exploratory Objectives ...............................................................................................26
3.2.
Estimands ....................................................................................................................26
3.3.
Endpoints ....................................................................................................................26
3.3.1. 
Primary Endpoints (Safety) ........................................................................................26
3.3.2. 
Exploratory Endpoints (Pharmacokinetics and Pharmacodynamics) .........................26
4.
STUDY DESIGN .......................................................................................................27


Protocol 
C3300-101 
7 
4.1.
Overall Design ............................................................................................................27
4.1.1. 
Study Periods ..............................................................................................................27
4.1.1.1. 
Screening Period .........................................................................................................27
4.1.1.2. 
Clinic Period ...............................................................................................................28
4.1.1.3. 
Follow-up Period ........................................................................................................28
4.2.
Scientific Rationale for Study Design ........................................................................28
4.2.1. 
Subject Input into Design ...........................................................................................29
4.3.
Justification for Dose ..................................................................................................29
4.4.
End of Study Definition ..............................................................................................29
5.
STUDY POPULATION .............................................................................................30
5.1.
Inclusion Criteria ........................................................................................................30
5.1.1. 
Age ..............................................................................................................................30
5.1.2. 
Type of Subject and Disease Characteristics ..............................................................30
5.1.3. 
Weight .........................................................................................................................30
5.1.4. 
Sex ..............................................................................................................................30
5.1.5. 
Informed Consent .......................................................................................................31
5.2.
Exclusion Criteria .......................................................................................................31
5.2.1. 
Medical Conditions .....................................................................................................31
5.2.2. 
Prior/Concomitant Therapy ........................................................................................31
5.2.3. 
Prior/Concurrent Clinical Study Experience ..............................................................31
5.2.4. 
Diagnostic Assessments ..............................................................................................32
5.2.5. 
Other Exclusions .........................................................................................................32
5.3.
Lifestyle Considerations .............................................................................................32
5.3.1. 
Meals and Dietary Restrictions ...................................................................................32
5.3.2. 
Caffeine, Alcohol, and Tobacco .................................................................................32
5.3.3. 
Activity .......................................................................................................................33
5.4.
Screen Failures ............................................................................................................33
6.
STUDY INTERVENTION ........................................................................................34
6.1.
Study Interventions Administered ..............................................................................34
6.1.1. 
Dosing Instructions .....................................................................................................35
6.2.
Preparation/Handling/Storage/Accountability ............................................................35
6.3.
Measures to Minimize Bias: Randomization and Blinding ........................................36
6.3.1. 
Randomization ............................................................................................................36


Protocol 
C3300-101 
8 
6.3.2. 
Blinding ......................................................................................................................36
6.4.
Study Intervention Compliance ..................................................................................37
6.5.
Concomitant Therapy .................................................................................................37
6.5.1. 
Rescue Medicine .........................................................................................................37
6.6.
Dose Modification ......................................................................................................37
6.7.
Intervention After the End of the Study .....................................................................37
7.
DISCONTINUATION OF STUDY INTERVENTION AND SUBJECT 
DISCONTINUATION/WITHDRAWAL ..................................................................38
7.1.
Discontinuation of Study Intervention ........................................................................38
7.1.1. 
Study Suspension or Termination ...............................................................................38
7.2.
Subject Discontinuation/Withdrawal from the Study .................................................38
7.3.
Lost to Follow-up .......................................................................................................39
8.
STUDY ASSESSMENTS AND PROCEDURES ......................................................40
8.1.
Efficacy Assessments .................................................................................................40
8.2.
Safety Assessments .....................................................................................................40
8.2.1. 
Physical Examinations ................................................................................................40
8.2.2. 
Vital Signs ..................................................................................................................41
8.2.3. 
Electrocardiograms .....................................................................................................41
8.2.4. 
Clinical Safety Laboratory Assessments ....................................................................41
8.3.
Adverse Events and Serious Adverse Events .............................................................42
8.3.1. 
Time Period and Frequency for Collecting AE and SAE Information .......................42
8.3.2. 
Method of Detecting AEs and SAEs ..........................................................................43
8.3.3. 
Follow-up of AEs and SAEs .......................................................................................43
8.3.4. 
Regulatory Reporting Requirements for SAEs ...........................................................43
8.3.5. 
Pregnancy ...................................................................................................................44
8.3.6. 
Cardiovascular and Death Events ...............................................................................44
8.3.7. 
Disease-related Events and/or Disease-related Outcomes Not Qualifying as 
AEs or SAEs ...............................................................................................................44
8.3.8. 
Adverse Events of Special Interest .............................................................................44
8.4.
Treatment of Overdose ...............................................................................................44
8.5.
Pharmacokinetics ........................................................................................................44
8.6.
Pharmacodynamics .....................................................................................................45
8.7.
Genetics ......................................................................................................................45


Protocol 
C3300-101 
9 
8.8.
Biomarkers ..................................................................................................................46
8.9.
Immunogenicity Assessments ....................................................................................46
8.10.
Medical Resource Utilization and Health Economics ................................................46
9.
STATISTICAL CONSIDERATIONS .......................................................................47
9.1.
Statistical Hypotheses .................................................................................................47
9.2.
Sample Size Determination ........................................................................................47
9.3.
Populations for Analyses ............................................................................................47
9.4.
Statistical Analyses .....................................................................................................47
9.4.1. 
General Considerations ...............................................................................................47
9.4.2. 
Primary Endpoints Analyses .......................................................................................48
9.4.3. 
Secondary Endpoint Analysis .....................................................................................48
9.4.4. 
Exploratory Endpoints Analyses ................................................................................48
9.4.5. 
Safety Analyses ..........................................................................................................49
9.4.6. 
Other Analysis/Analyses ............................................................................................49
9.5.
Interim Analyses .........................................................................................................49
9.6.
Data Monitoring Committee (DMC) ..........................................................................50
10.
SUPPORTING DOCUMENTATION AND OPERATIONAL 
CONSIDERATIONS ..................................................................................................51
10.1.
Appendix 1: Regulatory, Ethical, and Study Oversight Considerations ....................51
10.1.1. 
Regulatory and Ethical Considerations ......................................................................51
10.1.2. 
Financial Disclosure ...................................................................................................51
10.1.3. 
Informed Consent Process ..........................................................................................51
10.1.4. 
Data Protection ...........................................................................................................52
10.1.5. 
Committee Structure ...................................................................................................52
10.1.6. 
Dissemination of Clinical Study Data ........................................................................52
10.1.7. 
Data Quality Assurance ..............................................................................................53
10.1.8. 
Source Documents ......................................................................................................53
10.1.9. 
Study and Site Start and Closure ................................................................................54
10.1.10. 
Publication Policy .......................................................................................................54
10.2.
Appendix 2: Clinical Laboratory Tests .......................................................................55
10.3.
Appendix 3: Adverse Events: Definitions and Procedures for Recording, 
Evaluating, Follow-up, and Reporting ........................................................................57
10.3.1. 
Definition of AE and TEAE .......................................................................................57


Protocol 
C3300-101 
10 
10.3.1.1. 
AE Definition ..............................................................................................................57
10.3.1.2. 
TEAE Definition .........................................................................................................57
10.3.1.3. 
Events Meeting the AE Definition .............................................................................57
10.3.1.4. 
Events NOT Meeting the AE Definition ....................................................................58
10.3.2. 
Definition of SAE .......................................................................................................58
10.3.3. 
Recording and Follow-up of AE and/or SAE .............................................................59
10.3.3.1. 
AE and SAE Recording ..............................................................................................59
10.3.3.2. 
Assessment of Intensity/Severity ................................................................................60
10.3.3.3. 
Assessment of Causality .............................................................................................60
10.3.3.4. 
Follow-up of AEs and SAEs .......................................................................................61
10.3.4. 
Reporting of SAEs ......................................................................................................61
10.3.4.1. 
SAE Reporting to Ironwood Global Patient Safety via an Electronic Data 
Collection Tool ...........................................................................................................61
10.3.4.2. 
SAE Reporting to Ironwood Global Patient Safety via Paper SAE Report 
Form ............................................................................................................................62
10.4.
Appendix 4: Contraceptive Guidance and Collection of Pregnancy 
Information .................................................................................................................63
10.4.1. 
Definitions ..................................................................................................................63
10.4.1.1. 
Women of Childbearing Potential (WOCBP) ............................................................63
10.4.1.2. 
Women Not Considered Women of Childbearing Potential ......................................63
10.4.2. 
Contraception Guidance .............................................................................................63
10.4.2.1. 
Collection of Pregnancy Information .........................................................................64
10.5.
Appendix 5: Abbreviations .........................................................................................65
10.6.
Appendix 6: Protocol Signatures ................................................................................68
10.6.1. 
Sponsor Signature .......................................................................................................68
10.6.2. 
Investigator Signature .................................................................................................69
10.7.
Appendix 7: Protocol Amendment History ................................................................70
11.
REFERENCES ...........................................................................................................80


Protocol 
C3300-101 
11 
LIST OF TABLES
Table 1: 
Schedule of Activities .................................................................................................17
Table 2:
Protocol-Required Safety Laboratory Assessments ...................................................56
Table 3: 
List of Abbreviations ..................................................................................................65
LIST OF FIGURES
Figure 1: 
Schematic of Study Design .........................................................................................16


Protocol 
C3300-101 
12 
1.
PROTOCOL SUMMARY
1.1. 
Synopsis 
1.1.1. 
Protocol Title
A Phase 1 Placebo-controlled Study of the Safety and Tolerability of Rectally Administered 
Single Ascending Doses of 
in Healthy Volunteers 
1.1.2. 
Short Title 
A Phase 1 Study of Single Ascending Doses of 
in Healthy Volunteers 
1.1.3. 
Rationale 
is being developed for the treatment of bladder pain associated with interstitial 
cystitis/bladder pain syndrome (IC/BPS). 
is a novel, 13-amino-acid, guanylate 
cyclase C (GC C) agonist peptide. GC C, the target of IW 3300, is predominantly expressed on 
the luminal surface of the small and large intestines. When GC C receptors are stimulated, 
intracellular cyclic guanosine monophosphate (cGMP) is secreted across the basolateral 
membrane of colonic epithelial cells in the submucosa by multidrug resistance proteins MRP4 
and MRP5, decreasing the activity of afferent nerve fibers located in the colonic wall, resulting 
in reduced visceral pain, which ultimately produces an analgesic effect in other organs of the 
abdominopelvic region via action mediated through the common afferent pathways.(1, 2) 
Experiments conducted in animals indicate that colonic hypersensitivity induces persistent 
hypersensitivity of bladder afferent pathways in the absence of bladder pathology.(2) 
Hypersensitivity of bladder afferent pathways resembles the symptoms observed in patients with 
interstitial cystitis/bladder pain syndrome (IC/BPS). Afferent neurons are known to have 
peripheral endings in both the colon and the bladder, and the axons of colonic and bladder 
neurons travel through the same splanchnic and pelvic nerves. These nonclinical observations 
support the concept of cross-organ sensitization and suggest that GC C agonists such as 
may be useful clinically for the treatment of bladder pain associated with IC/BPS. 
has shown beneficial effects in reducing visceral pain in several animal models, 
including models of colonic pain and models of extra-intestinal chronic pelvic pain following 
intracolonic or intrarectal administration. 
(3 g/kg/day), administered either 
intracolonically or orally to female rats, reduced endometriosis-induced mechanical hind paw 
allodynia. When administered intracolonically (1, 3, 10 g/kg/day), 
reduced 
endometriosis-induced vulvar pain in a dose-dependent manner. In a rat model of chronic 
radiation proctopathy induced either by fractionated dose (total dose 48 Gray) or single high dose 
(25 Gray) of radiation, 
(3 g/kg/day) administered intracolonically reversed colorectal 
and bladder hypersensitivity. 
(3 g/kg/day) administered intrarectally reduced vaginal 
hypersensitivity in adult female mice previously exposed to neonatal vaginal irritation. In a rat 
model of early life stress, 
(3 g/kg/day) administered intracolonically reduced bladder 
and mechanical hind paw allodynia in adult male rats. 
is a new chemical entity that has similar activity to the naturally occurring peptide 
hormones guanylin and uroguanylin, which are key regulators of electrolytes. Activation of 
GC C expressed on the luminal surface of the intestinal epithelium leads to increased 




Protocol 
C3300-101 
14 
1.1.4. 
Objectives and Endpoints 
Objective 
Endpoints 
Primary 
To assess the safety and tolerability of single 
ascending doses of 
administered rectally 
via enema in healthy volunteers. 
Incidence of treatment-emergent adverse events 
(TEAEs) 
Incidence of treatment-emergent serious adverse 
events (TESAEs) 
Exploratory
To summarize the single-dose pharmacokinetics 
(PK) of 
administered rectally via enema 
in healthy volunteers. 
PK parameters 
Total amount of 
in stool (concentration 
and percent recovery) 
To summarize the single-dose pharmacodynamics 
(PD) of 
administered rectally via enema 
in healthy volunteers based on bladder and bowel 
assessments. 
Change from baseline in bowel movement (BM) 
frequency during the Clinic Period 
Change from baseline in stool consistency (Bristol 
Stool Form Scale [BSFS] score) during the Clinic 
Period 
Change from baseline in urinary frequency during 
the Clinic Period for a 24 hour duration 
Change from baseline in urinary urgency during 
the Clinic Period for a 24 hour duration 
Change from baseline in nocturia during the 
Clinic Period for a 24 hour duration 
Change from baseline in pain or burning in 
bladder or pelvic area during the Clinic Period for 
a 24 hour duration 
1.1.5. 
Overall Design 
This is a Phase 1, single-center, randomized, double-blind, placebo-controlled, single-ascending-
dose study assessing the safety, tolerability, and PK of 
administered rectally as a low-
volume enema in healthy adult volunteers. Each cohort will progress through 3 study periods: 
(1) Screening Period, (2) Clinic Period, and (3) Follow-up Period; these periods and the 
progression between cohorts are illustrated in the study schematic (Figure 1).
This first-in-human study in healthy adult volunteers will assess all subjects for safety, PK, and 
effect on stool frequency/form and urination. 
In Cohort 1, sentinel dosing will be implemented. If no safety signals are noted in the first 
2 subjects (randomized 1:1 to 
or placebo) following an observation period of at least 
4 hours postdose, the remaining 6 subjects in Cohort 1 will be dosed. For all subsequent cohorts, 
a Dose Escalation Committee will conduct blinded reviews of all the safety/tolerability 
parameters through Discharge (Day 2) from the prior dosing cohort in order to make decisions 
regarding dose escalation. The Dose Escalation Committee will include sponsor and contract 
research organization (CRO) representatives. 


Protocol 
C3300-101 
15 
1.1.6. 
Disclosure Statement 
This is a single-ascending-dose safety and tolerability study with up to 6 treatments (placebo and 
up to 5 dose levels of IW 3300) that is subject- and investigator-blinded. 
1.1.7. 
Number of Subjects 
A maximum of 40 subjects will be randomized in the study (up to 5 cohorts of 8 subjects each); 
within each cohort, 6 subjects will be randomized to 
and 2 subjects will be randomized 
to placebo. 
1.1.8. 
Intervention Groups and Duration 
The study will evaluate single ascending doses of 
in a double-blind manner. The 
8 subjects within each cohort will be randomized to receive a single dose of 
(6 subjects) or placebo (2 subjects), administered rectally (as a low-volume [20 mL] enema) 
following a fast of at least 6 hours (refer to the Schedule of Activities [SoA] for additional details 
regarding dosing instructions). The planned cohorts are: 
 Cohort 1: 100 g 
or matching placebo single rectal dose 
 Cohort 2: 300 g 
or matching placebo single rectal dose 
 Cohort 3: 900 g 
or matching placebo single rectal dose 
 Cohort 4: 2500 g 
or matching placebo single rectal dose 
 Cohort 5 
0 g 
or matching placebo single rectal dose 
(specific dose level to be determined after safety reviews of previous cohorts; doses 
higher than 2500 g will not be tested) 
In Cohort 1, sentinel dosing will be implemented. The first 2 subjects will be randomized in a 
1:1 ratio to 
or placebo and dosed at least 4 hours before the remaining subjects in the 
cohort. If no safety signals are noted following an observation period of at least 4 hours postdose, 
the remaining 6 subjects in Cohort 1 will be dosed. Dosing in Cohorts 2 through 5 will only 
proceed following a review of the prior dosed cohort(s), including adverse events (AEs), clinical 
laboratory test results, vital signs, and 12-lead ECGs. The determination of dose escalation will 
be made at a meeting of the Dose Escalation Committee. 
Treatment duration will be 1 day; subjects will be followed in the Phase 1 clinical research unit 
(CRU) for approximately 24 hours after dosing and contacted by phone for follow-up 
approximately 2 weeks after dosing. Total subject participation will be 22 to 45 days, including 
the Screening, Clinic, and Follow-up Periods. 
1.1.9. 
Data Monitoring Committee: No 
The study will utilize a Dose Escalation Committee to review safety data for each cohort in order 
to make decisions about dose escalation. 


Protocol 
C3300-101 
16 
1.2.
Schema
Figure 1: 
Schematic of Study Design 
 
Dose
Escalation
Screening
Visit
(Day -28
to Day -8)
Check-in
(Day -1)
Discharge
(Day 2)
Follow-up
Phone Call
(Day 14 +3)
Dosing
(Day 1)
7 to 27 days
3 days
12 days
Screening Period
Clinic Period
Follow-up Period
Dose Escalation
Review


Protocol 
 
C3300-101 
 
17 
 
1.3. 
Schedule of Activities 
Table 1: 
Schedule of Activities 
 
Screening 
Period 
Clinic Period 
Follow-up 
Period 
Study 
 
Visit/Day 
 
Screening Visit 
Day 
to 
Day 8 
Check-in 
Day 1 
Dosing 
Day 1 
Discharge 
Day 2a
Follow-up 
Phone Call 
Day 14 
(+3 days)
Informed consent
X 
Eligibility criteria
X 
X 
Medical history 
X 
 
 
 
 
Demographics 
X 
 
 
 
 
Body weight & heightb
X 
X 
 
X 
 
Urine drug & alcohol screen
X 
X 
Urine cotinine test 
X 
 
 
 
 
Physical examinationc
X 
X 
 
X 
 
Vital signsd
X 
 
Pre:  0 
25m) 
Post: 0.5, 1, 2, 4, 6, and 
8h (±10m) 
24h post-Day 1 
dose (±25m) 
 
Triplicate 12-lead ECGe 
X 
X 
Pre 1h (±30m) 
Post: 2h (±30m) 
Xf 
 
Clinical chemistry, hematology, urinalysis
X 
X 
Pre: 0 
 
Post: 6h (±30m) 
24h post-Day 1 
dose (±15m) 
ESR and CRP
X 
24h post-Day 1 
dose (±15m) 


Protocol 
 
C3300-101 
 
18 
 
 
Screening 
Period 
Clinic Period 
Follow-up 
Period 
Study 
 
Visit/Day 
 
Screening Visit 
Day 
to 
Day 8 
Check-in 
Day 1 
Dosing 
Day 1 
Discharge 
Day 2a 
Follow-up 
Phone Call 
Day 14 
(+3 days) 
testing
X 
X 
HIV & hepatitis panel 
X 
 
 
 
 
BBMD training 
X 
 
 
 
 
BBMD dispensed
X 
X 
 
 
 
Daily BBMD completiong 
Day 8 through 
Day 2 
X 
X 
X 
 
BBMD collection and reviewh
 
X 
 
X 
 
Concomitant medications
X 
X 
X 
X 
X 
AE monitoring 
X 
X 
X 
X 
X 
Subject confinement to clinici 
 
X 
X 
X 
 
Start of predose fastj
 
 
Pre: 
 
 
 
Randomization
X 
Study drug administrationk 
 
 
X 
 
 
PK blood drawsl
 
 
Pre: 0 
 
Post: 0.5 (±2m), 1h 
(±10m), 2, 4, 6, and 8h 
(±5m) 
24h post-Day 1 
dose (±15m) 
 
Stool collectionm 
 
 
Post: 0h to Discharge 
 
Hemoccult testing
X 
Post: 0h to Discharge


Protocol 
 
C3300-101 
 
19 
 
 
Screening 
Period 
Clinic Period 
Follow-up 
Period 
Study 
 
Visit/Day 
 
Screening Visit 
Day 
to 
Day 8 
Check-in 
Day 1 
Dosing 
Day 1 
Discharge 
Day 2a 
Follow-up 
Phone Call 
Day 14 
(+3 days) 
Follow-up phone calln 
 
 
 
 
X 
AE=adverse event; BBMD=bladder and bowel movement diary; CRP=C-reactive protein; CRU=clinical research unit; ECG=electrocardiogram; 
ESR=erythrocyte sedimentation rate; HIV=human immunodeficiency virus; PK=pharmacokinetic; Post=postdose; Pre=predose; SARS CoV 2=severe acute 
respiratory syndrome coronavirus 2 
a Subjects will be discharged on Day 2 unless they have physical examination findings or laboratory abnormalities that are considered by the investigator to be 
clinically meaningful. In the event that a subject discontinues from the study, an early termination visit will be performed prior to discharge from the CRU. 
The early termination assessments will be the same as those conducted at Discharge (Day 2). Every effort should be made and documented to ensure that safety 
procedures scheduled for Discharge are performed at the early termination visit. 
b Height will only be measured at the Screening Visit. At Check-in (Day 
 2), subjects will be weighed in the morning upon awakening 
and before they ingest any water or food. 
c Rectal examination should be performed at each physical examination; the rectal examination at all timepoints should include a digital rectal examination and 
inspection of the perianal area for redness or irritation. 
d Vital signs will include oral temperature, blood pressure, and pulse rate. Side-lying blood pressure and pulse will be obtained at the 0.5 hour postdose timepoint 
on Day 1 (consistent with the position required following dosing). Blood pressure and pulse at the 2, 4, and 8 hour postdose timepoints should be preceded by 
at least 5 minutes of rest for the subject in a quiet setting without distractions (eg, television, cell phones). At all other timepoints (ie, predose and 1, 6, and 
24 hours postdose), orthostatic blood pressure and pul
 minutes before supine/semisupine blood 
pressure and pulse measurements are taken, then assume standing position for 3 minutes before standing blood pressure and pulse measurements are taken. If a 
subject has orthostatic symptoms upon standing (eg, palpitation, dizziness), they will be assisted and asked to lie down without waiting the 3 minutes for vital 
sign assessment. Orthostatic measurements may be taken at other vital signs collection timepoints if clinically indicated, at the discretion of the investigator. 
When applicable, vital sign measurements will be obtained immediately before blood draws. 
e ECGs should be obtained after the subject has been supine for at least 5 minutes. At each timepoint required, 3 individual ECG tracings should be obtained as 
closely as possible in succession, but no more than 2 minutes apart. The full set of triplicates should be completed in less than 4 minutes. 
f Procedure will be done in the morning shortly after the subject awakens but before the subject eats breakfast. 
g The BM portion of the BBMD will be completed by the subject on an event-driven basis. For subjects with Screening Visits earlier than Day
call subjects on Day 
to remind them to begin recording their BMs in the BBMD the following day, answer any questions, and provide replacement BBMD 
pages via email, if needed. Bladder and urination information in the BBMD will be collected on Day 1 predose and Day 2, with a 24-hour recall period at each 
timepoint. 
h CRU staff will review the subject’s Screening Period BBMD at Check-in to confirm that subjects have at least 3 bowel movements during the 7 days prior to 
Check-in (Days 
 
 bowel movements per day to be eligible for the study; CRU staff will collect the subject’s Clinic Period 
BBMD at Discharge. 
i Subjects will remain at the CRU from Check-in (Day
2).


Protocol 
 
C3300-101 
 
20 
 
j Subjects will fast for at least 6 hours prior to dosing on Day 1 and for at least 1 hour following dosing. 
k Study drug (
or placebo low-volume enema) will be administered rectally after a fast of at least 6 hours. Subjects will be encouraged to empty their 
bowels in the morning prior to dosing, if possible. Subjects will be instructed to lie on their left side with their left leg extended and their right leg slightly bent. 
CRU staff will remove the cap from the applicator tip and gently insert the tip into the subject’s rectum, then slowly squeeze the bottle to empty the contents 
into the rectum. After using the enema, subjects will lie on their left side for at least 30 minutes to allow the liquid to distribute throughout their intestines, 
followed by at least 30 additional minutes in the semisupine position. CRU staff will monitor subjects for leakage of the study drug from the rectum during the 
initial 30 minutes post administration. Subjects should avoid using the bathroom and hold in the enema for at least 1 hour. 
l PK blood draws at 0.5 and 1 hour postdose should be collected while the subject maintains the position noted in the dosing instructions above. 
m Beginning immediately after dosing on Day 1, all stool passed up to the time of Discharge on Day 2 will be collected and the wet weight will be measured, to 
calculate 
concentrations in stool. 
n CRU staff will contact subjects by phone for safety follow-up (at the discretion of the investigator, subjects may be requested to return to the CRU for their 
follow-up contact). 


Protocol 
C3300-101 
21 
2.
INTRODUCTION
is being developed for the treatment of bladder pain associated with IC/BPS. 
2.1.
Study Rationale
This clinical study is the first-in-human, single-ascending-dose, safety, and tolerability study 
with IW 3300. 
will be administered rectally as a low-volume (20 mL) enema; this route 
of administration was selected to target effects to the colon. Dosing for rectal administration will 
be initiated at 100 g and will not go higher than 2500 g, which is approximately 1/10 of the 
HED of the lowest NOAEL (24,000 g). 
2.2. 
Background 
3300 is a novel, 13-amino-acid, GC-C agonist peptide. GC-C, the target of IW 3300, is 
predominantly expressed on the luminal surface of the small and large intestines. When GC-C 
receptors are stimulated, intracellular cGMP is secreted across the basolateral membrane of 
colonic epithelial cells in the submucosa by multidrug resistance proteins MRP4 and MRP5, 
decreasing the activity of afferent nerve fibers located in the colonic wall, resulting in reduced 
visceral pain This ultimately produces an analgesic effect in other organs of the abdominopelvic 
region via action mediated through the common afferent pathways.(1, 2) 
Experiments conducted in animals indicate that colonic hypersensitivity induces persistent 
hypersensitivity of bladder afferent pathways in the absence of bladder pathology.(2) 
Hypersensitivity of bladder afferent pathways resembles the symptoms observed in patients with 
IC/BPS. Afferent neurons are known to have peripheral endings in both the colon and the 
bladder, and the axons of colonic and bladder neurons travel through the same splanchnic and 
pelvic nerves. These sensory afferents have cell bodies located within the thoracolumbar (TL) 
and lumbosacral (LS) dorsal root ganglia (DRG) and central projections in the dorsal horn of the 
corresponding regions of the spinal cord.(8) In animal models, daily administration of GC-C 
agonist linaclotide reversed persistent afferent bladder hypersensitivity. GC-C is restricted to the 
gastrointestinal (GI) tract and is not expressed in the bladder. These nonclinical observations 
support the concept of cross-organ sensitization and suggest that GC-C agonists such as 
may be useful clinically for the treatment of bladder pain associated with IC/BPS. 
has shown beneficial effects in reducing visceral pain in several animal models, 
including models of colonic pain and models of extra-intestinal chronic pelvic pain following 
intracolonic or intrarectal administration. 
(3 g/kg/day), administered either 
intracolonically or orally to female rats, reduced endometriosis-induced mechanical hind paw 
allodynia. When administered intracolonically (1, 3, 10 g/kg/day), 
reduced 
endometriosis-induced vulvar pain in a dose-dependent manner. In a rat model of chronic 
radiation proctopathy induced either by fractionated dose (total dose 48 Gray) or single high dose 
(25 Gray) of radiation, 
(3 g/kg/day) administered intracolonically reversed colorectal 
and bladder hypersensitivity. 
(3 g/kg/day) administered intrarectally reduced vaginal 
hypersensitivity in adult female mice previously exposed to neonatal vaginal irritation. In a rat 
model of early life stress, 
(3 g/kg/day) administered intracolonically reduced bladder 
and mechanical hind paw allodynia in adult male rats. 




Protocol 
C3300-101 
23 
2.3.
Benefit/Risk Assessment
While there were no clinical data available for 
at the outset of this first-in-human study, 
available nonclinical data with 
support a favorable risk/benefit profile. When 
administered orally, GC-C agonists may cause diarrhea and dehydration. This should be 
minimized in this study as 
is being administered intrarectally as a low-volume enema. 
More detailed information about the known and expected benefits and potential risks of 
may be found in the IB. 
2.3.1. 
Risk Assessment 
Potential Risk of 
Clinical Significance 
Summary of 
Data/Rationale for Risk 
Mitigation Strategy 
Study Intervention: IW 3300
Potential for diarrhea 
and secondary 
dehydration 
These are class effects of 
GC C agonism or effects 
observed in nonclinical 
safety pharmacology and 
toxicology studies of 
IW 3300. 
Intrarectal administration will likely decrease the 
risk of diarrhea and subsequent dehydration. The 
study design includes dose escalation to closely 
monitor subject safety and tolerability at each 
dose level before dosing with a higher dose; a 
Dose Escalation Committee will conduct blinded 
reviews of all the safety parameters of the dosing 
cohort through Discharge (Day 2) in order to 
make decisions regarding escalation to the next 
dose. Orthostatic vital sign measurements will be 
taken to evaluate the potential for volume 
depletion. The daily BM frequency and stool 
consistency will be monitored in real time to 
evaluate changes in bowel habits and the potential 
risk of diarrhea. Stopping rules will also be in 
place to evaluate continued dosing 
(Section 7.1.1); dosing within a cohort may be 
temporarily suspended in the event of a diarrhea 
or dehydration serious adverse event (SAE); 
1 subject with Grade 3 diarrhea (increase from 
baseline of >7 stools per day and/or watery stools 
[BSFS score of 7] resulting in a limiting of self-
care activities) or dehydration (hospitalization 
indicated); 
2 subjects with Grade 2 diarrhea 
(increase from baseline of 4 to 6  stools per day 
and/or watery stools [BSFS score of 7] resulting 
in a limiting of instrumental activities) or 
dehydration (intravenous fluids indicated). 


Protocol 
C3300-101 
24 
Potential Risk of 
Clinical Significance 
Summary of 
Data/Rationale for Risk 
Mitigation Strategy 
Unknown AEs, 
laboratory 
abnormalities, or 
other safety findings 
with a novel study 
drug 
This is a first-in-human 
study with 
and, 
as such, there are no 
clinical data available. 
The first cohort will feature sentinel dosing, 
whereby the first 2 subjects in Cohort 1 will be 
randomized in a 1:1 ratio to 
or placebo 
and dosed at least 4 hours before the remaining 
subjects in the cohort. Following from there, the 
study design includes dose escalation to closely 
monitor subject safety at each dose level before 
dosing with a higher dose; a Dose Escalation 
Committee will conduct blinded reviews of all the 
safety parameters of the dosing cohort through 
Discharge (Day 2) in order to make decisions 
regarding escalation to the next dose. Stopping 
rules are also in place to evaluate continued 
dosing within a cohort (Section 7.1.1); dosing 
within a cohort may be temporarily suspended in 
the event of an SAE considered related to study 
 subject with a Grade 3 (severe or 
medically significant) or higher AE considered 
related to study drug
 subjects with Grade 2 
(moderate) or higher AEs considered related to 
study drug. 
Study drug will be 
administered rectally. 
Rectally administered 
medications may 
potentially be associated 
with AEs such as local 
trauma/discomfort, 
irritation, or bleeding. 
The study excludes subjects for whom rectal 
administration would be anticipated to be a safety 
concern. The low-volume enema used in this 
study is expected to be a lower risk than higher 
volume enemas. Nonclinical toxicology studies of 
have shown no signs of local trauma 
following intrarectal administration. 
Study Procedures 
Venipuncture will be 
performed during the 
study. 
There is the risk of 
bleeding, bruising, 
hematoma formation, and 
infection at the 
venipuncture site. 
Only appropriately qualified personnel will 
perform the blood draw. Subjects will be 
monitored for these risks and managed 
appropriately. 
2.3.2. 
Benefit Assessment 
Benefits to individual subjects may include: 
 Medical evaluations/assessments associated with study procedures (eg, physical 
examinations, ECGs, laboratory evaluations, 
(COVID 19) diagnostic 
testing) 
 Contributing to the process of developing new therapies in an area of unmet need 


Protocol 
C3300-101 
25 
2.3.3. 
Overall Benefit: Risk Conclusion 
Taking into account the measures taken to minimize risk to subjects participating in this study, 
the potential risks identified in association with 
are justified by the anticipated benefits. 


Protocol 
C3300-101 
26 
3.
OBJECTIVES AND ENDPOINTS
3.1. 
Objectives 
3.1.1. 
Primary Objective 
The primary objective is to assess the safety and tolerability of single ascending doses of 
administered rectally via enema in healthy volunteers.
3.1.2. 
Exploratory Objectives 
The exploratory objectives are: 
 To summarize the single-dose PK of 
administered rectally via enema in 
healthy volunteers 
 To summarize the single-dose PD of 
administered rectally via enema in 
healthy volunteers based on bladder and bowel assessments 
3.2. 
Estimands 
Not applicable for this Phase 1 study. 
3.3. 
Endpoints 
3.3.1. 
Primary Endpoints (Safety)
The primary endpoints are: 
Incidence of TEAEs
 Incidence of TESAEs 
3.3.2. 
Exploratory Endpoints (Pharmacokinetics and Pharmacodynamics) 
The exploratory endpoints are: 
PK parameters
 Total amount of 
in stool (concentration and percent recovery) 
 Change from baseline in BM frequency during the Clinic Period 
 Change from baseline in stool consistency (BSFS score) during the Clinic Period 
Change from baseline in urinary frequency during the Clinic Period for a 24-hour 
duration 
 Change from baseline in urinary urgency during the Clinic Period for a 24-hour 
duration 
 Change from baseline in nocturia during the Clinic Period for a 24-hour duration 
 Change from baseline in pain or burning in bladder or pelvic area during the Clinic 
Period for a 24-hour duration 


Protocol 
C3300-101 
27 
4.
STUDY DESIGN
4.1. 
Overall Design 
This is a Phase 1, single-center, randomized, double-blind, placebo-controlled, single-ascending-
dose study assessing the safety, tolerability, and PK of 
administered rectally as a low-
volume enema in healthy adult volunteers. Each cohort will progress through 3 study periods: 
(1) Screening Period, (2) Clinic Period, and (3) Follow-up Period; these periods and the 
progression between cohorts are illustrated in the study schematic (Figure 1). 
This first-in-human study in healthy adult volunteers will assess all subjects for safety, PK, and 
effect on stool frequency/form and urination. 
The study will include up to 5 cohorts with 8 subjects per cohort; within each cohort, subjects 
will be randomized to receive a single dose of 
(6 subjects) or placebo (2 subjects), 
administered rectally (as detailed in Section 6.1.1). The planned cohorts are: 
 Cohort 1: 100 g 
or matching placebo single rectal dose 
 Cohort 2: 300 g 
or matching placebo single rectal dose 
 Cohort 3: 900 g 
or matching placebo single rectal dose 
 Cohort 4: 2500 g 
or matching placebo single rectal dose 
 Cohort 5 
0 g 
or matching placebo single rectal dose 
(specific dose level to be determined after safety reviews of previous cohorts; doses 
higher than 2500 g will not be tested) 
In Cohort 1 only, sentinel dosing will be implemented. If no safety signals are noted in the first 
2 subjects (randomized 1:1 to 
or placebo) following an observation period of at least 
4 hours postdose, the remaining 6 subjects in Cohort 1 will be dosed. For all subsequent cohorts, 
a Dose Escalation Committee will conduct blinded reviews of all the safety/tolerability 
parameters through Discharge (Day 2) from the prior dosing cohort in order to make decisions 
regarding dose escalation. The Dose Escalation Committee will include sponsor and CRO 
representatives. Dose escalation is detailed in Section 6.6. 
4.1.1. 
Study Periods 
4.1.1.1. 
Screening Period 
The Screening Period will begin with the signature of the informed consent form (ICF) at the 
Screening Visit (which can occur from Day 28 to Day 8) and will last 7 to 27 days. At the 
Screening Visit, subjects will undergo preliminary screening procedures to ensure that they meet 
the inclusion and exclusion criteria for the study. Eligible healthy volunteers will receive the 
bladder and bowel movement diary (BBMD), a paper-based diary to complete on an event-
driven basis (ie, for each BM) at home, and training on how to complete it to record their bladder 
and bowel habits. Subjects will complete the BBMD (BM portion only) daily only for the 7 days 
before Check-in on Day 1. The end of the Screening Period will coincide with the beginning of 
the Clinic Period at Check-in (Day 1). 


Protocol 
C3300-101 
28 
4.1.1.2. 
Clinic Period 
The Clinic Period will begin at Check-in to the Phase 1 CRU on Day 
(the day before dosing), 
and will end at the time of Discharge on Day 2 (the day after dosing). Following confirmation of 
eligibility at Check-in, subjects will be admitted to the CRU and will undergo baseline 
procedures. On the morning of Day 1 (note that there is no Day 0), subjects in each cohort will 
undergo predose assessments and will be randomized in a 3:1 ratio to receive IW 3300 or 
matching placebo after a fast of at least 6 hours. In Cohort 1 only, the first 2 subjects will be 
randomized in a 1:1 ratio to 
or placebo and dosed at least 4 hours before the remaining 
subjects in the cohort. 
Blood and stool samples for PK assessments will be collected at specified times predose (blood) 
and postdose (blood and stool) during the Clinic Period (see SoA in Table 1). Throughout the 
Clinic Period, safety assessments will be performed and subjects will continue to enter their BM 
information into the BBMD on an event-driven basis. Bladder and urination information in the 
BBMD will be collected on Day 1 predose and Day 2, with a 24 hour recall period at each 
timepoint. Subjects will be confined to the CRU from Check-in (Day 1) through 1 day of dosing 
until Discharge (Day 2), which will occur after completion of the assessments (at least 24 hours 
after administration of study drug) and at the investigator’s discretion. 
4.1.1.3. 
Follow-up Period 
The Follow-up Period will start immediately after subjects are discharged from the Phase 1 CRU 
on Day 2 and will last for 12 (+3) days. At the conclusion of the Follow-up Period (Day 14+3), 
the Phase 1 CRU will contact subjects for the Follow-up Phone Call (at the discretion of the 
investigator, subjects may be requested to return to the Phase 1 CRU for their follow-up contact). 
The Follow-up Phone Call will be considered the end of the study for that subject. 
4.2. 
Scientific Rationale for Study Design 
A randomized, double-blind, placebo-controlled study design was chosen to investigate the 
safety, PK, and PD of IW 3300, and to determine the tolerability of a range of doses of 
administered rectally as a low-volume enema. Subjects will be randomized within each cohort to 
ensure that the treatment groups are comparable and to minimize the potential for selection bias. 
The study will be double-blind to ensure that the subjects and CRU staff are unaware of the 
treatment assignment and to minimize the potential for bias in study assessments or AE 
reporting. Placebo was chosen as the control so that the rate of spontaneously occurring AEs can 
be determined and to reduce the potential for bias in the reporting of AEs. 
Subjects will complete the BM portion of the BBMD during the last 7 days of the Screening 
Period to establish a baseline without study drug. Subjects will be confined to the CRU for the 
duration of the Clinic Period, from Check-in (Day 1) through 1 day of dosing until Discharge 
(Day 2), which will occur after completion of the assessments (at least 24 hours after 
administration of study drug) and at the investigator’s discretion. CRU staff will contact subjects 
for the Follow-up Phone Call 12 (+3) days after Discharge for safety follow-up; at the discretion 
of the investigator, subjects may be requested to return to the CRU for their follow-up contact. 
Because this is the first-in-human study with IW 3300, Cohort 1 will feature sentinel dosing 
whereby the first 2 subjects will be randomized in a 1:1 ratio to receive 
or placebo, and 




Protocol 
C3300-101 
30 
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 of the following criteria apply: 
5.1.1. 
Age 
1. Subject must be 18 to 60 years of age, inclusive, at the time of signing the ICF. 
5.1.2. 
Type of Subject and Disease Characteristics 
2. Subject is medically healthy with no clinically significant findings during medical 
evaluation including physical examination, 12-lead ECG, and clinical laboratory tests 
(serum chemistry, hematology, urinalysis, and urine drug and alcohol screen). (NOTE: In 
making this determination, the investigator or designee will consider whether any finding 
could prevent the subject from performing any of the protocol-specified assessments, 
could represent a condition that would exclude the subject from the study, could represent 
a safety concern if the subject participates in the study, or could confound the study-
specified assessments). 
3. 
3 
3 per day; average 
BSFS score of >2 and <6, as reported in the BBMD during the 7 days before Check-in 
[Day 1]). 
4. Subject agrees to refrain from making any new or major lifestyle changes (eg, new diet or 
exercise regimen) from the Screening Visit until after the Follow-up Phone Call on 
Day 14. 
5. If subject is 
5 years of age, subject is compliant with colorectal cancer screening 
guidelines according to the ACG Clinical Guidelines: Colorectal Cancer Screening 
2021. (9) 
5.1.3. 
Weight 
6. Body mass index (BMI) within the range 18.5 to 35.0 kg/m2 (inclusive) at the Screening 
Visit. 
5.1.4. 
Sex 
7. Female subjects must be of nonchildbearing potential (ie, women who are 
postmenopausal [greater than 12 months without menses] or who have had a bilateral 
oophorectomy, bilateral salpingectomy, or hysterectomy). 
8. Male subjects and female partners are willing to use double-barrier method of 
contraception during the study (eg, diaphragm with spermicide plus a condom, condom 
with spermicide plus a diaphragm or cervical cap), from the Screening Visit until after 
the Follow-up Phone Call on Day 14. 


Protocol 
C3300-101 
31 
5.1.5. 
Informed Consent 
9. Capable of giving signed informed consent as described in Appendix 1/Section 10.1 
which includes compliance with the requirements and restrictions listed in the ICF and in 
this protocol. 
5.2. 
Exclusion Criteria 
Subjects are excluded from the study if any of the following criteria apply:
5.2.1. 
Medical Conditions 
1. Subject has evidence or history of clinically significant acute or chronic disease, or 
clinically significant illness within 30 days of the Screening Visit. 
2. Subject has a history of clinically significant hypersensitivity or allergies to any of the 
inactive ingredients contained in the active or placebo drug products. 
3. Subject has a history of any condition that would interfere with their ability to receive an 
enema.
4. Subject has a recent history of anal fissure, anal abscess, complicated hemorrhoids, or 
presence or history of inflammatory bowel disease. 
5.2.2. 
Prior/Concomitant Therapy 
5. Subject has used a prescription medication during the 14 days before Check-in (Day 1). 
Use of prescription medication, unless necessary to treat a medical emergency, is 
excluded during the study until after the Follow-up Phone Call on Day 14. 
6. Subject has used any over-the-counter medications, including laxatives, during the 7 days 
before Check-in (Day 1). Use of over-the-counter medication, including laxatives, is not 
permitted during the study until after Discharge on Day 2. (Note: Acetaminophen may be 
used at the investigator’s discretion, in consultation with the sponsor.) 
7. Subject has used herbal supplements from 7 days before Check-in (Day 1). Use of these 
supplements is not permitted during the study until after Discharge on Day 2. 
8. Subject has received a licensed or investigational vaccine during the 30 days before 
Check-in (Day 1) or is planning to receive any vaccine during the study (up to the 
Follow-up Phone Call on Day 14). 
9. Subject has received blood products during the 2 months before Check-in (Day 1). 
10. Subject has undergone a surgical procedure during the 30 days before Check-in (Day 1), 
other than minor dermatologic procedures, or has a history of surgery involving the GI 
tract or anal canal (with the exception of endoscopic procedures, appendectomy, and 
cholecystectomy). 
5.2.3.
Prior/Concurrent Clinical Study Experience
11. Subject has received any investigational drug during the 30 days or 5 half lives of that 
investigational drug (whichever is longer) before the Screening Visit, or is planning to 
receive another investigational drug at any time during the study. 


Protocol 
C3300-101 
32 
5.2.4. 
Diagnostic Assessments 
12. Subject has a 12-lead ECG demonstrating severe bradycardia (heart rate <40 beats per 
0 
0 msec for females at the 
Screening Visit or at Check-in on Day 1. 
13. Subject has elevated (>1.5 times the upper limit of normal as defined by the laboratory) 
levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine, 
or bilirubin at the Screening Visit or at Check-in on Day 1. 
14. Subject has confirmed or suspected infection with 
(COVID 19) at the 
Screening Visit or Check-in on Day 1, or was diagnosed with 
or had contact 
with a 
patient within 14 days of the Screening Visit. 
15. Subject has positive serology for human immunodeficiency virus (HIV) 1, HIV 2, or 
hepatitis B surface antigen (HBsAg), or positive for anti-HIV 1, anti-HIV 2, or 
anti hepatitis C virus (HCV) antibodies at the Screening Visit. 
16. Identification of an anatomical abnormality upon Screening rectal examination which 
will make enema administration difficult. 
5.2.5. 
Other Exclusions 
17. Subject has a history of difficulty receiving an enema. 
18. Subject has a history of alcohol or drug addiction during the year before the Screening 
Visit, or has a positive drug or alcohol screen at the Screening Visit or Check-in on 
Day 1. 
19. Subject has ingested any alcohol-containing foods or beverages during the 48 hours 
before Check-in (Day 1). Ingestion of alcohol-containing foods or beverages is excluded 
during the study until after the Follow-up Phone Call on Day 14. 
20. Subject has donated blood products during the 6 weeks before Check-in (Day 1). 
21. Subject is involved in the conduct and administration of this study as an investigator, 
subinvestigator, study coordinator, other study staff, or sponsor staff. 
5.3. 
Lifestyle Considerations 
5.3.1. 
Meals and Dietary Restrictions 
1. Subjects agree to refrain from starting a new diet during the study as described in 
Section 5.1. 
2. Subjects will fast for at least 6 hours prior to dosing on Day 1 and for at least 1 hour 
following dosing. 
5.3.2. 
Caffeine, Alcohol, and Tobacco 
1. Subjects will abstain from ingesting any alcohol-containing foods or beverages from 
48 hours before Check-in (Day 1) until after the Follow-up Phone Call as described in 
Section 5.2. 


Protocol 
C3300-101 
33 
2. Subjects are permitted to ingest caffeine- or xanthine-containing products (eg, coffee, tea, 
cola drinks, and chocolate), but should limit 
3 cups per day. 
3. Subjects who use tobacco products will be instructed that use of nicotine-containing 
products (including nicotine patches) will not be permitted while they are in the CRU. 
5.3.3. 
Activity 
1. Subjects agree to refrain from making any new or major lifestyle changes (eg, new diet or 
exercise regimen) as described in Section 5.1. 
2. Subjects must follow contraception requirements outlined in Section 5.1. 
3. Prior to dosing, subjects will be encouraged to empty their bowels, if possible. 
4. After dosing, subjects will lie on their left side for at least 30 minutes (including for vital 
signs collection), followed by at least 30 additional minutes in the semisupine position as 
outlined in Section 6.1.1. Subjects should avoid using the bathroom and hold in the 
enema for at least 1 hour. 
5. For vital signs collection at 2, 4, and 8 hours postdose, subjects should rest for at least 
5 minutes prior to pulse and blood pressure measurement, in a quiet setting without 
distractions (eg, television, cell phones). 
6. For vital signs collection predose and 1, 6, and 24 hours postdose, subjects must lie 
quietly for 5 minutes before supine/semisupine blood pressure and pulse measurements 
are taken, then assume a standing position for 3 minutes before standing blood pressure 
and pulse measurements are taken, as described in Section 8.2.2. If a subject has 
orthostatic symptoms upon standing (eg, palpitation, dizziness), they will be assisted and 
asked to lie down without waiting the 3 minutes for vital sign assessment. At the 
discretion of the investigator, orthostatic measurements may be taken at other vital signs 
collection timepoints if clinically indicated. 
5.4. 
Screen Failures 
Screen failures are defined as subjects who consent to participate in the clinical study but are not 
subsequently randomized. 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 SAE. 
Individuals who do not meet the criteria for participation in this study (screen failure) may be 
rescreened following consultation with the sponsor. Rescreened subjects should be assigned a 
separate subject number for each screening; once a subject number has been assigned it must not 
be reused. 




Protocol 
C3300-101 
35 
Packaging and 
Labeling 
Study Intervention will be provided in 
filled syringes. Each syringe will be 
labeled as required per country 
requirement. 
Study Intervention will be provided in 
filled syringes. Each syringe will be 
labeled as required per country 
requirement. 
IMP=investigational medical product; NIMP=non-investigational medical product; TBD=to be determined 
6.1.1. 
Dosing Instructions 
Subjects will receive a single dose of study drug (
or placebo low-volume enema) in the 
CRU on Day 1. Study drug will be administered rectally after a fast of at least 6 hours. Subjects 
will be encouraged to empty their bowels in the morning prior to dosing, if possible. Subjects 
will be instructed to lie on their left side with their left leg extended and their right leg slightly 
bent. CRU staff will remove the cap from the applicator tip and gently insert the tip into the 
subject’s rectum, then slowly squeeze the bottle to empty the contents into the rectum. After 
using the enema, subjects will lie on their left side for at least 30 minutes to allow the liquid to 
distribute throughout their intestines, followed by at least 30 additional minutes in the 
semisupine position. CRU staff will monitor subjects for leakage of the study drug from the 
rectum during the initial 30 minutes post administration. Subjects should avoid using the 
bathroom and hold in the enema for as long as possible (at least 1 hour). 
CRU staff will record the date and time of study drug administration in the electronic case report 
form (eCRF). CRU staff will also record (as yes/no) if there was study drug leakage from the 
rectum in the 30 minutes following drug administration. 
6.2. 
Preparation/Handling/Storage/Accountability 
All study drug substance will be stored in the locked, limited access, temperature-controlled 
pharmacy accessible only to pharmacy staff. 
drug substance will be protected from 
moisture and light and stored between 25°C to 15°C. Prior to compounding, 
drug 
substance will be thawed for a minimum of 30 minutes prior to use. 
The investigational drug product will be compounded on the day of administration and stored at 
room temperature prior to dosing (continuous mixing is not required). Compounding must occur 
in accordance with the instructions provided in the Pharmacy Manual. Specifically designated 
unblinded CRU pharmacy staff will be responsible for preparing dosing for each cohort and 
providing doses to the study coordinator to administer to subjects (see Section 6.1.1 for dosing 
instructions). 
Storage and accountability of study drug should adhere to the following: 
1. 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. 
2. 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 accordance with 
the labeled storage conditions. 
3. The investigator, institution, or the head of the medical institution (where applicable) is 
responsible for study drug accountability, reconciliation, and record maintenance (ie, 
receipt, reconciliation, and final disposition records). 


Protocol 
C3300-101 
36 
4. Further guidance and information for the final disposition of unused study drug are 
provided in the Pharmacy Manual. 
6.3. 
Measures to Minimize Bias: Randomization and Blinding 
6.3.1.
Randomization 
The subject identification (SID) number will be assigned in an ascending sequential order (eg, 
beginning with 001, 002, etc.) at the time the subject signs the ICF. The subject will retain the 
same unique SID number throughout the Clinic and Follow-up Periods. 
Subjects who meet all of the inclusion criteria and none of the exclusion criteria will be 
randomized into the study on Day 1. A total of up to 40 subjects (up to 5 cohorts of 8 subjects 
each) will be randomized in a 3:1 ratio to receive 
or placebo, for each cohort, as 
detailed in Section 6. Randomization numbers encoding the subjects’ treatment assignments will 
be based on a randomization schedule that is computer-generated prior to the study, by an 
independent statistician from the CRO who is not otherwise associated with the study. 
6.3.2. 
Blinding
The investigator and all other CRU staff, sponsor study personnel, and the subject will remain 
blinded to individual subject treatment assignments throughout the study, except as noted below. 
The Dose Escalation Committee, which will include sponsor and CRO representatives, will 
conduct blinded reviews of all the safety/tolerability parameters of the dosing cohort through 
Discharge (Day 2) in order to make decisions regarding dose escalation. 
Specific designated personnel in the Ironwood may be unblinded to the treatment assignment of 
individual subjects for regulatory reporting purposes. All other sponsor study personnel, except 
as described, will remain blinded until the study is complete and the database is locked, unless 
warranted by emerging safety or tolerability issues. 
The designated independent study statistician, who will not be involved in study data analysis 
and interpretation, will have access to the randomization schedule including treatment 
assignments. 
Specifically designated unblinded CRU pharmacy staff will be responsible for preparing dosing 
for each cohort and providing doses to the study coordinator to administer to subjects. The 
investigator (except as detailed here) and the remaining CRU staff will be blinded as to 
treatment. In the event of a Quality Assurance audit, the auditor(s) will be allowed access to 
unblinded study intervention records at the CRU to verify that randomization/dispensing has 
been done accurately. 
Site unblinding of a subject’s treatment assignment is restricted to emergency situations that 
necessitate identifying the study drug for the welfare of the subject. Subject safety must always 
be the first consideration in making such a determination. If the investigator decides that 
unblinding is warranted, the investigator, or person designated by the investigator, should contact 
the sponsor’s medical monitor directly to discuss the need for emergency unblinding. Individual 
sealed unblinding envelopes, which can be opened to identify the treatment assignment for an 
individual subject in an emergency, will be provided to and retained by the CRU pharmacist. The 
reason for breaking the blind will be recorded. 


Protocol 
C3300-101 
37 
6.4.
Study Intervention Compliance
Subjects will receive study drug directly from the investigator or designee, under medical 
supervision. The date and time of each dose administered in the clinic will be recorded in the 
source documents and recorded in the eCRF. The dose of study drug and study subject 
identification will be confirmed at the time of dosing by a member of the CRU staff other than 
the person administering the study intervention. 
6.5. 
Concomitant Therapy 
Subjects must abstain from taking prescription medications during the 14 days before Check-in 
(Day 1) until after completion of the Follow-up Phone Call on Day 14; over-the-counter 
medications (including laxatives, vitamins, and dietary or herbal supplements) during the 7 days 
before Check-in (Day 1) until after Discharge on Day 2; and vaccines during the 30 days before 
Check-in (Day 1) until after completion of the Follow-up Phone Call on Day 14. 
Acetaminophen at doses of 3 grams/day is permitted for use during the study at the 
investigator’s discretion, in consultation with the medical monitor. 
Any medication (including over-the-counter or prescription medicines, vitamins, and/or herbal 
supplements) or vaccine that the subject is receiving at the time of Screening or receives during 
the study must be recorded along with: 
 Reason for use 
 Dates of administration, including start and end dates 
 Dosage information, including dose and frequency 
The medical monitor should be contacted if there are any questions regarding concomitant or 
prior therapy. 
6.5.1. 
Rescue Medicine 
Not applicable to this study of healthy volunteers. 
6.6. 
Dose Modification 
The decision to proceed to the next dose level of 
(either an increase or a decrease) will 
be made by the Dose Escalation Committee, based on blinded safety and tolerability data 
obtained through Discharge on Day 2 in all subjects at the prior dose level. 
Dosing at a given dose level may be temporarily suspended for any of the reasons listed in 
Section 7.1.1. 
6.7. 
Intervention After the End of the Study 
There is no study intervention following the end of the study. 
Subjects with ongoing AEs or other safety concerns will be followed until resolution or until no 
further queries or follow-up actions are required; at the discretion of the investigator, subjects 
with ongoing safety concerns may have discharge delayed if necessary for continued monitoring. 


Protocol 
C3300-101 
38 
7.
DISCONTINUATION OF STUDY INTERVENTION AND 
SUBJECT DISCONTINUATION/WITHDRAWAL 
7.1. 
Discontinuation of Study Intervention 
It is not anticipated that it will be necessary for a subject to permanently discontinue (definitive 
discontinuation) study intervention during this single-dose study. In rare instances, a subject may 
withdraw from the study prior to completion of the Clinic Period; see the SoA (Table 1) for data 
to be collected at the time of discontinuation and follow-up and for any further evaluations that 
need to be completed. If a subject is discontinuing due to an AE, the AE must be followed until it 
is resolved, stable, or judged by the investigator to be not clinically significant. 
7.1.1. 
Study Suspension or Termination 
Dosing at a given dose level may be temporarily suspended if any of the following occur within 
a cohort: 
 
1 subject with an SAE considered related to study drug 
 
1 subject with a Common Terminology Criteria for Adverse Events (CTCAE) 
Grade 3 or higher AE considered related to study drug (CTCAE v5.0 will be used; 
grading criteria are summarized in Appendix 3/Section 10.3.3) 
 
2 subjects with CTCAE Grade 2 or higher AEs considered related to study drug 
The Dose Escalation Committee should consider the benefit-risk ratio in their determination to 
suspend dosing temporarily or permanently. If the benefit-risk is considered unacceptable and 
the effect is considered related to active study drug by the Dose Escalation Committee, dosing at 
a given dose level will be stopped, and no additional cohorts will be dosed at the same or higher 
dose level (a lower dose could still be studied). 
The sponsor may permanently terminate the study, or a component of the study, at any time. 
Reasons for termination may include, but are not limited to: 
Death, SAE, or other safety finding considered to be related to 
that may 
preclude further dosing. 
7.2. 
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. 
 At the time of discontinuation from the study, if possible, an early discontinuation 
visit should be conducted, as shown in the SoA (Table 1). 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. 
 The subject will be permanently discontinued both from the study intervention and 
from the study at that time. 


Protocol 
C3300-101 
39 
 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. 
7.3. 
Lost to Follow-up 
A subject will be considered lost to follow-up if he or she repeatedly fails to return for scheduled 
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, reschedule the missed visit as soon as 
possible, 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 and reason for discontinuation will be “lost to follow-up.” 
Discontinuation of the site or of the study as a whole are handled as part of 
Appendix 1/Section 10.1. 


Protocol 
C3300-101 
40 
8.
STUDY ASSESSMENTS AND PROCEDURES
 Study procedures and their timing are summarized in the SoA (Table 1). 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. 
8.1. 
Efficacy Assessments 
Efficacy will not be evaluated in this Phase 1 study in healthy volunteers. 
8.2. 
Safety Assessments 
Planned timepoints for all safety assessments are provided in the SoA (Table 1). 
8.2.1. 
Physical Examinations 
A complete physical examination will be performed as outlined in the SoA, by the investigator or 
a licensed health professional listed on Form FDA 1572. 
 A complete physical examination will include, at a minimum, assessments of the 
general appearance of the subject and the HEENT (head, eyes, ears, nose, and throat), 
cardiac, respiratory, GI, musculoskeletal, neurological, and dermatological systems. 
Rectal examination should be performed at each physical examination; the rectal 
examination at all timepoints should include a digital rectal examination and 
inspection of the perianal area for redness or irritation. 
 Height (only at Screening) and weight will also be measured and recorded. 
 Investigators should pay special attention to clinical signs related to previous serious 
illnesses, which will be reason for exclusion from the study. 
 Any physical examination abnormality that the investigator considers to be 
potentially clinically significant and changed from baseline will be reported as an AE. 


Protocol 
C3300-101 
41 
8.2.2. 
Vital Signs 
Vital signs will be performed as outlined in the SoA (Table 1). 
 Oral temperature, pulse rate, and blood pressure will be assessed. 
 Blood pressure and pulse measurements will be assessed with a completely 
automated device. Manual techniques will be used only if an automated device is not 
available. 
 Blood pressure and pulse at 0.5 hours postdose on Day 1 will be assessed in a 
side lying position (consistent with the position required following dosing). 
 Blood pressure and pulse at the 2, 4, and 8 hour postdose timepoints should be 
preceded by at least 5 minutes of rest for the subject in a quiet setting without 
distractions (eg, television, cell phones). 
 At all other timepoints, blood pressure and pulse measurements will be assessed in a 
supine and standing position (for calculation of orthostatic measurements). For 
orthostatic blood pressure and pulse measurements, the subject must lie quietly 
for 5 minutes before supine/semisupine blood pressure and pulse measurements are 
taken, then assume a standing position for 3 minutes before standing blood pressure 
and pulse measurements are taken. If a subject has orthostatic symptoms upon 
standing (eg, palpitation, dizziness), they will be assisted and asked to lie down 
without waiting the 3 minutes for vital sign assessment. At the discretion of the 
investigator, orthostatic measurements may be taken at other vital signs collection 
timepoints if clinically indicated.
 When applicable, vital signs measurements will be obtained immediately before 
blood draws. 
8.2.3. 
Electrocardiograms 
Triplicate 12-lead ECG will be obtained as outlined in the SoA (Table 1) using an 
ECG machine that automatically calculates the heart rate and measures PR, QRS, QT, 
and QTcF intervals. 
 ECGs should be obtained after the subject has been supine for at least 5 minutes. 
At each time point at which triplicate ECG are required, 3 individual ECG tracings 
should be obtained as closely as possible in succession, but no more than 2 minutes 
apart. The full set of triplicates should be completed in less than 4 minutes. 
8.2.4. 
Clinical Safety Laboratory Assessments 
 See Appendix 2/Section 10.2 for the list of clinical laboratory tests to be performed 
and the SoA (Table 1) for the timing and frequency. All protocol-required laboratory 
assessments must be conducted in accordance with the laboratory manual and the 
SoA. 
 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 


Protocol 
C3300-101 
42 
case report form (CRF). The laboratory reports must be filed with the source 
documents. 
 All laboratory tests with values considered clinically significantly abnormal during 
participation in the study or within 14 days after the 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. 
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 (eg, SAE 
or AE or dose modification), then the results must be recorded in the eCRF. 
8.3. 
Adverse Events and Serious Adverse Events 
The definitions of AEs and SAEs can be found in Appendix 3/Section 10.3. 
AEs will be reported by the subject (or, when appropriate, by a caregiver, surrogate, or the 
subject's legally authorized representative). 
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, considered related to the study intervention or study procedures, or that 
caused the subject to discontinue study drug (see Section 7). 
8.3.1. 
Time Period and Frequency for Collecting AE and SAE Information 
All AEs and SAEs will be collected from the signing of the ICF until completion of study 
participation (ie, early termination or Follow-up Phone Call) at the timepoints specified in the 
SoA (Table 1). 
Any medical condition that is present at the time that a randomized subject is screened and does 
not deteriorate (worsen in severity and/or frequency) should be recorded as Medical History and 
not as an AE. However, if the subject’s condition deteriorates at any time during the study, it will 
be recorded as an AE. AEs characterized as intermittent require documentation of onset and 
duration of each episode. Pretreatment AEs will be collected and captured in the subject’s source 
documentation from the time the subject signs the ICF until the subject receives study drug. 
Pretreatment AEs in randomized subjects will additionally be entered on the AE page of the 
subject’s eCRF. Laboratory abnormalities, changes in vital signs, and physical examination 
findings should be considered AEs and reported on the AE page of the subject's eCRF only if the 
investigator considers them clinically significant and/or they necessitate intervention. 
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 Appendix 3/Section 10.3. The 
investigator will submit any updated SAE data to the sponsor within 24 hours of it being 
available. 


Protocol 
C3300-101 
43 
Investigators are not obligated to actively seek AEs or SAEs after the conclusion of 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 intervention 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 AEs and SAEs, and the 
procedures for completing and transmitting SAE reports are provided in 
Appendix 3/Section 10.3. 
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. Subjects with ongoing AEs or other safety concerns will be followed 
until resolution or until no further queries or follow-up actions are required. 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 7.3). Further information on follow-up procedures is provided in 
Appendix 3/Section 10.3.3. 
8.3.4. 
Regulatory Reporting Requirements for SAEs 
 Prompt notification by the investigator to the sponsor of an SAE is essential so that 
legal obligations and ethical responsibilities towards the safety of subjects and the 
safety of a study intervention under clinical investigation are met. 
 The sponsor has a legal responsibility to notify both the local regulatory authority and 
other regulatory agencies about the safety of a study intervention under clinical 
investigation. The sponsor will comply with country-specific regulatory requirements 
relating to safety reporting to the regulatory authority, institutional review boards 
(IRBs)/independent ethics committees (IECs), and investigators. 
 For all studies except those utilizing medical devices, investigator safety reports must 
be prepared for suspected unexpected serious adverse reactions (SUSARs) according 
to local regulatory requirements and sponsor policy and forwarded to investigators as 
necessary. 
 An investigator who receives an investigator safety report describing an SAE or other 
specific safety information (eg, summary or listing of SAEs) from the sponsor will 
review and then file it along with the IB and will notify the IRB/IEC, if appropriate 
according to local requirements. 


Protocol 
C3300-101 
44 
8.3.5. 
Pregnancy 
This study is enrolling males and females of non-childbearing potential. 
 Details of all pregnancies in female subjects and female partners of male subjects will 
be collected after the start of study intervention and either until completion of the 
Follow-up Phone Call or until the details regarding the pregnancy outcome are 
reported. The CRU should make a reasonable effort to follow any pregnant patients 
until delivery or end of the pregnancy. 
 If a pregnancy is reported, the investigator should inform the sponsor within 24 hours 
of learning of the pregnancy and should follow the procedures outlined in 
Appendix 4/Section 10.4. 
 Abnormal pregnancy outcomes (eg, spontaneous abortion, fetal death, stillbirth, 
congenital anomalies, ectopic pregnancy) are considered SAEs. 
8.3.6. 
Cardiovascular and Death Events 
Cardiovascular and death events should be reported according to the definitions and reporting 
procedures for AEs and SAEs outlined in Appendix 3/Section 10.3. 
8.3.7. 
Disease-related Events and/or Disease-related Outcomes Not Qualifying as AEs 
or SAEs 
Not applicable to this study in healthy volunteers. 
8.3.8. 
Adverse Events of Special Interest 
Not applicable. 
8.4. 
Treatment of Overdose 
For this study, any dose of study drug greater than 1 dose of study drug within a 24-hour time 
period will be considered an overdose. 
The sponsor does not recommend specific treatment for an overdose. 
In the event of an overdose, the investigator should: 
1. Report any case of overdose to the sponsor and use their clinical judgment to treat the 
case of overdose as needed with the appropriate general supportive measures. Subjects 
will be monitored for potential development of diarrhea and dehydration and, if present, 
will be managed accordingly. 
2. Document the quantity of the excess dose as well as the duration of the overdose in the 
CRF. 
8.5. 
Pharmacokinetics 
Blood and feces for the determination of 
in plasma and stool, respectively, will be 
collected from all dosed subjects during the Clinic Period at timepoints specified in the SoA 
(Table 1). Instructions for the collection and handling of biological samples will be provided by 
the sponsor. The actual date and time (24-hour clock time) of each sample will be recorded. 


Protocol 
C3300-101 
45 
The following PK parameters will be calculated using a noncompartmental approach: 
 Area under the plasma concentration time curve from time zero to the time at which 
the last measurable concentration is observed (AUC0-t) 
Area under the plasma concentration time curve from time zero to time infinity 
(AUC0- ) 
Maximum plasma concentration (Cmax) 
Time to maximum plasma concentration (Tmax) 
 Terminal elimination half-life (t1/2) 
The wet weight of stool collected during the postdose collection period prior to Discharge will be 
measured and the total amount of 
in stool will be calculated, including concentration of 
in stool (ng/g), and percent recovery. 
Genetic analyses will not be performed on these samples. Subject confidentiality will be 
maintained. At visits during which blood samples for the determination of multiple aspects of 
will be taken, 1 sample of sufficient volume can be used. 
Drug concentration information that may unblind the study will not be reported to investigative 
sites or blinded personnel until the study has been unblinded. 
8.6. 
Pharmacodynamics 
Subjects will enter BM-related information into a paper diary, the BBMD, on an event-driven 
basis (ie, following each BM) in accordance with the instructions in the SoA (Table 1). 
The BM-related information will include the day and time of BMs and a report of stool 
consistency for each BM using the BSFS (1=Separate hard lumps like nuts [difficult to pass] to 
7=Watery, no solid pieces [entirely liquid]). 
Subjects will enter bladder information into the BBMD, which will include the following 
questions: 
In the past 24 hours… 
Did you have to urinate more frequently than normal? (yes/no) 
 How many times did you urinate? (0 to 2; 3 to 6; 7 to 10; or 10+ times) 
 Did you feel the strong need to urinate with little or no warning? (yes/no) 
 Did you have to get up to urinate during the night more frequently than usual? 
(yes/no) 
 How many times did you have to get up at night to urinate? (0, 1, 2, 3+ times) 
 Did you have pain or burning in your bladder or pelvic area? (yes/no) 
8.7. 
Genetics 
Genetics are not evaluated in this study; no samples will be collected for genetics research. 


Protocol 
C3300-101 
46 
8.8.
Biomarkers
Biomarkers are not evaluated in this study; no samples will be collected for biomarker research. 
8.9.
Immunogenicity Assessments
Immunogenicity is not evaluated in this study; no samples will be collected to evaluate 
antibodies to IW 3300. 
8.10. 
Medical Resource Utilization and Health Economics 
Medical resource utilization and health economics parameters are not evaluated in this study. 


Protocol 
C3300-101 
47 
9.
STATISTICAL CONSIDERATIONS
9.1. 
Statistical Hypotheses 
No hypothesis testing is planned. 
9.2.
Sample Size Determination
No statistical sample size determination process was performed. The sample size chosen for each 
cohort (8 subjects) is considered sufficient for evaluation of safety, tolerability, PK, and PD. 
9.3. 
Populations for Analyses 
The following populations are defined: 
Population
Description
Screened Analysis Set 
The Screened Analysis Set consists of all subjects who have signed 
informed consent. 
ITT Set 
The ITT Set consists of all subjects in the Screened Analysis Set who 
have been randomized to a treatment regimen. Analysis will be 
performed according to the allocated treatment regimen regardless of 
the treatment regimen actually received. 
Safety Analysis Set 
The Safety Analysis Set consists of all subjects who receive any 
amount of study drug. Analysis will be performed according to the 
treatment actually received regardless of the allocated treatment. 
PK Analysis Set 
The PK Analysis Set consists of all subjects in the Safety Analysis Set 
who have at least 1 postdose PK assessment. Analysis will be 
performed according to the treatment actually received regardless of 
the allocated treatment. 
PD Analysis Set 
The PD Analysis Set consists of all subjects in the Safety Analysis Set 
who have at least 1 postdose BBMD entry in the Clinic Period. 
Analysis will be performed according to the treatment actually 
received regardless of the allocated treatment. 
9.4. 
Statistical Analyses 
The statistical analysis plan (SAP) will be finalized prior to the clinical database lock and will 
include a more technical and detailed description of the statistical analyses described in this 
section. 
9.4.1. 
General Considerations 
For summaries by treatment group, data from subjects who receive placebo in different cohorts 
can be combined and presented as one treatment group (referred to as “treatment” and 
“placebo”), as appropriate. 
Continuous variables will be summarized using descriptive statistics (N, mean, SD, median, 
minimum, and maximum). Categorical variables will be summarized using the count and 


Protocol 
C3300-101 
48 
proportion of subjects in each category. Furthermore, PK data will be summarized using 
descriptive statistics, geometric mean, and coefficient of variation (
) of the geometric mean. 
9.4.2. 
Primary Endpoints Analyses 
The primary endpoint analysis will be based on the Safety Analysis Set. 
AE verbatim terms will be coded in the electronic data capture (EDC) system against the most 
current version of the Medical Dictionary for Regulatory Activities (MedDRA) available at the 
start of the study. 
TEAEs are defined in Section 10.3.1. The TEAEs and TESAEs for 1 calendar day postdose will 
be summarized by system organ class, preferred term, and treatment (or cohort). 
9.4.3. 
Secondary Endpoint Analysis 
Not applicable. 
9.4.4. 
Exploratory Endpoints Analyses 
PK analyses will be based on the PK Analysis Set. 
Plasma concentrations of 
will be summarized by treatment (or cohort) at each 
assessment timepoint; additionally, a listing of plasma concentrations will be provided. PK 
parameters (as described in Section 8.5) will also be calculated for each subject and summarized 
by treatment, if systemic levels of 
are measurable/as data permit ( 1 plasma 
concentration above the lower limit of quantitation [LLOQ] is necessary for calculation of Cmax 
and Tmax; 3 plasma concentrations above LLOQ are necessary for calculation of AUC0-t, 
AUC0 , and t1/2). 
The total amount of 
in stool collected during the postdose collection period prior to 
Discharge will be summarized by treatment, including the concentration (ng/g; based on the wet 
weight of stool) and the percent recovery. Subjects who do not have a BM during the stool 
collection period will not be included in the analysis of 
in stool. 
The analysis of the BBMD parameters will be based on the PD Analysis Set.
BBMD parameters (BM frequency, stool consistency, urinary frequency, urinary urgency, and 
nocturia) will be summarized by treatment (or cohort) for each study period (Screening Period 
and Clinic Period) using descriptive statistics and changes from baseline will be calculated
whenever it is appropriate. 
Baseline BM values will be derived from the BBMD data collected in the Screening Period, 
specifically, the period of time from 7 days prior to Check-in up to the time of randomization on 
Day 1. The baseline BM frequency rate (BMs per day) will be derived based on the number of 
BMs the subject reports during this period. The baseline stool consistency will be calculated as 
the average of the nonmissing BSFS scores associated with the BMs reported by the subject 
during this period. 
The subjects reported assessments for urination category parameters (urinary frequency, urinary 
urgency, and nocturia) in the 24-hour period prior to drug administration on Day 1 will be used 
to establish the baseline for each urinary parameter. The subjects reported assessment of pain or 


Protocol 
C3300-101 
49 
burning in the bladder or pelvic area in the 24-hour period prior to drug administration on Day 1 
will be used to establish the baseline for this parameter. 
The Clinic Period values will be derived from the BBMD data collected after randomization in 
the Clinic Period, specifically, the period of time from Day 1 (postrandomization) through Day 2 
(Discharge). The Clinic Period BM frequency rate will be derived based on the number of BMs 
the subject reports during this period. The Clinic Period stool consistency score is the mean of 
the nonmissing BSFS scores associated with the BMs reported by the subject during this period. 
The subjects reported assessments for urination category parameters (urinary frequency, urinary 
urgency, and nocturia) in the 24-hour period after dosing on Day 1 will be used to establish the 
postbaseline assessments for each parameter. Subjects reported assessment of pain or burning in 
the bladder or pelvic area in the 24-hour period after dosing on Day 1 will be used to establish 
the postbaseline assessments for this parameter. 
For the continuous variables, change from baseline will be defined as the Clinic Period value 
minus the baseline value. For the categorical variables, category shift from baseline to the Clinic 
Period will be assessed. 
9.4.5. 
Safety Analyses 
All safety analyses will be made on the Safety Analysis Set. 
Summaries for the safety parameters corresponding to the primary endpoints (including TEAEs 
and TESAEs) are described in Section 9.4.2 above. Additionally, listings will be provided for 
severe AEs, drug-related AEs, SAEs, and AEs leading to study discontinuation. 
Descriptive statistics and changes from baseline at each assessment timepoint will be presented 
by treatment (or cohort) for the Clinic Period for: 
 Clinical laboratory values (in standard units) for each hematology, chemistry, and 
urinalysis parameter 
 Vital signs (ie, pulse rate, systolic and diastolic blood pressure, oral temperature, and 
body weight) 
 ECG parameters 
 Additionally, the overall 12-lead ECG interpretation (normal, abnormal not 
clinically significant, or abnormal clinically significant) will be summarized. 
9.4.6. 
Other Analysis/Analyses 
Not applicable. 
9.5. 
Interim Analyses 
No interim analysis is planned for the study. Interim safety reviews are detailed in Section 6.6. 


Protocol 
C3300-101 
50 
9.6.
Data Monitoring Committee (DMC)
This study will not utilize a DMC. A Dose Escalation Committee will be utilized to review 
safety data for each cohort in order to make decisions about dose escalation, as described in 
Section 6.6. 


Protocol 
C3300-101 
51 
10.
SUPPORTING DOCUMENTATION AND OPERATIONAL 
CONSIDERATIONS 
10.1. 
Appendix 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 GCP Guidelines 
 Applicable laws and regulations 
The protocol, protocol amendments, ICF, IB, and other relevant documents (eg, advertisements) 
must be submitted to an IRB/IEC by the investigator and reviewed and approved by the IRB/IEC 
before the study is initiated. 
Any amendments to the protocol will require IRB/IEC 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/IEC annually or 
more frequently in accordance with the requirements, policies, and procedures 
established by the IRB/IEC 
 Notifying the IRB/IEC of SAEs or other significant safety findings as required by 
IRB/IEC procedures 
 Providing oversight of the conduct of the study at the site and adherence to 
requirements of 21 CFR, ICH guidelines, the IRB/IEC, European regulation 
536/2014 for clinical studies (if applicable), and all other applicable local regulations 
10.1.2. 
Financial Disclosure 
Investigators and subinvestigators will provide the sponsor with sufficient, accurate financial 
information as requested to allow the sponsor to submit complete and accurate financial 
certification or disclosure statements to the appropriate regulatory authorities. Investigators are 
responsible for providing information on financial interests during the course of the study and for 
1 year after completion of the study.
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. 


Protocol 
C3300-101 
52 
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 Part 50, local regulations, ICH guidelines, Health Insurance Portability 
and Accountability Act (HIPAA) requirements, where applicable, and the IRB/IEC 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 reconsented 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. 
Subjects who are rescreened are required to sign a new ICF. 
10.1.4. 
Data Protection 
All parties will comply with all applicable laws, including laws regarding the implementation of 
organizational and technical measures to ensure protection of participant data. 
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 laws. 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 ICF. 
The subject must be informed that his/her medical records may be examined by clinical quality 
assurance auditors or other authorized personnel appointed by the sponsor, by appropriate 
IRB/IEC members, and by inspectors from regulatory authorities. 
10.1.5. 
Committee Structure 
Key study personnel, including sponsor and CRO representatives, and their contact details will 
be provided prior to the Site Initiation Visit. 
The safety of this study will be closely monitored on an ongoing basis by sponsor 
representatives. A Dose Escalation Committee composed of sponsor and CRO representatives 
will make decisions about dose escalation, as detailed in Section 6.6. 
10.1.6. 
Dissemination of Clinical Study Data 
The sponsor will make study results available through www.ClinicalTrials.gov, or other public 
registries, in accordance with applicable local laws and regulations. 


Protocol 
C3300-101 
53 
10.1.7. 
Data Quality Assurance 
All subject data relating to the study will be recorded on the eCRF unless transmitted to the 
sponsor or designee electronically (eg, laboratory data). The investigator is responsible for 
verifying that data entries are accurate and correct by electronically signing the eCRF. 
The investigator must maintain accurate documentation (source data) that supports the 
information entered in the eCRF. 
The investigator must permit study-related monitoring, audits, IRB/IEC review, and regulatory 
agency inspections and provide direct access to source data documents. 
Monitoring details describing strategy (eg, 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 on-site 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 (eg, CROs). 
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 the longer of: 
 2 years after the last marketing authorization for the study drug has been approved or 
the sponsor has discontinued its research with respect to the study drug, or 
 Such longer period as required by applicable US regulatory requirements or by US 
law 
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. 
Definition of what constitutes source data can be found in the Monitoring Plan. 
Description of the use of the EDC system is documented in the Data Management Plan. 


Protocol 
C3300-101 
54 
10.1.9. 
Study and Site Start and Closure 
The first act of recruitment is the date of first subject consent and 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. The study site 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/IEC or local health authorities, the sponsor's procedures, or GCP guidelines 
 Inadequate recruitment of subjects by the investigator 
 Discontinuation of further study intervention development 
If the study is prematurely terminated or suspended, the sponsor shall promptly inform the 
investigator, the IEC/IRB, the regulatory authorities, and any CRO(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 ensure appropriate subject therapy 
and/or follow-up. 
Study termination is also provided for in the clinical study agreement. If there is any conflict 
between the contract and this protocol, the contract will control as to termination rights. 
10.1.10. 
Publication Policy 
The results of this study may be published or presented at scientific meetings. The sponsor will 
comply with the requirements for publication of study results. Any publication of data from the 
study by the investigator will be subject to mutual agreement between the investigator and the 
Sponsor. 


Protocol 
C3300-101 
55 
10.2.
Appendix 2: Clinical Laboratory Tests
All protocol-required laboratory assessments, as defined below, must be conducted in 
accordance with the laboratory manual and the Schedule of Activities. 
 The tests detailed in Table 2 will be performed by the local laboratory, with the 
exception of the point-of-care hemoccult tests and ESR which will be sent to an 
outside laboratory. 
 Protocol-specific requirements for inclusion or exclusion of subjects are detailed in 
Section 5 of the protocol. 
 Additional tests may be performed at any time during the study as determined 
necessary by the investigator or required by local regulations. 
 The investigator may consider repeating a laboratory test for any subject who has an 
abnormal laboratory test result (eg, positive hemoccult test, out-of-range chemistry 
test, abnormal urine culture) at Discharge (Day 2). The decision to repeat a laboratory
test would be based on the investigator's clinical judgment after discussion with the 
sponsor's medical monitor and depending on the clinical context. 
 Pregnancy testing is not required, as women of childbearing potential are not eligible 
for the study (Section 5). 


Protocol 
C3300-101 
56 
Table 2: 
Protocol-Required Safety Laboratory Assessments 
Laboratory 
Assessments 
Parameters 
Hematology 
Platelet count 
RBC Indices: 
MCV 
MCH 
MCHC 
 Reticulocytes 
WBC Count with Differential: 
Neutrophils 
Lymphocytes 
Monocytes 
Eosinophils 
Basophils 
RBC count 
Hemoglobin 
Hematocrit 
Clinical 
Chemistrya 
Blood urea 
nitrogen (BUN) 
Potassium 
AST 
Total and direct 
bilirubin 
Creatinine 
Sodium 
ALT 
Total protein 
Glucose 
Calcium 
Alkaline 
phosphatase 
Magnesium 
Chloride 
Albumin 
Bicarbonate 
Phosphate 
Cholesterol 
Uric acid 
 
 
Routine Urinalysis 
 Specific gravity 
 pH, glucose, protein, blood, and ketones, by dipstick 
 Microscopic examination (if blood or protein is abnormal) 
 Urine culture (if urinary tract infection is suspected) 
Inflammatory 
markers 
 ESR (analyzed at an outside laboratory) 
 CRP 
Hemoccult testing 
Point-of-care hemoccult test 
Other Screening 
Tests 
Alcohol and drug screen (to include at minimum: amphetamines, barbiturates, 
cocaine, opiates, cannabinoids, and benzodiazepines). 
Urine cotinine test 
testing (Screening and Check-in) 
HIV antibody and hepatitis panel (HBsAg, and HCV antibody) 
a All events of ALT >3×upper limit of normal (ULN) and bilirubin >
ALT >3×ULN and international normalized ratio (INR) >1.5, if INR measured which may indicate severe liver 
injury (possible Hy’s Law), must be reported as an SAE (excluding studies of hepatic impairment or cirrhosis). 
Investigators must document their review of each laboratory safety report. 
Laboratory/analyte results that could unblind the study will not be reported to CRU staff or other 
blinded personnel until the study has been unblinded. 


Protocol 
C3300-101 
57 
10.3.
Appendix 3: Adverse Events: Definitions and Procedures for 
Recording, Evaluating, Follow-up, and Reporting 
10.3.1. 
Definition of AE and TEAE 
10.3.1.1. AE Definition 
 An AE is any untoward medical occurrence in a patient or clinical study subject, 
temporally associated with the use of study intervention, whether or not considered 
related to the study intervention. 
 NOTE: An AE can therefore be any unfavorable and unintended sign (including an 
abnormal laboratory finding), symptom, or disease (new or exacerbated) temporally 
associated with the use of study intervention. 
10.3.1.2. TEAE Definition 
 A TEAE is an event that emerges, or a pre-existing event that worsens, any time after 
the initiation of the first dosing of the investigational product. 
10.3.1.3. Events Meeting the AE Definition 
 Any abnormal laboratory test results (hematology, clinical chemistry, or urinalysis) or 
other safety assessments (eg, ECG, radiological scans, vital signs measurements), 
including those that worsen from baseline, considered clinically significant in the 
medical and scientific judgment of the investigator (ie, not related to progression of 
underlying disease). 
 Exacerbation of a chronic or intermittent pre-existing condition including either an 
increase in frequency and/or intensity of the condition. 
 New conditions detected or diagnosed after study intervention administration even 
though it may have been present before the start of the study. 
Signs, symptoms, or the clinical sequelae of a suspected drug-drug interaction. 
 Signs, symptoms, or the clinical sequelae of a suspected overdose of either study 
intervention or a concomitant medication. Overdose per se will not be reported as an 
AE/SAE unless it is an intentional overdose taken with possible suicidal/self-harming 
intent. Such overdoses should be reported regardless of sequelae. 
 Specific Situations 
AEs that are secondary to other AEs: In general, AEs that are secondary to other 
AEs (eg, cascade events or clinical sequelae) should be identified by their primary 
cause, with the exception of severe or serious secondary events. If a medically 
significant secondary AE that is separated in time from the initiating event occurs, 
the event may be recorded as an independent event on the AE eCRF after 
consultation with the sponsor. All AEs should be recorded separately if it is not 
clear as to whether the events are associated. 


Protocol 
C3300-101 
58 
Persistent AEs: A persistent AE is one that extends continuously, without 
resolution, between subject evaluation time points. If the severity changes 
throughout the duration of the persistent AE, record the severity as it changes. 
Recurrent AEs: A recurrent AE is one that resolves between subject visits and 
subsequently recurs. Each recurrence of the AE should be recorded as a separate 
AE on the AE eCRF. 
10.3.1.4. Events NOT Meeting the AE Definition 
 Any clinically significant abnormal laboratory findings or other abnormal safety 
assessments which are associated with the underlying disease, unless judged by the 
investigator to be more severe than expected for the subject’s condition. 
 The disease/disorder being studied or expected progression, signs, or symptoms of the 
disease/disorder being studied, unless more severe than expected for the subject’s 
condition. 
 Medical or surgical procedure (eg, endoscopy, appendectomy): the condition that 
leads to the procedure is the AE. 
 Situations in which an untoward medical occurrence did not occur (social and/or 
convenience admission to a hospital). 
 Anticipated day-to-day fluctuations of pre-existing disease(s) or condition(s) present 
or detected at the start of the study that do not worsen. 
10.3.2. 
Definition of SAE
If an event is not an AE per definition above (Section 10.3.1), then it cannot be an SAE even if 
serious conditions are met (eg, hospitalization for signs/symptoms of the disease under study, 
death due to progression of disease). 
An SAE is defined as any untoward medical occurrence that, at any dose: 
1. Results in death
2. Is life-threatening 
The term “life-threatening” in the definition of “serious” refers to an event in which 
the subject was at risk of death at the time of the event. It does not refer to an event, 
which hypothetically might have caused death, if it were more severe. 
3. Requires inpatient hospitalization or prolongation of existing hospitalization
In general, hospitalization signifies that the subject has been detained (usually 
involving at least an overnight stay) at the hospital or emergency ward for 
observation and/or treatment that would not have been appropriate in the physician’s 
office or outpatient setting. Complications that occur during hospitalization are AEs. 
If a complication prolongs hospitalization or fulfills any other serious criteria, the 
event is serious. When in doubt as to whether “hospitalization” occurred or was 
necessary, the AE should be considered serious. 


Protocol 
C3300-101 
59 
 Hospitalization for elective treatment of a pre-existing condition that did not worsen 
from baseline is not considered an AE. 
4. Results in persistent disability/incapacity 
 The term disability means a substantial disruption of a person’s ability to conduct 
normal life functions. 
 This definition is not intended to include experiences of relatively minor medical 
significance such as uncomplicated headache, nausea, vomiting, diarrhea, influenza, 
and accidental trauma (eg, sprained ankle) which may interfere with or prevent 
everyday life functions but do not constitute a substantial disruption. 
5. Is a congenital anomaly/birth defect 
6. Other situations: 
 Medical or scientific judgment should be exercised in deciding whether SAE 
reporting is appropriate in other situations such as important medical events that may 
jeopardize the subject or may require medical or surgical intervention to prevent one 
of the other outcomes listed in the above definition. These events should usually be 
considered serious. 
 Examples of such events include invasive or malignant cancers, intensive treatment in 
an emergency room or at home for allergic bronchospasm, blood dyscrasias or 
convulsions that do not result in hospitalization, or development of drug dependency 
or drug abuse. 
10.3.3. 
Recording and Follow-up of AE and/or SAE 
10.3.3.1. AE and SAE Recording 
 When an AE/SAE occurs, it is the responsibility of the investigator to review all 
documentation (eg, hospital progress notes, laboratory reports, and diagnostics 
reports) related to the event. 
 The investigator will then record all relevant AE/SAE information in the CRF. 
 It is not acceptable for the investigator to send photocopies of the subject's medical 
records to Ironwood Global Patient Safety in lieu of completion of the SAE 
Form/AE/SAE CRF page.
 There may be instances when copies of medical records for certain cases are 
requested by Ironwood Global Patient Safety. In this case, all subject identifiers, with 
the exception of the subject number, will be redacted on the copies of the medical 
records before submission to Ironwood Global Patient Safety. 
 The investigator will attempt to establish a diagnosis of the event based on signs, 
symptoms, and/or other clinical information. Whenever possible, the diagnosis (not 
the individual signs/symptoms) will be documented as the AE/SAE. 


Protocol 
C3300-101 
60 
 For subjects who fail screening and have an SAE, only the SAE will be collected in 
CRF. 
10.3.3.2. Assessment of Intensity/Severity 
The investigator will make an assessment of intensity for each AE and SAE reported during the 
study and assign it to 1 of the following categories, according to the National Cancer Institute 
Common Terminology Criteria for Adverse Events [NCI-CTCAE]):
 Grade 1: Mild; asymptomatic or mild symptoms; clinical or diagnostic observations 
only; intervention not indicated. 
Grade 2: Moderate; minimal, local or noninvasive intervention indicated; limiting 
age-appropriate instrumental Activities of Daily Living (ADL).
Grade 3: Severe or medically significant but not immediately life-threatening; 
hospitalization or prolongation of hospitalization indicated; disabling; limiting self 
care ADL. 
 Grade 4: Life threatening consequences; urgent intervention indicated. 
 Grade 5: Death related to AE.
A semicolon indicates “or” within the description of the grade. 
ADL are defined as follows: Instrumental ADL refer to preparing meals, shopping for groceries 
or clothes, using the telephone, managing money, etc. Self care ADL refer to bathing, dressing 
and undressing, feeding self, using the toilet, taking medications, and not bedridden. 
An event is defined as “serious” when it meets at least 1 of the predefined outcomes as described 
in the definition of an SAE, NOT when it is rated as severe. 
10.3.3.3. Assessment of Causality
 The investigator is obligated to assess the relationship between study intervention and 
each occurrence of each AE/SAE.
 A “reasonable possibility” of a relationship conveys that there are facts, evidence, 
and/or arguments to suggest a causal relationship, rather than a relationship cannot be 
ruled out. 
The investigator will use clinical judgment to determine the relationship.
 Alternative causes, such as underlying disease(s), concomitant therapy, and other risk 
factors, as well as the temporal relationship of the event to study intervention 
administration will be considered and investigated. 
The investigator will also consult IB and/or Product Information, for marketed 
products, in his/her assessment. 
 For each AE/SAE, the investigator must document in the medical notes that he/she 
has reviewed the AE/SAE and has provided an assessment of causality. 
 There may be situations in which an SAE has occurred and the investigator has 
minimal information to include in the initial report to the sponsor. However, it is very 


Protocol 
C3300-101 
61 
important that the investigator always make an assessment of causality for every 
event before the initial transmission of the SAE data to the sponsor. 
 The investigator may change his/her opinion of causality in light of follow-up 
information and send an SAE follow-up report with the updated causality assessment. 
 The causality assessment is one of the criteria used when determining regulatory 
reporting requirements. 
10.3.3.4. Follow-up of AEs and SAEs 
 The investigator is obligated to perform or arrange for the conduct of supplemental 
measurements and/or evaluations as medically indicated or as requested by the 
sponsor to elucidate the nature and/or causality of the AE or SAE as fully as possible. 
This may include additional laboratory tests or investigations, histopathological 
examinations, or consultation with other health care professionals. 
 If a subject dies during participation in the study or during a recognized follow-up 
period, the investigator will provide the sponsor with a copy of any postmortem 
findings including histopathology. 
 New or updated information will be recorded in the originally completed CRF. 
 The investigator will submit any updated SAE data to the sponsor within 24 hours of 
receipt of the information. 
10.3.4. 
Reporting of SAEs 
10.3.4.1. SAE Reporting to Ironwood Global Patient Safety via an Electronic Data 
Collection Tool 
 The primary mechanism for reporting an SAE to the Sponsor will be the electronic 
data collection tool. 
 If the electronic system is unavailable, then the site will use the paper SAE data 
collection tool (see next section) in order to report the event within 24 hours. 
 The site will enter the SAE data into the electronic system as soon as it becomes 
available. 
 After the study is completed, the electronic data collection tool will be taken offline 
to prevent the entry of new data or changes to existing data. 
 If the site receives a report of a new SAE from a study subject or receives updated 
data on a previously reported SAE after the electronic data collection tool has been 
taken offline, then the site can report this information on a paper SAE form (see next 
section) or to the medical monitor/SAE coordinator by telephone. 
 Contacts for SAE reporting can be found in the study contact list. 


Protocol 
C3300-101 
62 
10.3.4.2. SAE Reporting to Ironwood Global Patient Safety via Paper SAE Report Form 
 If the electronic data collection system is not available, email transmission of the SAE 
report form is the preferred method to transmit this information to the medical 
monitor and SAE coordinator (ICSRoperations@ironwoodpharma.com). If email is 
not available, the SAE report form should be transmitted via facsimile. 
 In rare circumstances and in the absence of email or facsimile equipment, notification 
by telephone is acceptable with a copy of the SAE data collection tool sent by 
overnight mail or courier service. 
 Initial notification via telephone does not replace the need for the investigator to 
complete and sign the SAE report form within the designated reporting time frames. 
 Contacts for SAE reporting can be found in the study contact list. 


Protocol 
C3300-101 
63 
10.4.
Appendix 4: Contraceptive Guidance and Collection of Pregnancy 
Information 
10.4.1. 
Definitions 
10.4.1.1. Women of Childbearing Potential (WOCBP) 
A woman is considered fertile following menarche and until becoming postmenopausal unless 
permanently sterile (see below). 
If fertility is unclear (eg, amenorrhea in adolescents or athletes) and a menstrual cycle cannot be 
confirmed before first dose of study intervention, additional evaluation should be considered. 
10.4.1.2. Women Not Considered Women of Childbearing Potential 
Women in the following categories are not considered WOCBP: 
1. Premenarchal 
2. Premenopausal female with 1 of the following: 
 Documented hysterectomy 
 Documented bilateral salpingectomy 
 Documented bilateral oophorectomy 
For individuals with permanent infertility due to an alternate medical cause other than the 
above, (eg, mullerian agenesis, androgen insensitivity), investigator discretion should be 
applied to determining study entry. 
Note: Documentation can come from the site personnel’s: review of the subject’s medical 
records, medical examination, or medical history interview. 
3. Postmenopausal female 
A postmenopausal state is defined as no menses for 12 months without an alternative 
medical cause. 
Females on hormonal replacement therapy (HRT) and whose menopausal status is in 
doubt will be required to use one of the non-estrogen hormonal highly effective 
contraception methods if they wish to continue their HRT during the study. 
Otherwise, they must discontinue HRT to allow confirmation of postmenopausal 
status before study enrollment. 
10.4.2. 
Contraception Guidance 
Male subjects and female partners must use double-barrier method of contraception (ie, a 
combination of male condom with either cervical cap, diaphragm, or sponge with spermicide) 
during the study, until after the Follow-up Phone Call on Day 14. 
Women of childbearing potential are excluded from the study. 


Protocol 
C3300-101 
64 
10.4.2.1. Collection of Pregnancy Information 
10.4.2.1.1. Male subjects with partners who become pregnant 
 The investigator will attempt to collect pregnancy information on any male subject’s 
female partner who becomes pregnant while the male subject is in this study. This 
applies only to male subjects who receive study intervention. 
 After obtaining the necessary signed informed consent from the pregnant female 
partner directly, the investigator will record pregnancy information on the appropriate 
form and submit it to the sponsor within 24 hours of learning of the partner’s 
pregnancy. The female partner will also be followed to determine the outcome of the 
pregnancy. Information on the status of the mother and child will be forwarded to the 
sponsor. Generally, the follow-up will be no longer than 6 to 8 weeks following the 
estimated delivery date. Any termination of the pregnancy will be reported regardless 
of fetal status (presence or absence of anomalies) or indication for the procedure.
10.4.2.1.2. Female subjects who become pregnant 
 The investigator will collect pregnancy information on any female subject who 
becomes pregnant while participating in this study. The initial information will be 
recorded on the appropriate form and submitted to the sponsor within 24 hours of 
learning of a subject's pregnancy. 
 The subject will be followed to determine the outcome of the pregnancy. The 
investigator will collect follow-up information on the subject and the neonate and the 
information will be forwarded to the sponsor. Generally, follow-up will not be 
required for longer than 6 to 8 weeks beyond the estimated delivery date. Any 
termination of pregnancy will be reported, regardless of fetal status (presence or 
absence of anomalies) or indication for the procedure. 
 While pregnancy itself is not considered to be an AE or SAE, any pregnancy 
complication or elective termination of a pregnancy for medical reasons will be 
reported as an AE or SAE. A spontaneous abortion (occurring at <22 weeks 
gestational age) or still birth (occurring at >22 weeks gestational age) is always 
considered to be an SAE and will be reported as such. Any poststudy pregnancy-
related SAE considered reasonably related to the study intervention by the 
investigator will be reported to the sponsor as described in Section 8.3.4. While the 
investigator is not obligated to actively seek this information in former study subjects, 
he or she may learn of an SAE through spontaneous reporting. 
 Any female subject who becomes pregnant while participating in the study be 
withdrawn from the study. 


Protocol 
C3300-101 
65 
10.5.
Appendix 5: Abbreviations
Table 3: 
List of Abbreviations 
Abbreviation 
Definition 
ADLs 
Activities of Daily Living 
AE 
adverse event 
ALT 
alanine aminotransferase 
AST 
aspartate aminotransferase 
AUC0-  
Area under the plasma concentration time curve from time zero to time 
infinity 
AUC0-t 
Area under the plasma concentration time curve from time zero to the time at 
which the last measurable concentration is observed 
BBMD 
bladder and bowel movement diary
BM 
bowel movement 
BMI 
body mass index 
BSFS 
Bristol Stool Form Scale 
BUN 
blood urea nitrogen 
CFTR 
cystic fibrosis transmembrane conductance regulator 
cGMP 
cyclic guanosine monophosphate 
CIOMS 
Council for International Organizations of Medical Sciences 
Cmax 
maximum plasma concentration 
CONSORT 
Consolidated Standards of Reporting Trials 
CRF 
case report form 
CRO 
contract research organization 
CRP 
C-reactive protein 
CRU 
clinical research unit 
CTCAE 
Common Terminology Criteria for Adverse Events 
 
coefficient of variation 
DMC 
data monitoring committee 
DRG 
dorsal root ganglia 
eCRF 
electronic case report form 
EDC 
electronic data capture 
ESR 
erythrocyte sedimentation rate 
GC-C 
guanylate cyclase C 


Protocol 
C3300-101 
66 
Abbreviation 
Definition 
GI 
Gastrointestinal 
HBsAg 
hepatitis B surface antigen  
HCV 
hepatitis C virus 
HED 
human equivalent dose 
HEENT 
head, eyes, ears, nose, and throat 
HIPAA 
Health Insurance Portability and Accountability Act 
HIV 
human immunodeficiency virus 
HRT
hormonal replacement therapy
IB 
investigator’s brochure 
IC/BPS 
interstitial cystitis/bladder pain syndrome 
ICF 
informed consent form 
IEC 
independent ethics committee 
IMP 
investigational medical product 
INR 
international normalized ratio 
IRB 
institutional review board 
LLOQ 
lower limit of quantitation 
LS 
Lumbosacral 
MCH 
mean corpuscular hemoglobin 
MCHC 
mean corpuscular hemoglobin concentration 
MCV 
mean corpuscular volume 
MedDRA 
Medical Dictionary for Regulatory Activities 
NCI-CTCAE
National Cancer Institute Common Terminology Criteria for Adverse Events 
NIMP
noninvestigational medical product
NOAEL 
no-observed-adverse-effect level 
PD 
pharmacodynamic(s) 
PK 
pharmacokinetic(s) 
PKGII 
protein kinase G type II 
RBC 
red blood cell 
SAE 
serious adverse event 
SAP 
statistical analysis plan 
SARS CoV 2 
severe acute respiratory syndrome coronavirus 2 
SID 
subject identification 


Protocol 
C3300-101 
67 
Abbreviation 
Definition 
SoA 
schedule of activities 
SUSAR 
suspected unexpected serious adverse reaction 
t1/2
terminal elimination half-life 
TEAE 
treatment-emergent adverse event 
TESAE 
treatment-emergent serious adverse event 
TL 
Thoracolumbar 
Tmax 
time to maximum plasma concentration 
ULN
upper limit of normal
WBC 
white blood cell 
WOCBP 
woman/women of childbearing potential 




Protocol 
C3300-101 
69 
10.6.2. 
Investigator Signature 
Protocol Title: 
A Phase 1 Placebo-controlled Study of the Safety and 
Tolerability of Rectally Administered Single Ascending Doses 
of 
in Healthy Volunteers
Short Title:  
A Phase 1 Study of Single Ascending Doses of 
in 
Healthy Volunteers 
Protocol Number: 
C3300 101 
Version Number
/Amendment Number: 
Version 4/Amendment 3 
Final Date:  
10 January 2022 
I have read the protocol described above. I agree to comply with all applicable regulations and to 
conduct the study as described in the protocol. 
Investigator Name:
 
Signed:  
Date: 


Protocol 
C3300-101 
70 
10.7.
Appendix 7: Protocol Amendment History
The Protocol Amendment Summary of Changes Table summarizing the current amendment is 
located directly before the Table of Contents. Prior protocol amendments are summarized below. 
Amendment 1 (22 September 2021) 
Overall Rationale for the Amendment:
Updates to the safety and pharmacodynamic (PD) assessments were made based on FDA 
comments and recommendations. 
Section # and Name 
Description of Change 
Brief Rationale 
Substantial/ 
Nonsubstantial 
1.3 (Schedule of 
Activities); 
5.3.3 (Activity); 
8.2.2 (Vital Signs) 
Timepoints for vital signs 
collection on Days 1 and 2 were 
changed to align with PK blood 
sample collection timepoints: 
Day 1 Predose: 0 
 
Day 1 Postdose: 0.5, 1, 2, 4, 6, 
and 8h (±10m) 
Day 2: 24h post-Day 1 dose 
(±25m) 
Corresponding footnote specifies 
that the 0.5 hour postdose 
assessment on Day 1 will be 
obtained in the side-lying position 
(consistent with the position 
required following dosing); the 
2, 4, and 8 hour postdose 
timepoints should be preceded by 
at least 5 minutes of rest for the 
subject in a quiet setting without 
distractions (eg, television, cell 
phones); and at all other 
timepoints (or if clinically 
indicated), orthostatic 
measurements will be obtained 
(supine/semisupine measurements 
 minutes, 
then standing measurements after 
3 minutes in the standing 
position). 
Change to vital signs 
collection times was 
requested by FDA. 
Granularity regarding 
position for collection 
of blood pressure and 
pulse was added to 
account for additional 
collection times, as 
orthostatic 
measurements were not 
needed at every 
timepoint in order to 
evaluate the potential 
for volume depletion, 
unless clinically 
indicated. 
Substantial 


Protocol 
C3300-101 
71 
Section # and Name 
Description of Change 
Brief Rationale 
Substantial/ 
Nonsubstantial 
1.3 (Schedule of 
Activities) 
Timepoints for clinical chemistry, 
hematology, and urinalysis on 
Days 1 and 2 were updated to add 
a Day 1 predose (0 
timepoint and clarify the Day 2 
timing (24h post-Day 1 dose 
[±15m]). 
Change to clinical 
chemistry and 
urinalysis collection 
times was requested by 
FDA; addition of 
Day 1 predose 
timepoint establishes a 
clear baseline for 
laboratory assessments. 
Substantial 
1.3 (Schedule of 
Activities); 
8.5 
(Pharmacokinetics); 
9.4.4 (Exploratory 
Endpoints Analysis) 
The end of the stool collection 
period was changed from 24h 
postdose to the time of Discharge 
on Day 2. 
The corresponding analysis was 
clarified to note that subjects who 
do not have a bowel movement 
(BM) during the stool collection 
period will not be included in the 
analysis of 
concentration in the stool. 
Clarification on the 
handling of subjects 
with no stool sample 
for assessment of 
in stool was 
requested by FDA; the 
collection timing was 
changed in order to 
minimize missing data. 
Substantial 
8.6 
(Pharmacodynamics) 
The following bladder 
assessments were added to the PD 
assessments: day and time of 
urination, urinary urgency using a 
5-point scale (0=no urgency to 
4=urge incontinence), and 
nocturia (the number of 
awakenings to urinate each 
night). 
Addition of bladder 
assessments was 
requested by FDA. 
Substantial 
1 (Synopsis, 
Objectives and 
Endpoints); 
3.1.2 (Exploratory 
Objectives); 
3.3.2 (Exploratory 
Endpoints); 
9.4.4 (Exploratory 
Endpoints Analysis) 
Edited exploratory PD objective 
to specify that it is based on 
bladder and bowel assessments. 
Added exploratory PD endpoints: 
Change from baseline in urinary 
frequency during the Clinic 
Period 
Change from baseline in urinary 
urgency during the Clinic Period 
Change from baseline in nocturia 
during the Clinic Period 
To reflect addition of 
bladder assessments 
Substantial 
9.4.4 (Exploratory 
Endpoints Analysis) 
Described the derivation/ 
calculation of baseline and Clinic 
Period values for bladder 
assessments. 
To reflect addition of 
bladder assessments 
and endpoints 
Substantial 


Protocol 
C3300-101 
72 
Section # and Name 
Description of Change 
Brief Rationale 
Substantial/ 
Nonsubstantial 
Throughout
The bowel movement diary 
(BMD) was renamed the bladder 
and bowel movement diary 
(BBMD). Descriptions of the 
diary specify that bladder 
symptoms will be collected along 
with BMs/bowel habits. 
To reflect addition of 
bladder assessments 
Substantial 
1.3 (Schedule of 
Activities); 
4.1.1.2 (Clinic 
Period); 
8.6 
(Pharmacodynamics) 
BBMD will be completed by the 
subject throughout the Screening 
and Clinic Periods, rather than by 
the subject during the Screening 
Period and clinical research unit 
(CRU) staff during the Clinic 
Period. Added BBMD dispensed 
at Check-in and BBMD collection 
at Discharge to account for the 
subject completing the BBMD 
during the Clinic Period. 
To reflect addition of 
bladder assessments, 
which cannot be 
assessed by CRU staff 
Substantial 
1.3 (Schedule of 
Activities); 
8.2.1 (Physical 
Examination) 
The physical examination 
instructions were updated to 
require a digital rectal 
examination as part of each 
physical examination (rather than 
at the Screening Visit only). 
FDA recommended 
performing digital 
rectal examination 
during all physical 
examinations. 
Substantial 
1.3 (Schedule of 
Activities); 
5.3.1 (Meals and 
Dietary Restrictions) 
The predose fast instructions were 
updated to change completion of 
fasting on Day 1 from 4 hours 
postdose to at least 1 hour 
postdose. 
Mitigate risk of volume 
depletion and potential 
AEs due to prolonged 
fasting. 
Substantial 
1.3 (Schedule of 
Activities); 
5.3.3 (Activity); 
6.1.1 (Dosing 
Instructions) 
Additional detail was added to the 
study drug administration 
instructions to note that subjects 
will be encouraged to empty their 
bowels in the morning prior to 
dosing, if possible. 
May help subjects 
avoid using the 
bathroom and hold in 
the enema for at least 
1 hour postdose. 
Substantial 
1.3 (Schedule of 
Activities) 
The window for the 
pharmacokinetic (PK) blood draw 
at 1 hour postdose was changed to 
(±10m). 
To allow for orthostatic 
vital signs to be taken 
after the hour of 
restricted activity 
postdose (30 minutes 
lying on side followed 
by 30 minutes in 
semisupine position), 
but before the PK 
blood draw 
Substantial 


Protocol 
C3300-101 
73 
Section # and Name 
Description of Change 
Brief Rationale 
Substantial/ 
Nonsubstantial 
5.2 (Exclusion 
Criteria) 
Exclusion #4 was updated to 
specifically exclude subjects with 
presence or history of 
inflammatory bowel disease. 
FDA recommended 
excluding subjects with 
presence or history of 
inflammatory bowel 
disease. 
Substantial 
10.2 (Appendix 2: 
Clinical Laboratory 
Tests) 
Under Routine Urinalysis, added 
urine culture (if urinary tract 
infection is suspected). 
FDA recommended 
collecting data for 
urinary tract infections 
(including urine 
culture). 
Substantial 
10.3.4 (Reporting of 
SAEs) 
Serious adverse event (SAE) 
reporting method was changed 
from paper via email (primary) or 
fax (secondary) to the electronic 
data collection tool as primary 
method with paper (SAE Report 
Form) as secondary. 
Correction 
Substantial 
10.2 (Appendix 2: 
Clinical Laboratory 
Tests) 
Clinical laboratory tests will be 
performed by a central laboratory, 
with the exception of the 
Check in (Day 1) laboratory 
assessment, which will be 
performed at a local laboratory 
Correction (original 
protocol stated that all 
tests would be 
performed at the local 
laboratory)
Nonsubstantial 
9.3 (Populations for 
Analyses) 
An Intent-to-treat (ITT) Set was 
added, consisting of all subjects 
in the Screened Analysis Set who 
have been randomized to a 
treatment regimen. Analysis will 
be performed according to the 
allocated treatment regimen 
regardless of the treatment 
regimen actually received. 
Preference to present 
disposition table based 
on randomized 
subjects. 
Nonsubstantial 
10.1.6 
(Dissemination of 
Clinical Study Data) 
Removed EudraCT from the list 
of public registries where results 
will be made available. 
The Sponsor does not 
plan to post this study 
on EudraCT. 
Nonsubstantial 
Throughout 
Minor editorial and document 
formatting revisions 
Minor corrections and 
clarifications 
Nonsubstantial 


Protocol 
C3300-101 
74 
Amendment 2 (24 November 2021) 
Section # and Name 
Description of Change 
Brief Rationale 
Substantial/ 
Nonsubstantial 
Section 1.1.5 
(Overall Design) 
and 
Section 4.1 (Overall 
Design) 
The following sentence was 
deleted: 
Following blinded review, safety 
data for individual subjects may 
be unblinded as described below. 
The Dose Escalation 
Committee will remain 
blinded to the treatment 
assignments of 
individual subjects 
throughout the study. 
In accordance with 
Section 6.3.2, the 
treatment assignments 
of individual subjects 
will only be unblinded 
to a sponsor 
representative for 
regulatory reporting 
purposes or if 
warranted by emerging 
safety or tolerability 
issues. 
Substantial 
Section 1.1.4 
(Objectives and 
Endpoints), 
Section 3.3.2 
(Exploratory 
Endpoints), and 
Section 9.4.4 
(Exploratory 
Endpoints Analyses) 
The 3 exploratory endpoints 
related to bladder function were 
modified to specify the time 
period as a 
 
during the Clinic Period: 
 Change from baseline in 
urinary frequency during the 
Clinic Period 
duration 
 Change from baseline in 
urinary urgency during the 
Clinic Period for a 24 hour 
duration 
 Change from baseline in 
nocturia during the Clinic 
 
An additional exploratory 
endpoint was added: 
Change from baseline in pain or 
burning in bladder or pelvic area 
during the Clinic Period for a 
 
To reflect these changes, the 
statistical analysis sections related 
to bladder function were updated. 
This change adds 
additional specificity to 
the exploratory 
endpoint description 
and allows for the 
evaluation of bladder 
pain and burning in 
addition to function. 
Substantial 


Protocol 
C3300-101 
75 
Section # and Name 
Description of Change 
Brief Rationale 
Substantial/ 
Nonsubstantial 
Section 1.3 (Schedule 
of Activities/Table 1 
[Footnote a]) 
The following sentence was 
added: 
The early termination 
assessments will be the same as 
those conducted at Discharge 
(Day 2). 
Clarification was made 
to specify which 
assessments would be 
performed in the event 
of a subject’s early 
termination. 
Substantial 
Section 1.3 (Schedule 
of Activities/Table 1 
[Footnote c]) and 
Section 10.2 
(Appendix 2: Clinical 
Laboratory 
Tests/Table 2) 
In Table 1, the “urine drug and 
alcohol screen” was renamed 
“drug and alcohol screen”. A new 
table footnote (Footnote c) was 
added: 
The alcohol screen at Check-in 
(Day 
but all other drug and alcohol 
screens will be via urine tests. 
In Table 2, the following text was 
added: 
Note: The alcohol screen at 
Check-in (Day 
breath test, but all other drug and 
alcohol screens will be via urine 
tests. 
This change was made 
due to operational 
feasibility. 
Substantial 
Section 1.3 (Schedule 
of Activities/Table 1 
[Footnote h], 
Section 4.1.1.1 
(Screening Period), 
Section 4.2 
(Scientific Rationale 
for Study Design), 
and Section 8.6 
(Pharmacodynamics) 
1. The bladder and urination 
portion of the Bladder and Bowel 
Movement Diary (BBMD) will 
not be completed on 
Days 
 
This bladder 
and urination portion of the 
BBMD will be collected on Day 1 
(predose) and Day 2 with a 
24 
timepoint. 
2. In the BBMD, the specific 
questions related to bladder 
function were updated. 
This change was made 
to better characterize 
any potential changes 
in bladder function 
and/or potential urinary 
symptoms following 
dosing and reduce 
subject burden.  
Substantial 
Section 5.1 (Inclusion 
Criteria) 
The following inclusion criterion 
was added: 
Subject is compliant with 
colorectal cancer screening 
guidelines according to the ACG 
Clinical Guidelines: Colorectal 
 
This change was made 
to ensure that subjects 
are in compliance with 
current colorectal 
cancer screening 
guidelines and to avoid 
potential confounding 
factors. 
Substantial 


Protocol 
C3300-101 
76 
Section # and Name 
Description of Change 
Brief Rationale 
Substantial/ 
Nonsubstantial 
Section 7.1.1 (Study 
Suspension or 
Termination) 
The following sentence was 
deleted: 
Treatment assignments for the 
affected subjects will be 
unblinded to a designated 
sponsor representative not 
otherwise involved in the study, to 
evaluate whether or not to 
continue dosing in that cohort or 
to proceed with any further dose 
escalation in the remaining 
cohorts. 
In accordance with 
Section 6.3.2, the 
treatment assignments 
of individual subjects 
will only be unblinded 
to a sponsor 
representative for 
regulatory reporting 
purposes. The Dose 
Escalation Committee 
will remain blinded to 
the treatment 
assignments of 
individual subjects 
throughout the study. 
Substantial 
Section 7.1.1 (Study 
Suspension or 
Termination) 
The following phrase in 
strikethrough was removed: 
The sponsor may permanently 
terminate the study, or a 
component of the study, at any 
time. Reasons for termination 
may include, but are not limited 
to: 
• Death, SAE, or other safety 
finding considered to be 
related to 
that may 
preclude further dosing., in the 
opinion of the sponsor 
The rules for 
permanently 
terminating the study 
were modified. The 
determination to 
terminate the study will 
be based on the 
predefined stopping 
rules and evaluation of 
relatedness and balance 
risk benefit by the dose 
escalation committee. 
Substantial 
Section 6.3.2 
(Blinding) 
The last sentence in the second 
paragraph was deleted: 
Following blinded review, safety 
data for individual subjects may 
be unblinded as described in 
Section 
. 
In accordance with the 
change in 
Section 1.1.5/Overall 
Design, Section 4.1, 
and Section 7.1.1, this 
sentence is no longer 
relevant. 
Substantial 


Protocol 
C3300-101 
77 
Section # and Name 
Description of Change 
Brief Rationale 
Substantial/ 
Nonsubstantial 
Section 6.1.1 (Dosing 
Instructions) and 
Section 1.3 (Schedule 
of Activities/Table 1 
[Footnote l]) 
The following information was 
added: 
Following study drug 
administration, clinical research 
unit (CRU) staff will monitor 
subjects for leakage of the study 
drug from the rectum during the 
initial 30 minutes post 
administration. 
CRU staff will record (as yes/no) 
if there was study drug leakage 
from the rectum in the 30 minutes 
following drug administration. 
Leakage following 
dose administration 
will be monitored and 
recorded in order to 
more fully characterize 
study drug exposure.  
Substantial 
Section 9.3 
(Populations for 
Analyses) 
The Pharmacokinetic (PK) and 
Pharmacodynamic (PD) Analysis 
Set definitions were changed. 
The PK and PD analyses will be 
performed according to the 
treatment actually received 
regardless of the allocated 
treatment, rather than according 
to the allocated treatment 
regimen regardless of the 
treatment regimen actually 
received. 
These changes correct 
errors in the 
definitions. 
Substantial 
Section 1.1.5 
(Overall Design), 
Section 4.1 (Overall 
Design), 
Section 6.3.2 
(Blinding), and 
Section 10.1.5 
(Committee 
Structure) 
The text in strikethrough was 
deleted form the description of 
the Dose Escalation membership: 
The Dose Escalation Committee 
will include sponsor and contract 
research organization (CRO) 
representatives (including the 
sponsor medical monitor, sponsor 
clinical operations lead, sponsor 
safety physician, the principal 
investigator, and others as 
necessary). 
This change allows for 
additional flexibility in 
staffing the committee. 
Nonsubstantial 


Protocol 
C3300-101 
78 
Section # and Name 
Description of Change 
Brief Rationale 
Substantial/ 
Nonsubstantial 
Section 1.1.3 
(Rationale) and 
Section 2.2 
(Background) 
The text in underline was added: 
This is supported by the data 
investigational delayed release 
formulation of another GC-C 
agonist, linaclotide, in healthy 
in subjects with irritable bowel 
syndrome with constipation 
), and in 
subjects with irritable bowel 
syndrome with diarrhea 
(Study 
. 
Two additional studies 
were referenced to 
provide additional 
context and to support 
the potential lower 
likelihood of diarrhea 
with colonic release 
formulations of GC-C 
agonists. 
Nonsubstantial 
Section 2.3.1 (Risk 
Assessment) 
Under the risk mitigation for 
“Potential for diarrhea and 
secondary dehydration diarrhea”, 
the CTCAE criteria for Grade 2 
and Grade 3 diarrhea were 
corrected. 
The change corrects an 
error in the description. 
Nonsubstantial 
Section 7.1.1 (Study 
Suspension and 
Termination) 
The following text in italic was 
added: 
The Dose Escalation Committee 
should consider the benefit-risk 
ratio in their determination to 
suspend dosing temporarily or 
permanently. If the benefit-risk is 
considered unacceptable and the 
effect is considered related to 
active study drug by the Dose 
Escalation Committee, dosing at a 
given dose level will be stopped, 
and no additional cohorts will be 
dosed at the same or higher dose 
level (a lower dose could still be 
studied). 
This addition further 
describes the intention 
of the Dose Escalation 
Committee review. 
Nonsubstantial 
Section 9.3 
(Populations for 
Analyses) 
The following sentence was 
deleted: 
If any subjects are found to have 
incomplete data, a decision will 
be made on a case-by-case basis 
as to their inclusion in the 
analysis, but they will be 
presented in the listings. 
The specific rules for 
handling incomplete 
data will be described 
in the Statistical 
Analysis Plan that will 
be finalized before 
database lock. 
Nonsubstantial 


Protocol 
C3300-101 
79 
Section # and Name 
Description of Change 
Brief Rationale 
Substantial/ 
Nonsubstantial 
Section 10.7 
(Protocol 
Amendment History) 
The contents of 
Amendment # 1 were moved to 
this new section showing the 
protocol amendment history. 
This section was 
created as per the 
document template. 
Nonsubstantial 
Throughout 
Minor editorial and document 
formatting revisions 
Minor corrections and 
clarifications were 
made. 
Nonsubstantial 


Protocol 
C3300-101 
80 
11.
REFERENCES
1. 
Castro J, Harrington AM, Hughes PA, Martin CM, Ge P, Shea CM, et al. Linaclotide 
Inhibits Colonic Nociceptors and Relieves Abdominal Pain via Guanylate Cyclase-C and 
Extracellular Cyclic GMP. Gastroenterology. 2013;145(6):133
6. 
2. 
Grundy L, Harrington AM, Castro J, Garcia-Caraballo S, Deiteren A, Maddern J, et al. 
Chronic linaclotide treatment reduces colitis-induced neuroplasticity and reverses 
persistent bladder dysfunction. JCI Insight. 2018;3(19):e121841. 
3. 
Pfeifer A, Aszódi A, Seidler U, Ruth P, Hofmann F, Fässler R. Intestinal secretory 
defects and dwarfism in mice lacking cGMP-dependent protein kinase II. Science. 
1996;274(5295):208
. 
4. 
Forte, LR. Uroguanylin and guanylin peptides: pharmacology and experimental 
therapeutics. Pharmacol Ther. 2004;104(2):13
2. 
5. 
Schlossmann J, Feil R, Hofmann F. Insights into cGMP signalling derived from cGMP 
kinase knockout mice. Frontiers in Bioscience. 2005;10:127
9. 
6. 
Forte LR. Guanylin regulatory peptides: structures, biological activities mediated by 
cyclic GMP and pathobiology. Regul Pept. 1999;81(
):2
9. 
7. 
London RM, Krause WJ, Fan X, Eber SL, Forte LR. Signal transduction pathways via 
guanylin and uroguanylin in stomach and intestine. Am J Physiol. 1997;273(1 Pt 
1):G9
05. 
8. 
Grundy L, Brierley S. Cross-organ sensitization between the colon and bladder: to pee or 
not to pee? Am J Physiol Gastrointest Liver Physiol. 2018;314:G301–G8.
9. 
Shaukat A, Kahi CJ, Burke CA, Rabeneck L, Sauer BG, Rex DK. ACG Clinical 
Guidelines: Colorectal Cancer Screening 2021. Official journal of the American College 
of Gastroenterology | ACG. 2021;116(3):45
9. 
 



SAP_001.pdf: