Conditions: Healthy Volunteers
Keywords: healthy volunteers
Allocation: RANDOMIZED | Intervention model: SEQUENTIAL
Enrollment: 18
Healthy volunteers: yes

Brief summary:
This clinical study is designed as a multiple-ascending-dose, safety and tolerability study with IW-3300. The study drug will be administered as a low-volume \[20 mL\] enema. Study participants will be randomized in a 2:1 ratio to receive IW-3300 or placebo. Up to 3 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, multiple-ascending-dose study of IW-3300 administered rectally, once-daily, for 7 days as a low-volume enema in healthy adult participants. This study will assess the effect of IW-3300 on safety and tolerability.

The study includes up to 4 treatments: placebo and up to 3 dose levels of IW-3300 which will be determined after safety reviews of previous cohorts.

The 9 participants within each cohort will be randomized to receive IW-3300 (6 subjects) or placebo (3 participants), administered rectally (as a low-volume \[20 mL\] enema). Participants in each dosing cohort will progress through 3 study periods: (1) Screening Period, (2) Clinic Period, and (3) Follow-up Period. Treatment duration will be 7 days; participants will be followed in the Phase 1 clinical research unit (CRU) for the duration of dosing, until at least 24 hours after the last dose of study drug and contacted by phone for follow-up approximately 2 weeks after the last dose. Total participant participation will be 29 to 57 days, including the Screening, Clinic, and Follow-up Periods.

Arms:
arm 1: 100 μg dose of active drug (IW-3300) once daily for 7 days arm 2: matching placebo once daily for 7 days arm 3: 300 μg dose of active drug (IW-3300) once daily for 7 days arm 4: matching placebo once daily for 7 days arm 5: Active drug (IW-3300) once daily for 7 days arm 6: Matching placebo once daily for 7 days

Interventions: intervention 1: IW-3300 intervention 2: Placebo
Intervention descriptions: intervention 1: A dose of IW-3300 administered rectally (as a low-volume \[20 mL\] enema). intervention 2: A dose of placebo administered rectally (as a low-volume \[20 mL\] enema).

Protocol document:
Prot_000.pdf: 
Protocol 
C3300-102 
1 
TITLE PAGE 
Protocol Title: 
A Phase 1 Placebo-controlled Study of the Safety and 
Tolerability of Rectally Administered, Multiple-ascending 
Doses of IW-3300 in Healthy Subjects 
Short Title: 
A Phase 1 Study of Multiple-ascending Doses of IW-3300 in 
Healthy Subjects 
Protocol Number: 
C3300-102 
Version Number: 
Version 1 
Final Date: 
14 February 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 
C3300-102 
2 
TABLE OF CONTENTS 
TITLE PAGE ...................................................................................................................................1 
1.
PROTOCOL SUMMARY ............................................................................................8 
1.1. 
Synopsis ........................................................................................................................8 
Protocol Title ................................................................................................................8 
Short Title .....................................................................................................................8 
Rationale .......................................................................................................................8 
Objectives and Endpoints .............................................................................................8 
Overall Design ..............................................................................................................9 
Disclosure Statement ....................................................................................................9 
Number of Subjects ......................................................................................................9 
Intervention Groups and Duration ................................................................................9 
Data Monitoring Committee: No ................................................................................10 
1.2. 
Schema ........................................................................................................................10 
1.3. 
Schedule of Activities .................................................................................................11 
2.
INTRODUCTION ......................................................................................................15 
2.1. 
Study Rationale ...........................................................................................................15 
2.2. 
Background .................................................................................................................15 
2.3. 
Benefit/Risk Assessment ............................................................................................17 
2.3.1. 
Risk Assessment .........................................................................................................17 
2.3.2. 
Benefit Assessment .....................................................................................................18 
2.3.3. 
Overall Benefit: Risk Conclusion ...............................................................................18 
3.
OBJECTIVES AND ENDPOINTS ............................................................................19 
3.1. 
Objectives ...................................................................................................................19 
3.1.1. 
Primary Objective .......................................................................................................19 
3.1.2. 
Exploratory Objectives ...............................................................................................19 
3.2. 
Estimands ....................................................................................................................19 
3.3. 
Endpoints ....................................................................................................................19 
3.3.1. 
Primary Endpoints (Safety) ........................................................................................19 
3.3.2. 
Exploratory Endpoints ................................................................................................19 
4.
STUDY DESIGN .......................................................................................................20 
4.1. 
Overall Design ............................................................................................................20 


Protocol 
C3300-102 
3 
4.1.1. 
Study Periods ..............................................................................................................20 
4.1.1.1. 
Screening Period .........................................................................................................20 
4.1.1.2. 
Clinic Period ...............................................................................................................21 
4.1.1.3. 
Follow-up Period ........................................................................................................21 
4.2. 
Scientific Rationale for Study Design ........................................................................21 
4.2.1. 
Subject Input into Design ...........................................................................................22 
4.3. 
Justification for Dose ..................................................................................................22 
4.4. 
End of Study Definition ..............................................................................................22 
5.
STUDY POPULATION .............................................................................................23 
5.1. 
Inclusion Criteria ........................................................................................................23 
Age ..............................................................................................................................23 
Type of Subject and Disease Characteristics ..............................................................23 
Weight .........................................................................................................................23 
Sex ..............................................................................................................................23 
Informed Consent .......................................................................................................24 
5.2. 
Exclusion Criteria .......................................................................................................24 
Medical Conditions .....................................................................................................24 
Prior/Concomitant Therapy ........................................................................................24 
Prior/Concurrent Clinical Study Experience ..............................................................25 
Diagnostic Assessments ..............................................................................................25 
Other Exclusions .........................................................................................................25 
5.3. 
Lifestyle Considerations .............................................................................................26 
5.3.1. 
Meals and Dietary Restrictions ...................................................................................26 
5.3.2. 
Caffeine, Alcohol, and Tobacco .................................................................................26 
5.3.3. 
Activity .......................................................................................................................26 
5.4. 
Screen Failures ............................................................................................................26 
6.
STUDY INTERVENTION ........................................................................................27 
6.1. 
Study Interventions Administered ..............................................................................28 
6.1.1. 
Dosing Instructions .....................................................................................................28 
6.2. 
Preparation/Handling/Storage/Accountability ............................................................28 
6.3. 
Measures to Minimize Bias: Randomization and Blinding ........................................29 
6.3.1. 
Randomization ............................................................................................................29 
6.3.2. 
Blinding ......................................................................................................................29 


Protocol 
C3300-102 
4 
6.4. 
Study Intervention Compliance ..................................................................................30 
6.5. 
Concomitant Therapy .................................................................................................30 
6.5.1. 
Rescue Medicine .........................................................................................................31 
6.6. 
Dose Modification ......................................................................................................31 
6.7. 
Intervention After the End of the Study .....................................................................31 
7.
DISCONTINUATION OF STUDY INTERVENTION AND SUBJECT
DISCONTINUATION/WITHDRAWAL ..................................................................32 
7.1. 
Discontinuation of Study Intervention ........................................................................32 
7.1.1. 
Study Suspension or Termination ...............................................................................32 
7.2. 
Subject Discontinuation/Withdrawal from the Study .................................................33 
7.3. 
Lost to Follow-up .......................................................................................................33 
8.
STUDY ASSESSMENTS AND PROCEDURES ......................................................34 
8.1. 
Efficacy Assessments .................................................................................................34 
8.2. 
Safety Assessments .....................................................................................................34 
8.2.1. 
Physical Examinations ................................................................................................34 
8.2.2. 
Vital Signs ..................................................................................................................35 
8.2.3. 
Electrocardiograms .....................................................................................................35 
8.2.4. 
Clinical Safety Laboratory Assessments ....................................................................35 
8.2.5. 
Bladder and Bowel Movement Diary .........................................................................36 
8.3. 
Adverse Events and Serious Adverse Events .............................................................36 
8.3.1. 
Time Period and Frequency for Collecting AE and SAE Information .......................37 
8.3.2. 
Method of Detecting AEs and SAEs ..........................................................................37 
8.3.3. 
Follow-up of AEs and SAEs .......................................................................................37 
8.3.4. 
Regulatory Reporting Requirements for SAEs ...........................................................38 
8.3.5. 
Pregnancy ...................................................................................................................38 
8.3.6. 
Cardiovascular and Death Events ...............................................................................38 
8.3.7. 
Disease-related Events and/or Disease-related Outcomes Not Qualifying as 
AEs or SAEs ...............................................................................................................38 
8.3.8. 
Adverse Events of Special Interest .............................................................................38 
8.4. 
Treatment of Overdose ...............................................................................................39 
8.5. 
Pharmacokinetics ........................................................................................................39 
8.6. 
Microbiome .................................................................................................................39 
8.7. 
Pharmacodynamics .....................................................................................................40 


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8.8. 
Genetics ......................................................................................................................40 
8.9. 
Biomarkers ..................................................................................................................40 
8.10. 
Immunogenicity Assessments ....................................................................................40 
8.11. 
Medical Resource Utilization and Health Economics ................................................40 
9. 
STATISTICAL CONSIDERATIONS .......................................................................41 
9.1. 
Statistical Hypotheses .................................................................................................41 
9.2. 
Sample Size Determination ........................................................................................41 
9.3. 
Populations for Analyses ............................................................................................41 
9.4. 
Statistical Analyses .....................................................................................................41 
9.4.1. 
General Considerations ...............................................................................................41 
9.4.2. 
Primary Endpoints Analyses .......................................................................................42 
9.4.3. 
Secondary Endpoint Analysis .....................................................................................42 
9.4.4. 
Exploratory Endpoints Analyses ................................................................................42 
9.4.5. 
Safety Analyses ..........................................................................................................42 
9.4.6. 
Other Analysis/Analyses ............................................................................................43 
9.5. 
Interim Analyses .........................................................................................................43 
9.6. 
Data Monitoring Committee (DMC) ..........................................................................43 
10. 
SUPPORTING DOCUMENTATION AND OPERATIONAL 
CONSIDERATIONS ..................................................................................................44 
10.1. 
Appendix 1: Regulatory, Ethical, and Study Oversight Considerations ....................44 
10.1.1. 
Regulatory and Ethical Considerations ......................................................................44 
10.1.2. 
Financial Disclosure ...................................................................................................44 
10.1.3. 
Informed Consent Process ..........................................................................................44 
10.1.4. 
Data Protection ...........................................................................................................45 
10.1.5. 
Committee Structure ...................................................................................................45 
10.1.6. 
Dissemination of Clinical Study Data ........................................................................45 
10.1.7. 
Data Quality Assurance ..............................................................................................46 
10.1.8. 
Source Documents ......................................................................................................46 
10.1.9. 
Study and Site Start and Closure ................................................................................47 
10.1.10. 
Publication Policy .......................................................................................................47 
10.2. 
Appendix 2: Clinical Laboratory Tests .......................................................................48 
10.3. 
Appendix 3: Adverse Events: Definitions and Procedures for Recording, 
Evaluating, Follow-up, and Reporting ........................................................................50 


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10.3.1. 
Definition of AE and TEAE .......................................................................................50 
10.3.1.1. 
AE Definition ..............................................................................................................50 
10.3.1.2. 
TEAE Definition .........................................................................................................50 
10.3.1.3. 
Events Meeting the AE Definition .............................................................................50 
10.3.1.4. 
Events NOT Meeting the AE Definition ....................................................................51 
10.3.2. 
Definition of SAE .......................................................................................................51 
10.3.3. 
Recording and Follow-up of AE and/or SAE .............................................................52 
10.3.3.1. 
AE and SAE Recording ..............................................................................................52 
10.3.3.2. 
Assessment of Intensity/Severity ................................................................................53 
10.3.3.3. 
Assessment of Causality .............................................................................................53 
10.3.3.4. 
Follow-up of AEs and SAEs .......................................................................................54 
10.3.4. 
Reporting of SAEs ......................................................................................................54 
10.3.4.1. 
SAE Reporting to Ironwood Global Patient Safety via an Electronic Data 
Collection Tool ...........................................................................................................54 
10.3.4.2. 
SAE Reporting to Ironwood Global Patient Safety via Paper SAE Report 
Form ............................................................................................................................55 
10.4. 
Appendix 4: Contraceptive Guidance and Collection of Pregnancy 
Information .................................................................................................................56 
10.4.1. 
Definitions ..................................................................................................................56 
10.4.1.1. 
Women of Childbearing Potential (WOCBP) ............................................................56 
10.4.1.2. 
Women Not Considered Women of Childbearing Potential ......................................56 
10.4.2. 
Contraception Guidance .............................................................................................56 
10.4.2.1. 
Collection of Pregnancy Information .........................................................................57 
10.5. 
Appendix 5: Abbreviations .........................................................................................58 
10.6. 
Appendix 6: Protocol Signatures ................................................................................61 
10.6.1. 
Sponsor Signature .......................................................................................................61 
10.6.2. 
Investigator Signature .................................................................................................62 
11. 
REFERENCES ...........................................................................................................63 


Protocol 
 
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LIST OF TABLES 
Table 1: 
Schedule of Activities .................................................................................................11 
Table 2: 
Protocol-required Safety Laboratory Assessments .....................................................49 
Table 3: 
List of Abbreviations ..................................................................................................58 
 
LIST OF FIGURES 
Figure 1: 
Schematic of Study Design .........................................................................................10 
 


Protocol 
 
C3300-102 
 
8 
 
 
  
 
1. 
PROTOCOL SUMMARY 
1.1. 
Synopsis 
Protocol Title 
A Phase 1 Placebo-controlled Study of the Safety and Tolerability of Rectally Administered, 
Multiple-ascending Doses of IW-3300 in Healthy Subjects 
Short Title 
A Phase 1 Study of Multiple-ascending Doses of IW-3300 in Healthy Subjects 
Rationale 
The aim of this study is to assess the effects of IW‑3300 administered rectally, once-daily for 
7 days in healthy subjects. IW‑3300 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. Preclinical models provide preliminary evidence which suggests 
that 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. This decreases 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) Because of these analgesic effects, IW‑3300 is being 
developed as a rectally-administered therapeutic for the treatment of interstitial cystitis/bladder 
pain syndrome (IC/BPS), a chronic and debilitating condition for which there are no safe, 
effective treatment options. Data from this current study will be used to enable a Phase 2 safety, 
tolerability, and efficacy study of IW‑3300 in subjects with IC/BPS. More information on the 
pharmacology and safety of IW‑3300 is provided in Section 2. 
Objectives and Endpoints 
Objective 
Endpoints 
Primary 
To assess the safety and tolerability of 7-day 
dosing with multiple-ascending doses of IW‑3300 
administered rectally via enema in healthy 
subjects 
Incidence of treatment-emergent adverse events 
(TEAEs) 
Incidence of treatment-emergent serious adverse 
events (TESAEs) 
Exploratory 
To summarize the pharmacokinetics (PK) of 
7‑day dosing with IW‑3300 administered rectally 
via enema in healthy subjects 
PK parameters (IW‑3300 and metabolites in 
plasma) 
To explore potential changes to the microbiome 
with 7‑day dosing with IW‑3300 administered 
rectally via enema in healthy subjects 
Change in 16s RNA based analysis of gut 
microbiome composition at the genus level from 
baseline to 7 days post IW‑3300 administration 


Protocol 
 
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Overall Design 
This is a Phase 1, single-center, randomized, double-blind, placebo-controlled, multiple-
ascending-dose study of IW‑3300 administered rectally as a low-volume enema in healthy adult 
subjects. This study will assess the effect of IW‑3300 on safety, tolerability, and PK, and also 
explore the potential effect of IW‑3300 on gut microbiome composition. 
Subjects in 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). 
A Dose Escalation Committee will conduct blinded reviews of all the safety/tolerability 
parameters of the IW‑3300 100‑μg dosing cohort through Discharge (Day 8) in order to make a 
decision regarding dose escalation to the next IW‑3300 dosing cohort. The Dose Escalation 
Committee will include sponsor and contract research organization (CRO) representatives. 
Disclosure Statement 
This is a multiple-ascending-dose safety and tolerability study with 4 possible treatments 
(placebo, 2 dose levels of IW‑3300, and an optional 3rd dose level of IW‑3300) that is subject‑ 
and investigator‑blinded. 
Number of Subjects 
A total of up to 27 subjects will be randomized in the study (2 cohorts of 9 subjects each, and an 
optional 3rd cohort of 9 additional subjects). Within each cohort, 6 subjects will be randomized 
to IW‑3300 and 3 subjects will be randomized to placebo. 
Intervention Groups and Duration 
The study will evaluate multiple-ascending doses of IW‑3300 in a double-blind manner. The 
9 subjects within each cohort will be randomized to receive IW‑3300 (6 subjects) or placebo 
(3 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; Table 1] for additional details regarding dosing 
instructions) once daily for 7 days. The planned cohorts are: 
• Cohort 1: IW‑3300 100 μg or matching placebo rectal dose once daily 
• Cohort 2: IW‑3300 300 μg or matching placebo rectal dose once daily 
• Cohort 3 (optional): IW‑3300 >100 μg but <300 μg or matching placebo rectal dose 
once daily (decision to enroll this cohort will be determined after safety reviews of 
previous cohorts, in the case of intolerability at 300 μg but tolerability at 100 μg) 
Dosing in Cohort 2 and optional Cohort 3 will only proceed following a review of safety data 
from the previous 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 7 days; subjects will be followed in the Phase 1 clinical research unit 
(CRU) for the duration of dosing, until at least 24 hours after the last dose of study drug and 


Protocol 
 
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10 
 
 
  
 
contacted by phone for follow-up approximately 2 weeks after the last dose. Total subject 
participation will be 29 to 57 days, including the Screening, Clinic, and Follow-up Periods. 
Data Monitoring Committee: No 
The study will utilize a Dose Escalation Committee to review safety data for Cohort 1 in order to 
make decisions about dose escalation to Cohort 2. An optional cohort, Cohort 3, may be enrolled 
based on a review of safety data from Cohorts 1 and 2. 
1.2. 
Schema 
Figure 1: 
Schematic of Study Design 
Dose
Escalation
Screening
Visit
(Day -36
to Day -8)
Check-in
(Day -1)
Discharge
(Day 8)
Follow-up
Phone Call
(Day 21 +3)
Dosing
(Day 1)
7 to 35 days
9 days
13 days
Screening Period
Clinic Period
Follow-up Period
Dose Escalation
Review
Cohort 2
Cohort 1


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1.3. 
Schedule of Activities 
Table 1: 
Schedule of Activities 
Study Period→ 
Screening 
Period 
Clinic Period 
Follow-up 
Period 
Study 
Procedure↓ 
Visit/Day→ 
Screening 
Visit 
Day ‑36 to 
Day ‑8 
Check-in 
Day ‑1 
Dosing 
Day 1 
Days 2 
through 6 
Day 7 
Discharge 
Day 8a/ 
ET 
Follow-up 
Phone Call 
Day 21 
(+3 days) 
Informed consent 
X 
 
 
 
 
 
 
Eligibility criteria 
X 
X 
 
 
 
 
 
Medical history 
X 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
Body weight & heightb 
X 
 
Pre 
 
 
X 
 
Urine drug & alcohol screen 
X 
X 
 
 
 
 
 
Physical examinationc 
X 
X 
Pre 
Pre 
Pre 
X 
 
Rectal examinationd 
X 
X 
Pre 
Pre 
Pre 
X 
 
Vital signse 
X 
X 
Post: 0.5, 2, 4, 
and 8h (±10m) 
Days 3 
through 6 
Pre: 0 (≤25m) 
Post: 0.5, 2, 4, 
and 8h (±10m) 
 
 
Orthostatic blood pressure and 
pulsef 
 
 
Pre: 0 (≤25m) 
Post: 1 and 6h 
(±20m) 
Day 2 only 
Pre: 0 (≤25m) 
Pre: 0 (≤25m) 
Post: 1 and 6h 
(±20m) 
24h post-
Day 7 dose 
(±20m) 
 
Triplicate 12-lead ECGg 
X 
X 
Pre: 1h (±30m) 
Post: 2h (±30m) 
Pre: 1h 
(±30m) 
Post: 2h 
(±30m) 
Pre: 1h 
(±30m) 
Post: 2h 
(±30m) 
 
 


Protocol 
 
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Study Period→ 
Screening 
Period 
Clinic Period 
Follow-up 
Period 
Study 
Procedure↓ 
Visit/Day→ 
Screening 
Visit 
Day ‑36 to 
Day ‑8 
Check-in 
Day ‑1 
Dosing 
Day 1 
Days 2 
through 6 
Day 7 
Discharge 
Day 8a/ 
ET 
Follow-up 
Phone Call 
Day 21 
(+3 days) 
Clinical chemistry, hematology, 
urinalysis 
X 
X 
Pre: 0 (≤15m, 
≤30m for UA) 
Post: 6h (±30m) 
Day 4 only 
Pre: 0 (≤15m, 
≤30m for UA) 
 
X 
 
ESR and CRP blood drawh 
 
X 
 
 
 
X 
 
FSH 
X 
 
 
 
 
 
 
HCGi 
X 
X 
 
 
 
 
 
SARS‑CoV‑2 testing 
X 
X 
 
 
 
 
 
HIV & hepatitis panel 
X 
 
 
 
 
 
 
BBMD training 
X 
 
 
 
 
 
 
BBMD dispensed 
X 
X 
 
 
 
 
 
Daily BBMD completionj 
Day ‑8 through 
Day ‑2 
X 
X 
X 
X 
X 
 
BBMD collection and reviewk 
 
X 
 
 
 
X 
 
Concomitant medications 
X 
X 
X 
X 
X 
X 
X 
AE monitoring 
X 
X 
X 
X 
X 
X 
X 
Subject confinement to clinicl 
 
X 
X 
X 
X 
X 
 
Start of predose fastm 
 
 
Pre: ≥6h 
Pre: ≥6h 
Pre: ≥6h 
 
 
Randomization 
 
 
X 
 
 
 
 




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f 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 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 before blood draws. 
g 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. 
h Elevated inflammatory markers (ESR, CRP), and/or relevant symptoms (eg, rectal pain, hematochezia) may be suggestive of colonic inflammation; subjects 
with these signs or symptoms should undergo recto-sigmoidoscopy. If this is negative, a colonoscopy may be performed after a discussion between the 
investigator and the Sponsor. 
i HCG at Screening will be a blood-based test. HCG at Check-in/Day ‑1 will be a urine-based test. 
j 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 ‑9, CRU staff will 
call subjects on Day ‑9 to remind them to begin recording their BMs in the BBMD the following day, answer any questions, and provide additional BM pages 
via email, if needed. Bladder information in the BBMD will be collected predose on each dosing day, with a 24‑hour recall period at each timepoint. 
k 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 ‑8 through ‑2) and no more than 3 bowel movements per day to be eligible for the study; CRU staff will collect the subject’s Clinic Period 
BBMD at Discharge. 
l Subjects will remain at the CRU from Check-in (Day ‑1) through Discharge (Day 8). 
m Subjects will fast for at least 6 hours prior to dosing on Days 1 through 7 and for at least 1 hour following dosing. During these times, water is permitted. 
n Study drug (IW‑3300 or placebo low-volume enema) will be administered rectally once daily, after a fast of at least 6 hours; water is permitted. 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. Study drug will be dispensed in capped syringes with a separately packaged colon tip applicator. The CRU dosing staff will 
remove the cap from the syringe, attach the colon tip applicator, prime the syringe and applicator to 20 mL, and slowly administer by inserting the contents of 
the syringe into the rectum. After the full dose has been administered, 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). 
o 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. 
p At Check-in/Day ‑1, the subject will bring in a stool sample using the sample collection kit issued by the site. Between Day 7 (postdose) and Day 8 
(predischarge), the first stool passed will be collected. If no stool is passed during this time, the subject will be discharged with a stool collection kit and asked 
to return the sample to the CRU. 
q The predose hemoccult testing may be performed using a stool sample obtained any time from Check-in (Day ‑1) to predose. If the subject does not produce a 
stool sample, stool that is present on the Day 1 (predose) digital rectal examination may be used. The Discharge (Day 8) hemoccult testing may be performed 
using a stool sample obtained any time from postdose on Day 7 to Discharge (Day 8). If the subject does not produce a stool sample, stool that is present on the 
Discharge (Day 8) digital rectal examination may be used. 
r 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 
 
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2. 
INTRODUCTION 
IW‑3300 is being developed for the treatment of bladder pain associated with interstitial 
cystitis/bladder pain syndrome (IC/BPS). 
2.1. 
Study Rationale 
This clinical study is the second clinical study designed as a multiple-ascending-dose, safety, and 
tolerability study with IW‑3300. IW‑3300 will be administered rectally as a low-volume (20 mL) 
enema; this route of administration was selected to target the effects of IW‑3300 to the afferent 
nerve fibers in the colonic wall to reduce visceral pain in the pelvic region while minimizing the 
risk of diarrhea. Dosing for rectal administration will be initiated at 100 μg and will not go 
higher than 300 μg, which is approximately 80-fold lower than the human-equivalent dose 
[HED] based on body weight conversion of the lowest no adverse effect level (NOAEL) 
(24,000 μg) identified in nonclinical toxicology studies. 
2.2. 
Background 
IW‑3300 is being developed for the treatment of bladder pain associated with IC/BPS. IW‑3300 
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. 
Preclinical models provide preliminary evidence which suggests that 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. This decreases 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 
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 IW‑3300 may be useful clinically for the treatment of 
bladder pain associated with IC/BPS. 
IW‑3300 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. IW‑3300 (3 μg/kg/day), administered either 
intracolonically or orally to female rats, reduced endometriosis-induced mechanical hind paw 
allodynia. When administered intracolonically (1, 3, or 10 μg/kg/day), IW‑3300 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, IW‑3300 (3 μg/kg/day) administered intracolonically reversed colorectal 
and bladder hypersensitivity. IW‑3300 (3 μg/kg/day) administered intrarectally reduced vaginal 




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2.3. 
Benefit/Risk Assessment 
Available data with IW‑3300 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 
IW‑3300 is being administered intrarectally as a low-volume enema. More detailed information 
about the known and expected benefits and potential risks of IW‑3300 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 8) 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 bowel movement (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 
[Bristol Stool Form Scale {BSFS} score of 7] 
resulting in a limiting of self-care activities) or 
dehydration (hospitalization indicated); or 
≥3 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). 


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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 an early phase 
study with IW‑3300 and, 
as such, limited clinical 
data are available. 
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 8) 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 drug; ≥1 subject with a Grade 3 
(severe or medically significant) or higher AE 
considered related to study drug; or ≥3 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 (20 ml) enema used in 
this study is expected to be a lower risk than 
higher volume enemas. Nonclinical toxicology 
studies of IW‑3300 have shown no signs of local 
trauma following intrarectal administration. 
Study C3300‑101, a single-ascending dose 
clinical study testing IW‑3300 doses up to 
2500 μg in healthy subjects is currently ongoing. 
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 access to medical evaluations/assessments associated 
with study procedures (eg, physical examinations, ECGs, laboratory evaluations, SARS‑CoV‑2 
[severe acute respiratory syndrome coronavirus 2; COVID‑19] diagnostic testing). 
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 IW‑3300 are justified by the anticipated benefits 
to patients with IC/BPS. 


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3. 
OBJECTIVES AND ENDPOINTS 
3.1. 
Objectives 
3.1.1. 
Primary Objective 
To assess the safety and tolerability of 7-day dosing with multiple-ascending doses of IW‑3300 
administered rectally via enema in healthy subjects 
3.1.2. 
Exploratory Objectives 
The exploratory objectives are: 
• To summarize the PK of 7‑day dosing with IW‑3300 administered rectally via enema 
in healthy subjects 
• To explore potential changes to the microbiome with 7‑day dosing with IW‑3300 
administered rectally via enema in healthy subjects 
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 
The exploratory endpoints are: 
• PK parameters (IW‑3300 and metabolites in plasma) 
• Change in 16s RNA based analysis of gut microbiome composition at the genus level 
from baseline to 7 days post IW‑3300 administration 


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4. 
STUDY DESIGN 
4.1. 
Overall Design 
This is a Phase 1, single-center, randomized, double-blind, placebo-controlled, multiple-
ascending-dose study of IW‑3300 administered rectally, once-daily, for 7 days as a low-volume 
enema in healthy adult subjects. This study will assess the effect of IW‑3300 on safety, 
tolerability, and PK, and also explore the potential effect of IW‑3300 on gut microbiome 
composition. 
Subjects in each dosing 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). Treatment duration will be 7 days; subjects will 
be followed in the Phase 1 clinical research unit (CRU) for the duration of dosing, until at least 
24 hours after the last dose of study drug and contacted by phone for follow-up approximately 
14 days after the last dose. Total subject participation will be 29 to 57 days, including the 
Screening, Clinic, and Follow-up Periods. 
The study will evaluate multiple-ascending doses of IW‑3300 in a double-blind manner. The 
9 subjects within each cohort will be randomized to receive IW‑3300 (6 subjects) or placebo 
(3 subjects), administered rectally, once-daily for 7 days (as a low-volume [20 mL] enema) 
following a fast of at least 6 hours (refer to the SoA [Table 1] for additional details regarding 
dosing instructions). The planned cohorts are: 
• Cohort 1: IW‑3300 100 μg or matching placebo rectal dose once daily 
• Cohort 2: IW‑3300 300 μg or matching placebo rectal dose once daily 
• Cohort 3 (optional): IW‑3300 >100 μg but <300 μg or matching placebo rectal dose 
once daily (decision to enroll this cohort will be determined after safety reviews of 
previous cohorts, in the case of intolerability at 300 μg but tolerability at 100 μg) 
A Dose Escalation Committee will conduct blinded reviews of all the safety/tolerability 
parameters of the IW‑3300 100-μg dosing cohort through Discharge (Day 8) in order to make a 
decision regarding dose escalation to the next IW‑3300 dosing cohort. The Dose Escalation 
Committee will include sponsor and contract research organization (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 ‑36 to Day ‑8) and will last 7 to 35 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 subjects will receive the BM portion of 
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 bowel 
habits. Subjects will complete the BM portion of the BBMD daily only for the 7 days before 


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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). 
4.1.1.2. 
Clinic Period 
The Clinic Period will begin at Check-in to the Phase 1 CRU on Day ‑1 (the day before dosing), 
and will end at the time of Discharge on Day 8 (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 2:1 ratio to receive IW‑3300 or 
matching placebo after a fast of at least 6 hours. 
The assessments conducted during the Clinic Period are shown in the SOA (Table 1). Daily 
safety assessments will be performed. Subjects will continue to enter their BM information into 
the BBMD on an event-driven basis. The questions related to bladder/urinary symptoms in the 
BBMD will be collected predose on each dosing day, with a 24-hour recall period at each 
timepoint to ascertain any undesirable effects on the bladder or potential urinary symptoms. 
Blood samples for PK assessments will be collected at specified times predose (blood) and 
postdose (blood and stool). Subjects will be confined to the CRU from Check-in (Day ‑1) 
through Discharge (Day 8), which will occur after completion of the assessments (at least 
24 hours after the last 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 8 and will last until Day 21 (+3). On Day 21 (+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 
effects of IW‑3300 administered once daily for 7 days 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. 
To ensure a thorough assessment of safety, subjects will be confined to the CRU for the duration 
of the Clinic Period, from Check-in (Day ‑1) through until Discharge (Day 8), 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 on 
Day 21 (+3), conducted 14 days after the last dose, for safety follow-up; at the discretion of the 
investigator, subjects may be requested to return to the CRU for their follow-up contact. 
To evaluate any effects of IW‑3300 on bowel and bladder function, subjects will complete the 
BM portion of the BBMD on 7 consecutive days during the Screening Period to establish a 




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5. 
STUDY POPULATION 
Prospective approval of protocol deviations to recruitment and enrollment criteria, also known as 
protocol waivers or exemptions, is not permitted. 
5.1. 
Inclusion Criteria 
Subjects are eligible to be included in the study only if all of the following criteria apply: 
Age 
1. Subject is 18 to 60 years of age, inclusive, at the time of signing the ICF. 
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 [eg, incontinence, overactive bladder, prostatitis, urinary tract 
infection]). 
3. Subject has normal formed bowel movements (≥3 per week and ≤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 21. 
5. If subject is ≥45 years of age, subject is compliant with colorectal cancer screening 
guidelines according to the American College of Gastroenterology (ACG) Clinical 
Guidelines: Colorectal Cancer Screening 2021. (8) 
Weight 
6. Subject’s body mass index (BMI) is within the range 18.5 to 35.0 kg/m2 (inclusive) at the 
Screening Visit. 
Sex 
7. Male and female subjects 
a. Female subject is of nonchildbearing potential (ie, women who are postmenopausal 
[greater than 12 months without menses] or who have had a procedure such as a 
bilateral oophorectomy, bilateral salpingectomy, or hysterectomy). 
b. Subject is willing to abstain from sexual intercourse during the period of confinement 
in the CRU. 


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c. Male subject and female partner(s) are willing to use a 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 21. 
Informed Consent 
8. Subject is 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: 
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, active 
proctitis, or presence or history of ulcerative colitis or Crohn's disease. 
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 21. 
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 8. (Note: Acetaminophen may be 
used at the investigator’s discretion, in consultation with the sponsor, as per Section 6.5.) 
7. Subject has used vitamins, dietary, or 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 8. 
8. Subject has received a licensed vaccine during the 14 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 21). 
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 any surgery involving the 


Protocol 
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25 
GI tract or anal canal (with the exception of endoscopic procedures, appendectomy, and 
cholecystectomy). 
Prior/Concurrent Clinical Study Experience 
11. Subject has received any investigational drug or vaccine 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 (up to
the Follow-up Phone Call on Day 21).
Diagnostic Assessments 
12. Subject has a 12-lead ECG demonstrating severe bradycardia (heart rate <40 beats per
minute) or average QTcF ≥450 msec for males and ≥470 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 SARS‑CoV‑2 (COVID‑19) at the
Screening Visit or Check-in on Day ‑1, or was diagnosed with COVID‑19 or had contact
with a person with COVID‑19 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.
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 21.
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.


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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 as described in Section 6. During this time, water is permitted. 
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 on Day 21 as 
described in Section 5.2. 
2. Subjects are permitted to ingest caffeine- or xanthine-containing products (eg, coffee, tea, 
cola drinks, and chocolate), but should limit intake to ≤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, 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 sign collection during the Clinic Period, positional and activity restrictions at 
each timepoint are noted in the SoA (Table 1) and Section 8.2.2. 
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. 


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6. 
STUDY INTERVENTION 
Study intervention (also referred to as “study drug”) is defined as any investigational 
intervention(s), and may include marketed product(s) or placebo, intended to be administered to 
a study subject according to the study protocol. 
The study will evaluate multiple-ascending doses of IW‑3300 in a double-blind manner. The 
9 subjects within each cohort will be randomized to receive multiple doses of IW‑3300 
(6 subjects) or placebo (3 subjects), administered rectally (as a low-volume [20 mL] enema) 
following a fast of at least 6 hours. The planned cohorts are: 
• Cohort 1: IW‑3300 100 μg or matching placebo rectal dose once daily 
• Cohort 2: IW‑3300 300 μg or matching placebo rectal dose once daily 
• Cohort 3 (optional): IW‑3300 >100 μg but <300 μg or matching placebo rectal dose 
once daily (decision to enroll this cohort will be determined after safety reviews of 
previous cohorts, in the case of intolerability at 300 μg but tolerability at 100 μg) 
Dosing in Cohort 2 and optional Cohort 3 will only proceed following a review of safety data 
from the previous cohort(s), including 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 7 days; subjects will be followed in the Phase 1 CRU for the duration 
of dosing, until at least 24 hours after the last dose of study drug and contacted by phone for 
follow-up approximately 2 weeks after the last dose. Total subject participation will be 29 to 
57 days, including the Screening, Clinic, and Follow-up Periods. 




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moisture and light and stored between ‑25°C to ‑15°C. Prior to compounding, IW‑3300 drug 
substance will be thawed for a minimum of 30 minutes prior to use. 
The investigational drug product may be compounded on the day of administration and stored at 
room temperature prior to dosing (continuous mixing not required). If the investigational drug 
product is stored for more than 24 hours, it must be stored refrigerated (2 C to 8°C) for no longer 
than the product shelf life. Refrigerated drug product must be equilibrated to room temperature 
for at least 30 minutes prior to dosing. Compounding and storage of the drug product 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). 
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 27 subjects (up to 3 cohorts of 9 subjects 
each) will be randomized in a 2:1 ratio to receive IW‑3300 (N=6) or placebo (N=3), for each 
cohort, as detailed in Section 6.1. 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. The randomized treatment allocation for each subject will be contained in a sealed, opaque 
envelope opened at the time of the subject’s randomization. 
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. 


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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 8) in order to make decisions regarding subsequent dosing. 
Specific designated personnel in the Ironwood Global Patient Safety Group 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. 
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 in the next paragraph) 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. 
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 21; 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 8; and licensed vaccines during the 
14 days before Check-in (Day ‑1) until after completion of the Follow-up Phone Call on Day 21. 
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 


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• 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 subjects 
6.6. 
Dose Modification 
The decision to proceed to the 300‑μg dose level of IW‑3300 (Cohort 2) will be made by the 
Dose Escalation Committee, based on blinded safety and tolerability data obtained through 
Discharge on Day 8 in all subjects at the 100‑μg dose level (Cohort 1). The decision to proceed 
with an optional 3rd cohort will be made by the Dose Escalation Committee based on blinded 
safety and tolerability data for both Cohorts 1 and 2. The dose will be IW‑3300 >100 μg but 
<300 μg. 
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. 


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7.
DISCONTINUATION OF STUDY INTERVENTION AND
SUBJECT DISCONTINUATION/WITHDRAWAL
7.1. 
Discontinuation of Study Intervention
It may be necessary for a subject to permanently discontinue study drug. A subject will be 
discontinued from study drug in the event of: 
•
A confirmed pregnancy
•
A SAE considered by the investigator or Sponsor to be related to study drug
Dosing of study drug for an individual subject may be suspended temporarily as detailed in 
Section 7.1.1; if the subject is unable to resume dosing, they should follow the instructions for 
permanent discontinuation below. 
If study drug is permanently discontinued, then the subject will have the safety evaluations 
scheduled for Discharge (Day 8) performed. 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 study drug 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 may be temporarily suspended for either dose level if any of the following occur within a 
cohort: 
•
≥1 subject with an SAE considered related to study drug
•
≥1 subject with an National Cancer Institute Common Terminology Criteria for
Adverse Events (NCI-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)
•
≥3 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 ratio is considered unacceptable 
and the effect 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 IW‑3300 that may
preclude further dosing


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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 ET visit should be 
conducted, as shown in the SoA (Table 1). See the 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. 
• 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. 


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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 
(Table 1), 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 subjects. 
8.2. 
Safety Assessments 
Planned timepoints for all safety assessments are provided in the SoA (Table 1). 
8.2.1. 
Physical Examinations 
Physical examinations will be performed as outlined in the SoA (Table 1), 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, gastrointestinal, musculoskeletal, neurological, and 
dermatological systems. 
• Rectal examination at all timepoints should include a digital rectal examination and 
visual inspection of the perianal area for redness or irritation. If significant redness, 
rectal pain, fever, bleeding, or mucus is observed, anoscopy may be performed as part 
of the rectal examination. 
• On Days 1 through 7, the physical examination will be symptom-directed and 
conducted prior to study drug administration. 
• Height and weight will also be measured and recorded at the timepoints noted in the 
SoA (Table 1). 
• 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. 


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8.2.2. 
Vital Signs 
Vital signs will be performed as outlined in the SoA (Table 1). 
• Vital signs will include oral temperature, blood pressure, and pulse rate. 
• 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. 
• In a semisupine position, blood pressure and pulse will be obtained at the 0.5-hour 
postdose timepoint on Days 1 and 7 (consistent with the position required following 
dosing). 
• Blood pressure and pulse at the 2-, 4-, and 8-hour postdose timepoints on Days 1 and 
7, as well as predose on Days 3 through 6, 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, Day 1 predose and 1- and 6-hours postdose, Day 2 
predose, Day 7 predose and 1- and 6-hours postdose, and Day 8 [24‑hours post-Day 7 
dose]), orthostatic blood pressure and pulse measurements will be obtained. Subjects 
must lie quietly for ≥5 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 signs measurements will be obtained 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 of 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 ECGs 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. 


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• 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 
eCRF. 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 last dose of study drug should be 
repeated until the values return to normal or baseline or are no longer considered 
clinically significant by the investigator or medical monitor. 
− If such values do not return to normal/baseline within a period of time judged 
reasonable by the investigator, the etiology should be identified and the sponsor 
notified. 
− 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. 
8.2.5. 
Bladder and Bowel Movement Diary 
The BBMD will be completed in accordance with the instructions in the SoA (Table 1). 
Subjects will enter BM-related information into a paper diary, the BBMD, on an event-driven 
basis (ie, following each BM). The BM-related information will include the day and time of BMs 
and a report of stool consistency for each BM using the BSFS (8); 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) 
8.3. 
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 


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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. 
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. 




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8.4. 
Treatment of Overdose 
For this study, any dose that exceeds the dosage or frequency specified in the protocol 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 for the determination of IW‑3300 and metabolites in plasma 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. 
The following PK parameters will be calculated using a noncompartmental approach if a 
sufficient number of plasma samples exist above lower limit of quantification: 
• 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) 
Blood samples will be analyzed in a single batch after all subjects have completed the study. No 
individual drug concentration information will be shared until the study has been unblinded. 
8.6. 
Microbiome 
Gut microbiome will be assessed using stool samples collected in accordance with the SoA 
(Table 1). At Check-in/Day ‑1, the subject will bring in a stool sample using the sample 
collection kit issued by the site. Between Day 7 (postdose) through Day 8 (predischarge), the 
first stool passed will also be collected. If no stool is passed during this time, the subject will be 
discharged with a stool collection kit and asked to return the sample to the CRU. 
The stool collection kit is a tube that contains DNA stabilizer. An aliquot of stool (approximately 
1 teaspoonful) will be added to the tube and frozen for analysis. 


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8.7. 
Pharmacodynamics 
Pharmacodynamics are not evaluated in this study. 
8.8. 
Genetics 
Genetics are not evaluated in this study; no samples will be collected for genetics research. 
8.9. 
Biomarkers 
Biomarkers are not evaluated in this study; no samples will be collected for biomarker research. 
8.10. 
Immunogenicity Assessments 
Immunogenicity is not evaluated in this study; no samples will be collected to evaluate 
antibodies to IW‑3300. 
8.11. 
Medical Resource Utilization and Health Economics 
Medical resource utilization and health economics parameters are not evaluated in this study. 


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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 (9 subjects) is considered sufficient for evaluation of safety, tolerability, PK, and 
microbiome. 
9.3. 
Populations for Analyses 
The following populations are defined: 
Population 
Description 
Screened Set 
The Screened Set consists of all subjects who have signed 
informed consent. 
Intent-to-treat Set 
The Intent-to-treat Set consists of all subjects in the Screened 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. 
Microbiome Analysis Set 
The Microbiome Analysis Set consists of all subjects in the Safety 
Analysis Set who have at least 1 postdose microbiome 
assessment. 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 


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proportion of subjects in each category. Furthermore, PK data will be summarized using 
descriptive statistics, geometric mean, and coefficient of variation (CV%) 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 post last dose 
will be summarized by system organ class, preferred term, and treatment (or cohort). 
9.4.3. 
Secondary Endpoint Analysis 
Not applicable to this study 
9.4.4. 
Exploratory Endpoints Analyses 
PK analyses will be based on the PK Analysis Set. 
Plasma concentrations of IW‑3300 and metabolites will be summarized by treatment (or cohort) 
at each assessment timepoint; additionally, a listing of plasma concentrations will be provided. 
PK parameters (listed in Section 8.5) will also be calculated for each subject and summarized by 
treatment, if systemic levels of IW‑3300 and/or metabolites 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). Otherwise, the levels of IW‑3300 and/or metabolites 
will not be included for the analysis. 
The analysis of the microbiome at the genus level will be based on the Microbiome Analysis Set. 
The relative abundance of different phyla, class, order, family, and genera will be constructed 
and summarized by treatment (or cohort) at baseline and following 7 days of study drug 
administration using descriptive statistics. Changes from baseline will be calculated. 
9.4.5. 
Safety Analyses 
All safety analyses, including the BBMD analyses, will be based 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. 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, 
inflammatory marker, and urinalysis parameter 
• Vital signs (ie, pulse rate, systolic and diastolic blood pressure, oral temperature, and 
body weight) 


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• ECG parameters (ie, heart rate and measures of PR, QRS, QT, and QTcF intervals) 
− Additionally, the overall 12-lead ECG interpretation (normal, abnormal not 
clinically significant, or abnormal clinically significant) will be summarized. 
For the BBMD parameters, descriptive statistics at each assessment timepoint will be presented 
by treatment (or cohort) for each study period (Screening Period and Clinic Period): BM 
frequency, stool consistency, fecal incontinence, urinary frequency, urinary urgency, nocturia, 
and bladder/pelvic pain. 
BM-related baseline 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. Bladder-related baseline values will be derived from the BBMD data 
collected predose on Day 1 (with a 24-hour recall). The Clinic Period BM- and bladder-related 
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 8 (Discharge). 
9.4.6. 
Other Analysis/Analyses 
Not applicable to this study 
9.5. 
Interim Analyses 
No interim analysis is planned for the study. Interim safety reviews are detailed in Section 6.6. 
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. 


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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. 


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Subjects must be informed that their participation is voluntary. Subjects or their legally 
authorized representative will be required to sign a statement of informed consent that meets the 
requirements of 21 CFR 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. 


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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. 


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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. 


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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 SoA (Table 1). 
•
The laboratory tests are detailed in Table 2.
•
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 the Discharge (Day 8)/ET Visit. The decision to repeat
a laboratory test would be based on the investigator's clinical judgment.




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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. 


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− 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. 


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• 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 
not be immediately life‑threatening or result in death or hospitalization but 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. 


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• 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 one of the following categories, according to the 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. 
ADLs are defined as follows: Instrumental ADLs refer to preparing meals, shopping for 
groceries or clothes, using the telephone, managing money, etc. Self-care ADLs 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 


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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. 


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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. 


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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 21. 
Women of childbearing potential are excluded from the study. 


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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 ICF 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. 


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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 
CRO 
contract research organization 
CRP 
c-reactive protein 
CRU 
clinical research unit 
CV% 
coefficient of variation 
DMC 
data monitoring committee 
DR 
delayed release 
eCRF 
electronic case report form 
EDC 
electronic data capture 
ESR 
erythrocyte sedimentation rate 
ET 
early termination 
FSH 
follicle-stimulating hormone 
GC-C 
guanylate cyclase C 


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Abbreviation 
Definition 
GI 
gastrointestinal 
HBsAg 
hepatitis B surface antigen  
HCV 
hepatitis C virus 
HCG 
human chorionic gonadotropin 
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 
IBS-C 
irritable bowel syndrome with constipation 
IBS-D 
irritable bowel syndrome with diarrhea 
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 
MCH 
mean corpuscular hemoglobin 
MCHC 
mean corpuscular hemoglobin concentration 
MCV 
mean corpuscular volume 
MedDRA 
Medical Dictionary for Regulatory Activities 
MRP 
multidrug resistance protein 
NCI-CTCAE 
National Cancer Institute Common Terminology Criteria for Adverse Events  
NIMP 
noninvestigational medical product 
NOAEL 
no-observed-adverse-effect level 
PK 
pharmacokinetic(s) 
PKGII 
protein kinase G type II 
RBC 
red blood cell 
SAE 
serious adverse event 
SAP 
statistical analysis plan 


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Abbreviation 
Definition 
SARS‑CoV‑2 
severe acute respiratory syndrome coronavirus 2 
SID 
subject identification 
SoA 
schedule of activities 
SUSAR 
suspected unexpected serious adverse reaction 
t1/2 
terminal elimination half-life 
TBD 
to be determined 
TEAE 
treatment-emergent adverse event 
TESAE 
treatment-emergent serious adverse event 
Tmax 
time to maximum plasma concentration 
UA 
urinalysis 
ULN 
upper limit of normal 
WBC 
white blood cell 
WOCBP 
woman/women of childbearing potential 


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10.6. 
Appendix 6: Protocol Signatures 
10.6.1. 
Sponsor Signature 
Protocol Title: 
A Phase 1 Placebo-controlled Study of the Safety and 
Tolerability of Rectally Administered, Multiple-ascending 
Doses of IW-3300 in Healthy Subjects 
Short Title:  
A Phase 1 Study of Multiple-ascending Doses of IW-3300 in 
Healthy Subjects 
Protocol Number:  
C3300-102 
Version Number: 
Version 1 
Final Date: 
14 February 2022 
This clinical study protocol was subject to critical review and has been approved by the sponsor. 
 
 
 
{Electronic signature page appended} 
 
 
 
 
Head of Clinical Development and Medical 
Scientific Affairs 
Ironwood Pharmaceuticals, Inc. 
 
Date 


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10.6.2. 
Investigator Signature 
Protocol Title:  
A Phase 1 Placebo-controlled Study of the Safety and 
Tolerability of Rectally Administered, Multiple-ascending 
Doses of IW-3300 in Healthy Subjects 
Short Title:  
A Phase 1 Study of Multiple-ascending Doses of IW-3300 in 
Healthy Subjects 
Protocol Number:  
C3300-102 
Version Number: 
Version 1 
Final Date:  
14 February 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: 
 


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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):1334‑46. 
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):2082‑6. 
4. 
Forte, LR. Uroguanylin and guanylin peptides: pharmacology and experimental 
therapeutics. Pharmacol Ther. 2004;104(2):137‑62. 
5. 
Schlossmann J, Feil R, Hofmann F. Insights into cGMP signalling derived from cGMP 
kinase knockout mice. Frontiers in Bioscience. 2005;10:1279‑89. 
6. 
Forte LR. Guanylin regulatory peptides: structures, biological activities mediated by 
cyclic GMP and pathobiology. Regul Pept. 1999;81(1‑3):25‑39. 
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):G93‑105. 
8. 
Lewis SJ, Heaton KW. Stool form scale as a useful guide to intestinal transit time. Scand 
J Gastroenterol. 1997;32(9):920‑4. 
 


The data and information in this file that pertain to my line function are 
truthful and accurate.
Signature Page for VV-CLIN-004845 
v1.0
Approval
23-Feb-2022 22:11:16 GMT+0000

SAP_001.pdf: 
Statistical Analysis Plan 
 
C3300-102 
 
1 
 
TITLE PAGE 
 
Study Title: 
A Phase 1 Placebo‑controlled Study of the Safety and 
Tolerability of Rectally Administered Multiple Ascending 
Doses of IW‑3300 in Healthy Subjects 
Protocol Number: 
C3300-102 
Version: 
1.0 
Date of Plan: 
20 July 2022 
Study Drug: 
IW‑3300 
Study Phase: 
1 
Sponsor: 
Ironwood Pharmaceuticals, Inc. 
100 Summer Street, Suite 2300 
Boston, MA 02110 USA 
(617)‑621‑7722 
 


Statistical Analysis Plan 
 
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TABLE OF CONTENTS 
1. 
ABBREVIATIONS ......................................................................................................5 
2. 
INTRODUCTION ........................................................................................................7 
3. 
OBJECTIVES, ESTIMAND(S), AND ENDPOINTS .................................................8 
3.1. 
Objectives .....................................................................................................................8 
3.1.1. 
Primary Objective .........................................................................................................8 
3.1.2. 
Exploratory Objectives .................................................................................................8 
3.2. 
Estimand(s) ...................................................................................................................8 
3.3. 
Endpoints ......................................................................................................................8 
3.3.1. 
Safety Endpoints ...........................................................................................................8 
3.3.2. 
Pharmacokinetic Endpoints ..........................................................................................8 
3.3.3. 
Microbiome Exploratory Endpoints .............................................................................8 
4. 
STUDY DESIGN .........................................................................................................9 
4.1. 
General Description ......................................................................................................9 
4.1.1. 
Number of Subjects ......................................................................................................9 
4.1.2. 
Intervention Groups and Duration ................................................................................9 
4.2. 
Discussion of Study Design, Including Choice of Control Group .............................11 
4.3. 
Method of Assigning Subjects to Treatment Groups .................................................11 
4.4. 
Blinding ......................................................................................................................11 
4.5. 
Sample Size and Power Considerations .....................................................................12 
5. 
STATISTICAL ANALYSIS SETS ............................................................................13 
5.1. 
Screened Set ................................................................................................................13 
5.2. 
ITT Set ........................................................................................................................13 
5.3. 
Safety Analysis Set .....................................................................................................13 
5.4. 
PK Analysis Set ..........................................................................................................13 
5.5. 
Microbiome Analysis Set ...........................................................................................13 
6. 
STUDY SUBJECTS ...................................................................................................14 
6.1. 
Disposition of Subjects ...............................................................................................14 
6.2. 
Demographic and Other Baseline Characteristics ......................................................14 
6.3. 
Medical and Surgical History .....................................................................................14 
6.4. 
Prior Medications, Therapies, Procedures ..................................................................14 
6.5. 
Concomitant Medications, Therapies, and Procedures ...............................................15 


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6.6. 
Exposure to Study Drug ..............................................................................................15 
6.7. 
Protocol Deviations ....................................................................................................15 
7. 
SAFETY ANALYSIS ................................................................................................16 
7.1. 
Adverse Events ...........................................................................................................16 
7.2. 
Clinical Laboratory Data ............................................................................................17 
7.3. 
Vital Signs ..................................................................................................................19 
7.4. 
Electrocardiogram .......................................................................................................20 
7.5. 
Bladder and Bowel Movement Diary (BBMD) ..........................................................21 
7.5.1. 
BBMD Analysis..........................................................................................................22 
7.6. 
Microbiome .................................................................................................................22 
8. 
PHARMACOKINETIC ANALYSIS .........................................................................23 
9. 
DATA MONITORING/REVIEW COMMITTEE .....................................................24 
10. 
REFERENCES ...........................................................................................................25 
11. 
APPENDICES ............................................................................................................26 
11.1. 
Data Handling Conventions ........................................................................................26 
11.1.1. 
General Data Reporting Conventions .........................................................................26 
11.1.2. 
Repeated or Unscheduled Assessments of Safety Parameters ...................................26 
11.1.3. 
Definition of Visit Windows ......................................................................................26 
11.1.4. 
Missing Date of Adverse Events ................................................................................26 
11.1.5. 
Missing Date of Prior or Concomitant Medications, Therapies, and 
Procedures ...................................................................................................................26 
11.1.6. 
Character Values of Clinical Laboratory Variables ....................................................26 
11.2. 
Analysis Software .......................................................................................................27 
11.3. 
Schedule of Activities .................................................................................................28 
11.4. 
Bladder and Bowel Movement Diary .........................................................................32 
 


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LIST OF TABLES 
Table 1: 
Laboratory Assessments .............................................................................................17 
Table 2: 
Criteria for Potentially Clinically Significant Laboratory Tests .................................18 
Table 3: 
Criteria for Potentially Clinically Significant Vital Signs ..........................................20 
Table 4: 
Criteria Associated with Orthostatic Hypotension .....................................................20 
Table 5: 
Criteria for Potentially Clinically Significant ECG Values ........................................21 
Table 6: 
Coding of Special Character Values for Clinical Laboratory Results ........................27 
Table 7: 
Schedule of Activities .................................................................................................28 
 
LIST OF FIGURES 
Figure 1: 
Schematic of Study Design .........................................................................................10 
 


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1. 
ABBREVIATIONS 
Abbreviation 
Term 
ADaM 
Analysis Data Model (a CDISC standard) 
AE 
adverse event 
BBMD 
bladder and bowel movement diary 
BM 
bowel movement 
BMI 
body mass index 
BSFS 
Bristol Stool Form Scale 
CDISC 
Clinical Data Interchange Standards Consortium 
cGMP 
cyclic guanosine monophosphate 
CI 
confidence interval 
CRO 
clinical research organization 
CRP 
C-reactive protein 
CRU 
clinical research unit 
CTCAE 
Common Terminology Criteria for Adverse Events 
DMC 
data monitoring committee 
ECG 
electrocardiogram 
eCRF 
electronic case report form 
ESR 
erythrocyte sedimentation rate 
GC‑C 
guanylate cyclase C 
IC/BPS 
interstitial cystitis/bladder pain syndrome 
ICF 
informed consent form 
IPD 
important protocol deviations 
ITT 
intention-to-treat 
LLN 
lower limit of normal 
MedDRA 
Medical Dictionary for Regulatory Activities 
MRP 
multidrug resistance protein 
PCS 
potentially clinically significant  
PD 
pharmacodynamic 
PK 
pharmacokinetic 
PT 
preferred term  
QTcF 
QT Interval corrected for heart rate using Fridericia’s formula 
SAE 
serious adverse event 


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Abbreviation 
Term 
SAP 
statistical analysis plan 
SD 
standard deviation 
SE 
standard error  
SI 
Système International 
SOC 
system organ class 
SDTM 
Study Data Tabulation Model (a CDISC standard) 
TEAE 
treatment-emergent adverse event 
TESAE 
treatment-emergent serious adverse event 
ULN 
upper limit of normal 


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2. 
INTRODUCTION 
Study C3300‑102 is a Phase 1, single-center, randomized, double-blind, placebo-controlled, 
multiple-ascending-dose study of IW‑3300 administered rectally as a low-volume enema in 
healthy adult subjects. This study will assess the effect of IW‑3300 on safety, tolerability, and 
PK, and also explore the potential effect of IW‑3300 on gut microbiome composition. 
IW‑3300 is being developed as a rectally-administered therapeutic for the treatment of interstitial 
cystitis/bladder pain syndrome (IC/BPS), a chronic and debilitating condition for which there are 
no safe, effective treatment options. IW‑3300 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 (MRP)4 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. 
This statistical analysis plan (SAP) was developed in accordance with ICH E9 guideline. All 
decisions regarding final analysis, as defined in this SAP document, will be made prior to 
database lock and unblinding of the study data. Further information can be found in the protocol. 
This SAP describes the statistical analysis methods for the analysis of safety and 
pharmacodynamic (PD) data. The statistical analysis methods for the pharmacokinetics (PK) 
assessments will be described in a PK analysis plan. The data are collected as described in the 
following documents: 
• Study protocol version 1 dated 14 February 2022 
• Electronic case report form (eCRF) version 1.0 dated 11 May 2022. 
The final SAP must be finalized, approved by the sponsor, Ironwood Pharmaceuticals, Inc. and 
formally archived before database is locked. Deviations from the final approved plan will be 
noted in the clinical study report. 


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3. 
OBJECTIVES, ESTIMAND(S), AND ENDPOINTS 
3.1. 
Objectives 
3.1.1. 
Primary Objective 
To assess the safety and tolerability of 7-day dosing with multiple-ascending doses of IW‑3300 
administered rectally via enema in healthy subjects. 
3.1.2. 
Exploratory Objectives 
• To summarize the pharmacokinetics (PK) of 7‑day dosing with IW‑3300 
administered rectally via enema in healthy subjects. 
• To explore potential changes to the microbiome with 7‑day dosing with IW‑3300 
administered rectally via enema in healthy subjects 
3.2. 
Estimand(s) 
Not applicable for this Phase 1 study. 
3.3. 
Endpoints 
3.3.1. 
Safety Endpoints 
• Incidence of treatment-emergent adverse events (TEAEs) 
• Incidence of treatment-emergent serious adverse events (TESAEs) 
3.3.2. 
Pharmacokinetic Endpoints 
• 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 within a dosing interval (AUC0-tau) 
• 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) 
3.3.3. 
Microbiome Exploratory Endpoints 
• Change in 16s RNA based analysis of gut microbiome composition at the genus level 
from baseline to 7 days post IW‑3300 administration.   


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4. 
STUDY DESIGN 
4.1. 
General Description 
This is a Phase 1, single-center, randomized, double-blind, placebo-controlled, multiple-
ascending-dose study of IW‑3300 administered rectally as a low-volume enema in healthy adult 
subjects. This study will assess the effect of IW‑3300 on safety, tolerability, and PK, and also 
explore the potential effect of IW‑3300 on gut microbiome composition. 
Subjects in 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). 
A Dose Escalation Committee will conduct blinded reviews of all the safety/tolerability 
parameters of the IW‑3300 100‑μg dosing cohort through Discharge (Day 8) in order to make a 
decision regarding dose escalation to the next IW‑3300 dosing cohort. The Dose Escalation 
Committee will include sponsor and contract research organization (CRO) representatives. 
4.1.1. 
Number of Subjects 
A total of up to 27 subjects will be randomized in the study (2 cohorts of 9 subjects each, and an 
optional 3rd cohort of 9 additional subjects). Within each cohort, 6 subjects will be randomized 
to IW‑3300 and 3 subjects will be randomized to placebo. 
4.1.2. 
Intervention Groups and Duration 
The study will evaluate multiple-ascending doses of IW‑3300 in a double-blind manner. The 
9 subjects within each cohort will be randomized to receive IW‑3300 (6 subjects) or placebo 
(3 subjects), administered rectally (as a low-volume [20 mL] enema) following a fast of at least 
6 hours (refer to the Schedule of Activities [Table 7] for additional details regarding dosing 
instructions) once daily for 7 days. The planned cohorts are: 
• Cohort 1: IW‑3300 100 μg or matching placebo rectal dose once daily 
• Cohort 2: IW‑3300 300 μg or matching placebo rectal dose once daily 
• Cohort 3 (optional): IW‑3300 >100 μg but <300 μg or matching placebo rectal dose 
once daily (decision to enroll this cohort will be determined after safety reviews of 
previous cohorts, in the case of intolerability at 300 μg but tolerability at 100 μg) 
Dosing in Cohort 2 and optional Cohort 3 will only proceed following a review of safety data 
from the previous 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 7 days; subjects will be followed in the Phase 1 clinical research unit 
(CRU) for the duration of dosing, until at least 24 hours after the last dose of study drug and 
contacted by phone for follow-up approximately 2 weeks after the last dose. Total subject 
participation will be 29 to 57 days, including the Screening, Clinic, and Follow-up Periods. 
The schematic of the study design is presented below (Figure 1). 


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Figure 1: 
Schematic of Study Design 
 
Note:  An optional cohort, Cohort 3, may be enrolled based on a review of safety data from Cohorts 1 and 2. 
Dose
Escalation
Screening
Visit
(Day -36
to Day -8)
Check-in
(Day -1)
Discharge
(Day 8)
Follow-up
Phone Call
(Day 21 +3)
Dosing
(Day 1)
7 to 35 days
9 days
13 days
Screening Period
Clinic Period
Follow-up Period
Dose Escalation
Review
Cohort 2
Cohort 1


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4.2. 
Discussion of Study Design, Including Choice of Control Group 
A randomized, double-blind, placebo-controlled study design was chosen to investigate the 
effects of IW‑3300 administered once daily for 7 days 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. 
To ensure a thorough assessment of safety, subjects will be confined to the CRU for the duration 
of the Clinic Period, from Check-in (Day ‑1) through until Discharge (Day 8), 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 on 
Day 21 (+3), conducted 14 days after the last dose, for safety follow-up; at the discretion of the 
investigator, subjects may be requested to return to the CRU for their follow-up contact. 
To evaluate any effects of IW‑3300 on bowel and bladder function, subjects will complete the 
bowel movement (BM) portion of the bladder and bowel movement diary (BBMD) on 
7 consecutive days during the Screening Period to establish a baseline for bowel function without 
study drug. Similarly, subjects will provide answers to the bladder/urinary-symptom-related 
questions in the BBMD predose on Day 1, with a 24‑hour recall, to establish a baseline without 
study drug. 
Because this is the first multiple-dose study with IW‑3300 in humans, cohorts will be enrolled 
sequentially, following a safety review of prior dosed cohort(s) by a Dose Escalation Committee 
(Protocol Section 6.6). In addition, stopping criteria have been established to ensure that dosing 
at the same or higher dose levels will stop should a safety signal be detected 
(Protocol Section 7.1.1). In this case, a lower dose of IW‑3300 could be used. 
4.3. 
Method of Assigning Subjects to Treatment Groups 
Randomization numbers encoding the subjects’ treatment assignments will be based on a 
randomization schedule that is computer-generated; the randomization schedule will be 
generated prior to the study, by an independent statistician from a CRO who is not otherwise 
associated with the study. 
4.4. 
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 8) in order to make decisions regarding subsequent dosing. 
Specific designated personnel in the Ironwood Global Patient Safety Group may be unblinded to 
the treatment assignment of individual subjects for regulatory reporting purposes. All other 


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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. 
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 in the next paragraph) 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. 
4.5. 
Sample Size and Power Considerations 
No statistical sample size determination process was performed. The sample size chosen for each 
cohort (9 subjects) is considered sufficient for evaluation of safety, tolerability, PK, and 
microbiome. 


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5. 
STATISTICAL ANALYSIS SETS 
5.1. 
Screened Set 
The Screened Set consists of all subjects who signed the informed consent. 
5.2. 
ITT Set 
The ITT Set consists of all subjects in the Screened 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. 
5.3. 
Safety Analysis Set 
The Safety Analysis Set consists of all subjects who received any amount of study drug. Analysis 
will be performed according to the treatment actually received regardless of the allocated 
treatment. 
5.4. 
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. 
5.5. 
Microbiome Analysis Set 
The Microbiome Analysis Set consists of all subjects in the Safety Analysis Set who have at least 
1 postdose microbiome assessment. Analysis will be performed according to the treatment 
actually received regardless of the allocated treatment. 


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6. 
STUDY SUBJECTS 
All subjects on placebo arm will be pooled from available cohorts, whenever appropriate, to 
conduct analyses. 
6.1. 
Disposition of Subjects 
The number and percentage of subjects who were included in the Screened, ITT, Safety or 
Microbiome analysis sets will be summarized by treatment group (or cohort) and overall, except 
for the Screened Set, which will only be summarized overall. 
Some subjects may withdraw from the study prior to completion of the Clinic Period, or may 
discontinue study drug before study day 7. The number and percentage of subjects who 
completed or who prematurely discontinued from the study or from study drug will be presented 
for each treatment group (or cohort) and overall for the Safety Analysis Set. Reasons for 
premature discontinuation as recorded either on the termination page of the eCRF or on the 
adverse event eCRF will be summarized (number and percentage) by treatment group (or cohort) 
and overall. 
6.2. 
Demographic and Other Baseline Characteristics 
Descriptive summaries of demographic and baseline characteristics will be presented by 
treatment group (or cohort) and overall, for the Safety Analysis Set. 
The following demographic characteristics will be summarized in the tables: age (years and by 
category), sex, ethnicity, race, weight, height, and BMI. 
6.3. 
Medical and Surgical History 
Medical and surgical history will be collected at the Screening Visit and will be coded using 
MedDRA Version 24.0. A data listing will be provided for the Safety Analysis Set. 
The medical history will be summarized by system organ class (SOC) and preferred term (PT) 
for each treatment group and overall, for the Safety Analysis Set. 
6.4. 
Prior Medications, Therapies, Procedures 
Prior medications will be coded using the WHO Drug Dictionary dated Sept 2021. Prior 
therapies and procedures will be coded using MedDRA Version 24.0. 
Prior medications, therapies, and procedures are defined as that with a start date prior to the date 
of the first dose of study drug. 
The prior medication, therapy, and procedure usage will be summarized by the number and 
percentage of subjects in each treatment group and overall, within each preferred term for the 
Safety Analysis Set. Multiple uses of the same medication, therapy, or procedure by a subject in 
the same category will be counted only once. 
All prior medications, therapies, and procedures will be listed for the Safety Analysis Set. 


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6.5. 
Concomitant Medications, Therapies, and Procedures 
Concomitant medications will be coded using the WHO Drug Dictionary dated Sept 2021. 
Concomitant medications, therapies, and procedures are defined as those with a start date prior to 
the date of the first dose of study drug and continuing on or after the first dose of study drug, or 
with a start date between the dates of the first and last doses of study drug, inclusive. Any 
medication, therapy, or procedure with a start date after the date of the last dose of study drug 
will not be considered a concomitant medication, therapy, or procedure. 
The concomitant medication, therapy, and procedure and usage will be summarized by the 
number and percentage of subjects in each treatment group and overall, within each preferred 
term for the Safety Analysis Set. Multiple uses of the same medication, therapy, or procedure by 
a subject in the same category will be counted only once. 
All concomitant medications, therapies, and procedures will be listed for the Safety Analysis Set. 
6.6. 
Exposure to Study Drug 
A data listing will be created giving the date and time of dose administration for each subject in 
the Safety Analysis Set. 
6.7. 
Protocol Deviations 
Protocol deviations will be identified and recorded by the site and tracked separately from the 
clinical database. Clinical team members on the Ironwood Study Execution Team (SET) will 
determine which protocol deviations are important protocol deviations (IPDs) and will categorize 
IPDs based on a blinded review of all protocol deviations prior to database lock and unblinding. 
The SET members reviewing the protocol deviations will include the study clinical operations 
lead, the medical director, the study biostatistician, and other personnel as appropriate. 
The number and percentage of subjects with IPDs will be presented by treatment group and IPD 
category for the Safety Analysis Set. Protocol deviations and IPDs will be provided by subject in 
a data listing for the Safety Analysis Set. 
A separate listing of all inclusion/exclusion criteria deviations will be provided for all 
randomized subjects (ie, the ITT Set). 


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7. 
SAFETY ANALYSIS 
All safety parameters will be summarized using descriptive statistics. Safety analyses will be 
performed on the Safety Analysis Set. The safety parameters will include AEs, clinical 
laboratory evaluations, vital signs, and ECGs. For each laboratory parameters and ECG site 
interpretation, the last nonmissing assessment before the first dose of study drug will be used as 
the baseline for all analyses of that laboratory parameter or ECG interpretation. For numeric 
ECG measurements, the average of the last triplicate ECG before dosing will be used as baseline. 
For vital signs, the last supine measurement before dosing will be used as baseline. 
All safety analyses will be performed according to the treatment actually received regardless of 
the allocated treatment. All subjects on placebo arm will be pooled from available cohorts, 
whenever appropriate, to conduct analyses. 
7.1. 
Adverse Events 
AEs will be coded by SOC and PT using MedDRA Version 24.0. The study investigator will 
assess the severity of the AE and assign a grade using the Common Terminology Criteria for 
Adverse Events (CTCAE). An AE is considered a treatment-emergent AE (TEAE) if the AE 
started after initial study drug administration and within 1 day of the last dose of study drug. In 
addition, an AE that started before initial study drug but worsened after the first dosing of the 
study drug is also considered a TEAE. 
The number and percentage of subjects reporting TEAEs in each treatment group (or cohort) will 
be tabulated: 
• by SOC and PT 
• by SOC, PT, and CTCAE grade 
• by SOC, PT, and relationship to study drug. 
If a subject has more than 1 TEAE coded to the same PT, the subject will be counted only once 
for that PT by identifying the TEAE with the highest CTCAE grade and the closest relationship 
to study drug. 
The incidence of the following TEAEs will be summarized by PT: 
• Treatment-emergent Serious Adverse Events (TESAEs) 
All AE summary tables will be sorted alphabetically by SOC and each PT within the SOC will 
be sorted by decreasing frequency for the IW‑3300 group. 
A TEAE summary table with all the non-SAEs by SOC and PT will be generated to support the 
clinicaltrials.gov reporting requirements. 
AE listings will be presented for all subjects with AEs, all screened subjects with SAEs, all SAEs 
(regardless of treatment-emergent status), and subjects who died (if any), respectively. 


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7.2. 
Clinical Laboratory Data 
Descriptive statistics for clinical laboratory values (in SI units) and changes from baseline at 
each postbaseline time point will be presented by treatment group for each clinical laboratory 
parameter. All laboratory data will be listed for the Safety Analysis Set. 
Table 1: 
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 Chemistry 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 
 
 
Inflammatory 
markers 
• ESR (analyzed at an outside laboratory) 
• CRP 
Hemoccult testing 
• Point-of-care hemoccult test 
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) 
Other Screening 
Tests 
• Drug and alcohol screen (to include at minimum: amphetamines, barbiturates, 
cocaine, opiates, cannabinoids, and benzodiazepines) 
• FSH 
• HCG (blood-based test at Screening and urine-based test at Check-in/Day ‑1) 
• SARS‑CoV‑2 testing (Screening and Check-in/Day ‑1) 
• HIV antibody and hepatitis panel (HBsAg, and HCV antibody) 
For each clinical laboratory parameter, the number and percentage of subjects with potentially 
clinically significant (PCS) postbaseline clinical laboratory values will be tabulated by treatment 
group (or cohort). The criteria for PCS laboratory values are described in Table 2. The 
percentages will be calculated relative to the number of subjects with non-PCS baseline values 


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and at least 1 postbaseline assessment. The numerator will be the total number of subjects who 
had non-PCS values at baseline and who had at least 1 postbaseline PCS value. 
A supportive listing for subjects with PCS postbaseline values will be provided and will include 
the subject ID number, all lab values, both pre- and postbaseline, and a flag marking the PCS 
laboratory values. 
Table 2: 
Criteria for Potentially Clinically Significant Laboratory Tests 
Parameter 
SI Unit 
Lower Limit 
Higher Limit 
Chemistry 
Albumin 
g/L 
<0.9 × LLN 
>1.1 × ULN 
Alanine aminotransferase 
U/L 
— 
≥3 × ULN 
Alkaline phosphatase 
U/L 
— 
≥3 × ULN 
Aspartate aminotransferase 
U/L 
— 
≥3 × ULN 
Bicarbonate 
mmol/L 
<0.9 × LLN 
>1.1 × ULN 
Bilirubin, total 
μmol/L 
— 
>1.5 × ULN 
Calcium 
mmol/L 
<0.9 × LLN 
>1.1 × ULN 
Chloride 
mmol/L 
<0.9 × LLN 
>1.1 × ULN 
Cholesterol, total 
mmol/L 
— 
>1.6 × ULN 
Creatinine 
μmol/L 
— 
>1.3 × ULN 
Glucose 
mmol/L 
<0.8 × LLN 
>1.4 × ULN 
Magnesium 
mmol/L 
<0.9 × LLN 
>1.1 × ULN 
Phosphate 
mmol/L 
<0.9 × LLN 
>1.1 × ULN 
Potassium 
mmol/L 
<0.9 × LLN 
>1.1 × ULN 
Protein, total  
g/L 
<0.9 × LLN 
>1.1 × ULN 
Sodium 
mmol/L 
<0.9 × LLN 
>1.1 × ULN 
Urea nitrogen 
mmol/L 
— 
>1.2 × ULN 
Uric acid  
μmol/L 
<0.9 × LLN 
>1.1 × ULN 
Hematology 
Basophils, absolute cell count 
109/L 
— 
>3 × ULN 
Eosinophils, absolute cell count 
109/L 
— 
>3 × ULN 
Hematocrit 
Ratio 
<0.9 × LLN 
>1.1 × ULN 
Hemoglobin 
g/L 
<0.9 × LLN 
>1.1 × ULN 
Lymphocytes, absolute cell count 
109/L 
<0.8 × LLN 
>1.5 × ULN 
Mean corpuscular hemoglobin 
Pg 
— 
>3 × ULN 
Mean corpuscular hemoglobin concentration 
g/L 
— 
>3 × ULN 


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Parameter 
SI Unit 
Lower Limit 
Higher Limit 
Mean corpuscular volume 
fL 
<0.9 × LLN 
>1.1 × ULN 
Monocytes, absolute cell count 
109/L 
— 
>3 × ULN 
Neutrophils, absolute cell count 
109/L 
<0.8 × LLN 
>1.5 × ULN 
Platelet count 
109/L 
<0.5 × LLN 
>1.5 × ULN 
Red blood cell count 
1012/L 
<0.9 × LLN 
>1.1 × ULN 
White blood cell count 
109/L 
<0.7 × LLN 
>1.5 × ULN 
LLN: Lower limit of normal value provided by the laboratory 
ULN: Upper limit of normal value provided by the laboratory 
7.3. 
Vital Signs 
Descriptive statistics for body weight and vital signs (ie, oral temperature, supine systolic and 
diastolic blood pressure, and pulse) and changes from baseline values at each time point will be 
presented by treatment group, and all the data will be listed. If there are multiple measurements 
for the same time point, the first one will be used for the by-time point analysis. 
The number and percentage of subjects with PCS postbaseline vital signs will be tabulated by 
treatment group (or cohort). A vital sign value will be considered PCS if it meets both the 
observed value criterion and the change from baseline criterion. The criteria for PCS vital sign 
values are detailed in Table 3. The PCS percentages will be calculated relative to the number of 
subjects with baseline values and at least 1 assessment in the postbaseline period. The numerator 
will be the total number of subjects with available baseline values and at least 1 postbaseline 
PCS value. 
A supportive data listing for subjects with PCS postbaseline values will be provided, including 
the subject ID number, baseline and postbaseline values for each visit, and a flag indicating the 
PCS vital sign values. 


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Table 3: 
Criteria for Potentially Clinically Significant Vital Signs 
Vital Sign Parameter 
Flag 
Criteriaa 
Observed Value 
Change from Baseline 
Supine systolic blood pressure (mmHg) 
High 
≥180 
Increase of ≥20 
Low 
≤90 
Decrease of ≥20 
Supine diastolic blood pressure (mmHg) 
High 
≥105 
Increase of ≥15 
Low 
≤50 
Decrease of ≥15 
Supine pulse (beats per minute) 
High 
≥120 
Increase of ≥15 
Low 
≤50 
Decrease of ≥15 
Weight (kg) 
High 
- 
Increase of ≥7% 
Low 
- 
Decrease of ≥7% 
a A postbaseline value is considered as a PCS value if it meets the criteria for both observed value and change from 
baseline. 
In addition to supine measurements, at the predose measurement and at various postdose time 
points on days 1, 2, 7 and 8, standing blood pressure and pulse will be measured 3 minutes after 
the supine measurements are taken. The change in blood pressure from supine to standing, along 
with associated adverse events, will be used to assess orthostatic hypotension. The criteria for 
testing positive for orthostatic hypotension are presented in Table 4. 
Table 4: 
Criteria Associated with Orthostatic Hypotension 
Blood Pressure Parameter 
Blood Pressure Change from Supine to Standing  
Systolic blood pressure (mmHg) 
Decrease of ≥20 mmHg 
Diastolic blood pressure (mmHg) 
Decrease of ≥10 mmHg 
Subjects meeting either one of these two criteria and who also have an associated adverse event, 
as indicated on the eCRF, are considered to be positive for orthostatic hypotension. The number 
and percent of subjects who have an orthostatic hypotension event will be tabulated by treatment 
group (or cohort) and by period (baseline versus postbaseline). 
7.4. 
Electrocardiogram 
Triplicate 12-lead ECGs will be obtained as outlined in the Schedule of Activities. An average of 
the 3 consecutive ECG values for each parameter (heart rate, PR interval, QRS interval, QT 
interval, and QTc interval), or 1 or 2 values if all 3 are not available, will be used for generating 
the summary statistics. Descriptive statistics for ECG parameters and their changes from baseline 
at each assessment time point will be presented by treatment group. QTc interval will be 
calculated using Fridericia correction (QTcF=QT/(RR)1/3); if RR is not available, it will be 
replaced with 60/hr in the correction formula. ECG interpretation (designated as normal, 
abnormal not clinically significant, abnormal clinically significant) will be summarized by time 
point. A shift table from baseline to each visit for qualitative ECG results will be presented and 
all the data will be listed. 


Statistical Analysis Plan 
 
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21 
 
In addition, the number and percentage of subjects with PCS postbaseline ECG results will be 
tabulated by treatment group (or cohort). An ECG value will be considered PCS if it meets the 
observed value criterion. The criteria for PCS ECG values are detailed in Table 5. The 
percentages will be calculated relative to the number of subjects with available non-PCS baseline 
values and at least 1 postbaseline assessment. The potential numerator will be the total number of 
subjects with available non-PCS baseline values and at least 1 postbaseline PCS value in the 
corresponding postbaseline period. 
A supportive data listing for subjects with PCS postbaseline values will be provided, including 
the subject ID number, baseline and postbaseline values for each visit, and a flag indicating the 
PCS ECG values. 
Table 5: 
Criteria for Potentially Clinically Significant ECG Values 
ECG Parameter 
Unit 
Higher Limit 
QRS Interval 
msec 
≥150 
PR Interval 
msec 
≥250 
QTc Interval 
msec 
>500 
7.5. 
Bladder and Bowel Movement Diary (BBMD) 
The analysis of the bladder and bowel movement diary (BBMD parameters will be based on the 
Safety Analysis Set. Subjects will enter BM‑related information into a paper diary, the BBMD, 
on an event-driven basis (ie, following each BM) during the Screening Period (beginning at 
Day ‑8) and throughout the Clinic Period. The BM related information will include the day and 
time of BMs and a report of stool consistency for each BM using the Bristol Stool Form Scale 
(BSFS) (1=Separate hard lumps like nuts [difficult to pass], 2=sausage shaped but lumpy, 3=like 
a sausage but with cracks on surface, 4=like a sausage or snake, smooth and soft, 5=soft blobs 
with clear-cut edges (passed easily), 6=fluffy pieces with ragged edges, a mushy stool and 
7=Watery, no solid pieces [entirely liquid]). 
Subjects will complete the bladder portion of the BBMD in the morning of Dosing Day 1 (which 
represents the baseline responses) and each subsequent morning from Dosing Day 2 thru 
Discharge Day 8. The bladder diary 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 more than 10) 
• 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) 


Statistical Analysis Plan 
 
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22 
 
7.5.1. 
BBMD Analysis 
BM data will be summarized by treatment for the baseline period and for the post-baseline 
period covered by the 7 × 24 = 168 hours after the first dose of study drug using descriptive 
statistics. Change from baseline for the following BM parameters will be calculated: 
• BM frequency 
• Stool consistency (BSFS) 
The baseline period for BM data is exactly 1 week and consists of the seven 24-hour periods that 
end at the time of first dose. Baseline values will be derived from the BBMD data collected 
during that period. The baseline BM daily frequency rate (BMs per day) will be calculated as the 
number of BMs the subject reports during this period divided by 7. Baseline stool consistency 
will be calculated as the average of the non-missing BSFS scores associated with the BMs 
reported by the subject during this period. 
For BM data, the postdosing frequency and averages will be based on events and reports that 
occur during the seven 24-hour periods that start on Day 1at the time of dosing and end on 
Day 8. Change from baseline will be defined as the postdosing values minus the baseline value. 
The data will be provided in listings as well. 
The data regarding bladder symptoms and urination is categorical. The baseline values are 
simply the responses to the 6 relevant questions asked, predose, in the morning of Dosing Day 1, 
and the postdosing values are the responses to the BBMD on Days 2 through 8. 
For the bladder symptoms and urination data, shift tables by study day will be created for each of 
the 6 questions, showing the responses for the 24 hours before dosing (completed on the morning 
of dosing, Day 1) versus the responses for the seven postdose 24-hour periods. 
For the Day 1 pre-dosing Bladder and Urination data, summary analyses will only include diaries 
that were completed before dosing. Any diaries that were completed after the first dose will not 
be used. Similarly, for the Day 2 Bladder and Urination diary, only diaries that were completed 
between 20 and 32 hours after dosing will be used, to avoid overlap with pre-dosing and with 
Day 3 A similar 12 hour window will be used for the BBMD diaries for days 3 through 8. 
7.6. 
Microbiome 
Analysis of microbiome at the genus level will be based on the Microbiome Analysis Set. 
The relative abundance of different categories of microorganisms will be constructed and 
summarized by treatment and by period (baseline versus Clinic Period Day 7 or Day 8) using 
descriptive statistics. Changes from baseline will be calculated. 


Statistical Analysis Plan 
 
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8. 
PHARMACOKINETIC ANALYSIS 
Pharmacokinetic (PK) assessments and analyses will be described in a separate PK analysis plan. 


Statistical Analysis Plan 
 
C3300-102 
 
24 
 
9. 
DATA MONITORING/REVIEW COMMITTEE 
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. 


Statistical Analysis Plan 
 
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10. 
REFERENCES 
Not applicable 


Statistical Analysis Plan 
 
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26 
 
11. 
APPENDICES 
11.1. 
Data Handling Conventions 
11.1.1. 
General Data Reporting Conventions 
Continuous variables will be summarized using the following descriptive statistics:  n, mean, 
median, standard deviation (SD), minimum and maximum. Categorical variables will be 
summarized by the number of subjects (n) and the percent of subjects in each category. 
11.1.2. 
Repeated or Unscheduled Assessments of Safety Parameters 
If a subject has repeated assessments for a time point, any by-time point analyses will use the last 
result for that time point. 
11.1.3. 
Definition of Visit Windows 
Study day will be calculated in accordance with CDISC standards defined in the SDTM and 
ADaM implementation guides. The date of first dose is defined as study day 1, and the day 
before is study day ‑1. Here are the formulas appear below: 
• If the assessment date is on or after the date of first dose of study drug: 
Study day = assessment date – date of first dose + 1 
• If the assessment date is before the date of first dose of study drug: 
Study day = assessment date – date of first dose 
11.1.4. 
Missing Date of Adverse Events 
For AEs with partial start dates, nonmissing date elements, such as start year, will be used to 
determine if the AE is treatment-emergent or not. If a determination cannot be made using the 
nonmissing date elements as to when the AE occurred relative to study drug administration (eg, 
AE start day is missing and the year and month are the same as the year and month of the first 
dose of study drug), then the AE will be classified as treatment-emergent. 
To facilitate categorization of AEs as treatment-emergent, the start date will be imputed. The 
stop date will be imputed if it is needed to determine whether an AE is treatment emergent. 
11.1.5. 
Missing Date of Prior or Concomitant Medications, Therapies, and Procedures 
For prior or concomitant medications, therapies, and procedures including rescue medications, 
incomplete (ie, partially missing) start date and stop dates will be imputed following the same 
rules that are used for adverse events, as detailed in the previous section. 
11.1.6. 
Character Values of Clinical Laboratory Variables 
It is possible that the laboratory will report a non-numeric value for a clinical lab result that is 
normally provided as a numeric value. This can happen if the test result is below the level of 
quantification. In this situation, analyses that require a numeric result will use an appropriately 
determined value. For example, if the reported result for ALT is “<5”, any relevant analyses will 
use a value of 0. However, the actual values as reported by the laboratory will be stored in the 
laboratory database will be presented in data listings. 


Statistical Analysis Plan 
 
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Table 6: 
Coding of Special Character Values for Clinical Laboratory Results 
Clinical Laboratory Test 
Possible Results (in SI units) 
Coded Value for Analysis 
Chemistry: ALT 
<5 
0 
Chemistry: AST 
<5 
0 
Chemistry: Total Bilirubin 
<2 
0 
Urinalysis: Glucose 
≥55 
Positive 
≤0 
Negative 
Urinalysis: pH 
≥8.0 
8.0 
11.2. 
Analysis Software 
Statistical analyses will be performed using of SAS® 9.4 on a validated, suitably qualified 
environment.


Statistical Analysis Plan 
 
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11.3. 
Schedule of Activities 
Table 7: 
Schedule of Activities 
Study Period→ 
Screening 
Period 
Clinic Period 
Follow-up 
Period 
Study 
Procedure↓ 
Visit/Day→ 
Screening 
Visit 
Day ‑36 to 
Day ‑8 
Check-in 
Day ‑1 
Dosing 
Day 1 
Days 2 
through 6 
Day 7 
Discharge 
Day 8a/ 
ET 
Follow-up 
Phone Call 
Day 21 
(+3 days) 
Informed consent 
X 
 
 
 
 
 
 
Eligibility criteria 
X 
X 
 
 
 
 
 
Medical history 
X 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
Body weight & heightb 
X 
 
Pre 
 
 
X 
 
Urine drug & alcohol screen 
X 
X 
 
 
 
 
 
Physical examinationc 
X 
X 
Pre 
Pre 
Pre 
X 
 
Rectal examinationd 
X 
X 
Pre 
Pre 
Pre 
X 
 
Vital signse 
X 
X 
Post: 0.5, 2, 4, 
and 8h (±10m) 
Days 3 
through 6 
Pre: 0 (≤25m) 
Post: 0.5, 2, 4, 
and 8h (±10m) 
 
 
Orthostatic blood pressure and 
pulsef 
 
 
Pre: 0 (≤25m) 
Post: 1 and 6h 
(±20m) 
Day 2 only 
Pre: 0 (≤25m) 
Pre: 0 (≤25m) 
Post: 1 and 6h 
(±20m) 
24h post-
Day 7 dose 
(±20m) 
 
Triplicate 12-lead ECGg 
X 
X 
Pre: 1h (±30m) 
Post: 2h (±30m) 
Pre: 1h 
(±30m) 
Post: 2h 
(±30m) 
Pre: 1h 
(±30m) 
Post: 2h 
(±30m) 
 
 


Statistical Analysis Plan 
 
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Study Period→ 
Screening 
Period 
Clinic Period 
Follow-up 
Period 
Study 
Procedure↓ 
Visit/Day→ 
Screening 
Visit 
Day ‑36 to 
Day ‑8 
Check-in 
Day ‑1 
Dosing 
Day 1 
Days 2 
through 6 
Day 7 
Discharge 
Day 8a/ 
ET 
Follow-up 
Phone Call 
Day 21 
(+3 days) 
Clinical chemistry, hematology, 
urinalysis 
X 
X 
Pre: 0 (≤15m, 
≤30m for UA) 
Post: 6h (±30m) 
Day 4 only 
Pre: 0 (≤15m, 
≤30m for UA) 
 
X 
 
ESR and CRP blood drawh 
 
X 
 
 
 
X 
 
FSH 
X 
 
 
 
 
 
 
HCGi 
X 
X 
 
 
 
 
 
SARS‑CoV‑2 testing 
X 
X 
 
 
 
 
 
HIV & hepatitis panel 
X 
 
 
 
 
 
 
BBMD training 
X 
 
 
 
 
 
 
BBMD dispensed 
X 
X 
 
 
 
 
 
Daily BBMD completionj 
Day ‑8 through 
Day ‑2 
X 
X 
X 
X 
X 
 
BBMD collection and reviewk 
 
X 
 
 
 
X 
 
Concomitant medications 
X 
X 
X 
X 
X 
X 
X 
AE monitoring 
X 
X 
X 
X 
X 
X 
X 
Subject confinement to clinicl 
 
X 
X 
X 
X 
X 
 
Start of predose fastm 
 
 
Pre: ≥6h 
Pre: ≥6h 
Pre: ≥6h 
 
 
Randomization 
 
 
X 
 
 
 
 


Statistical Analysis Plan 
 
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30 
 
Study Period→ 
Screening 
Period 
Clinic Period 
Follow-up 
Period 
Study 
Procedure↓ 
Visit/Day→ 
Screening 
Visit 
Day ‑36 to 
Day ‑8 
Check-in 
Day ‑1 
Dosing 
Day 1 
Days 2 
through 6 
Day 7 
Discharge 
Day 8a/ 
ET 
Follow-up 
Phone Call 
Day 21 
(+3 days) 
Study drug administrationn 
 
 
X 
X 
X 
 
 
PK blood drawso 
 
 
Pre: 0 (≤15m) 
Post: 0.5 (±2m), 
1, 2, 4, 6, and 
8h (±20m) 
Pre: 0 (≤15m) 
Pre: 0 (≤15m) 
Post: 
0.5 (±2m), 1, 
2, 4, 6, and 8h 
(±20m) 
24h post-
Day 7 dose 
(±20m) 
 
Stool collectionp 
 
Check-in to Pre: 0h 
 
1 sample collected between 
Day 7 (postdose) and Day 8 
(predischarge) 
 
Hemoccult testingq 
 
X 
 
 
X 
 
 
Follow-up phone callr 
 
 
 
 
 
 
X 
AE=adverse event; BBMD=bladder and bowel movement diary; CRP=C-reactive protein; CRU=clinical research unit; ECG=electrocardiogram; 
ESR=erythrocyte sedimentation rate; ET=Early Termination; FSH=follicle-stimulating hormone; HCG: human chorionic gonadotropin; HIV=human 
immunodeficiency virus; PK=pharmacokinetic; Post=postdose; Pre=predose; SARS‑CoV‑2=severe acute respiratory syndrome coronavirus 2; UA=urinalysis 
a Subjects will be discharged on Day 8 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 ET Visit will be performed prior to discharge from the CRU. Every effort 
should be made and documented to ensure that safety procedures scheduled for Discharge are performed at the ET visit. 
b Height will only be measured at the Screening Visit. On Day 1 (predose) and Discharge (Day 8), subjects will be weighed in the morning upon awakening and 
before they ingest any water or food. 
c A complete physical examination will be conducted except on Days 1 through 7 when the physical examination will be symptom-directed and conducted prior 
to study drug administration. 
d Rectal examination at all timepoints should include a digital rectal examination and visual inspection of the perianal area for redness or irritation. If significant 
redness, rectal pain, fever, bleeding, or mucus is observed, anoscopy may be performed as part of the rectal examination. 
e Vital signs will include oral temperature, blood pressure, and pulse rate. In a semisupine position, blood pressure and pulse will be obtained at the 0.5-hour 
postdose timepoint on Days 1 and 7 (consistent with the position required following dosing). Blood pressure and pulse at the 2-, 4-, and 8-hour postdose 
timepoints on Days 1 and 7, as well as predose on Days 3 through 6, should be preceded by at least 5 minutes of rest for the subject in a quiet setting without 
distractions (eg, television, cell phones). When applicable, vital sign measurements will be obtained before blood draws. 


Statistical Analysis Plan 
 
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f 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 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 before blood draws. 
g 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. 
h Elevated inflammatory markers (ESR, CRP), and/or relevant symptoms (eg, rectal pain, hematochezia) may be suggestive of colonic inflammation; subjects 
with these signs or symptoms should undergo recto-sigmoidoscopy. If this is negative, a colonoscopy may be performed after a discussion between the 
investigator and the Sponsor. 
i HCG at Screening will be a blood-based test. HCG at Check-in/Day ‑1 will be a urine-based test. 
j 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 ‑9, CRU staff will 
call subjects on Day ‑9 to remind them to begin recording their BMs in the BBMD the following day, answer any questions, and provide additional BM pages 
via email, if needed. Bladder information in the BBMD will be collected predose on each dosing day, with a 24‑hour recall period at each timepoint. 
k 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 ‑8 through ‑2) and no more than 3 bowel movements per day to be eligible for the study; CRU staff will collect the subject’s Clinic Period 
BBMD at Discharge. 
l Subjects will remain at the CRU from Check-in (Day ‑1) through Discharge (Day 8). 
m Subjects will fast for at least 6 hours prior to dosing on Days 1 through 7 and for at least 1 hour following dosing. During these times, water is permitted. 
n Study drug (IW‑3300 or placebo low-volume enema) will be administered rectally once daily, after a fast of at least 6 hours; water is permitted. 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. Study drug will be dispensed in capped syringes with a separately packaged colon tip applicator. The CRU dosing staff will 
remove the cap from the syringe, attach the colon tip applicator, prime the syringe and applicator to 20 mL, and slowly administer by inserting the contents of 
the syringe into the rectum. After the full dose has been administered, 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). 
o 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. 
p At Check-in/Day ‑1, the subject will bring in a stool sample using the sample collection kit issued by the site. Between Day 7 (postdose) and Day 8 
(predischarge), the first stool passed will be collected. If no stool is passed during this time, the subject will be discharged with a stool collection kit and asked 
to return the sample to the CRU. 
q The predose hemoccult testing may be performed using a stool sample obtained any time from Check-in (Day ‑1) to predose. If the subject does not produce a 
stool sample, stool that is present on the Day 1 (predose) digital rectal examination may be used. The Discharge (Day 8) hemoccult testing may be performed 
using a stool sample obtained any time from postdose on Day 7 to Discharge (Day 8). If the subject does not produce a stool sample, stool that is present on the 
Discharge (Day 8) digital rectal examination may be used. 
r 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).


Statistical Analysis Plan 
 
C3300-102 
 
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11.4. 
Bladder and Bowel Movement Diary 
 
Protocol C3300‑102  
SCREENING PERIOD 
Subject Number: 001 - __ __ __ 
Study Visit: _____________________________ Date: ____________ _____, _______ 
 
Day of the week 
 
Month 
Day 
Year 
 
BLADDER AND BOWEL MOVEMENT DIARY 
Bowel Movements 
Did you have a Bowel Movement today? 
 Yes  No 
If yes, please complete the table below. 
Please complete one row below for each bowel movement (BM). If you have more than one BM in a 
day, please complete additional rows as needed. At the end of each day, please confirm that all BMs for 
that day have been entered and complete a row for each BM you may have forgotten to record. 
Time of Bowel Movement 
Describe the bowel movement form 
(Please refer to the Bristol Stool Form Scale) 
Initials 
______:______  AM   PM Type:  1 
 2 
 3 
 4 
 5 
 6 
 7 
 
______:______  AM   PM Type:  1 
 2 
 3 
 4 
 5 
 6 
 7 
 
______:______  AM   PM Type:  1 
 2 
 3 
 4 
 5 
 6 
 7 
 
______:______  AM   PM Type:  1 
 2 
 3 
 4 
 5 
 6 
 7 
 
______:______  AM   PM Type:  1 
 2 
 3 
 4 
 5 
 6 
 7 
 
______:______  AM   PM Type:  1 
 2 
 3 
 4 
 5 
 6 
 7 
 
 
Bladder and Urination – Not Applicable for Screening 


Statistical Analysis Plan 
 
C3300-102 
 
33 
 
Protocol C3300‑102 
CLINIC PERIOD 
Subject Number: 001 - __ __ __ 
Study Visit: _____________________________ Date: ____________ _____, _______ 
 
Day ‑1 through Day 8 
 
Month 
Day 
Year 
 
BLADDER AND BOWEL MOVEMENT DIARY 
Bladder and Urination 
Time of Completion: ______:______  AM  PM 
Please answer the questions below upon awakening for the day, thinking back over the past 24 hours 
 In the past 24 hours… 
 
1 Did you have to urinate more frequently than normal? 
 Yes  No 
2 How many times did you urinate? 
 0‑2 
 3‑6 
 7‑10  more than 10 
3 Did you feel the strong need to urinate with little or no 
warning? 
 Yes  No 
4 Did you have to get up to urinate during the night more 
frequently than usual? 
 Yes  No 
5 How many times did you have to get up at night to urinate?  0 
 1 
 2 
 3 or more 
6 Did you have pain or burning in your bladder or pelvic area?  Yes  No 
 
Bowel Movements 
Did you have a Bowel Movement today? 
 Yes  No 
If yes, please complete the table below. 
Please complete one row below for each bowel movement (BM). If you have more than one BM in a 
day, please complete additional rows as needed. At the end of each day, please confirm that all BMs for 
that day have been entered and complete a row for each BM you may have forgotten to record. 
Time of Bowel Movement 
Describe the bowel movement form 
(Please refer to the Bristol Stool Form Scale) 
Initials 
______:______  AM   PM Type:  1 
 2 
 3 
 4 
 5 
 6 
 7 
 
______:______  AM   PM Type:  1 
 2 
 3 
 4 
 5 
 6 
 7 
 
______:______  AM   PM Type:  1 
 2 
 3 
 4 
 5 
 6 
 7 
 
______:______  AM   PM Type:  1 
 2 
 3 
 4 
 5 
 6 
 7 
 
______:______  AM   PM Type:  1 
 2 
 3 
 4 
 5 
 6 
 7 
 
______:______  AM   PM Type:  1 
 2 
 3 
 4 
 5 
 6 
 7