Clinical trial NCT04150250
Conditions: Cholera
Sponsor: PATH
Start date: 2019-11-04; Completion date: 2020-07-27
Enrollment: 47 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
This trial was designed to assess safety and preliminary efficacy of oral doses of iOWH032 on diarrhea output and clinical symptoms after a cholera challenge in healthy adult participants.

Detailed description:
The study consists of a screening phase, an inpatient containment period with challenge with Vibrio cholerae on Day 1 followed by treatment with iOWH032 or placebo and a post-challenge observation period until discharge, an outpatient follow-up period of at least 28 days, and a final follow-up (by telephone) 6 months post-challenge (Day 180) for the collection of serious adverse events.

Participants will be randomized 1:1 to receive either iOWH032 500 mg every 8 hours for three days or matching placebo. Blinded therapeutic dosing will start at the onset of diarrhea or by 48 hours after ingesting the challenge inoculum of V. cholerae. The observation and management of cholera diarrhea and symptomatology will occur on an inpatient isolation research ward over a duration of approximately 11 days, including a three-day course of antibiotics to treat all participants prior to discharge from the inpatient unit.

Arms: arm 1: On Day 1, participants were challenged with 10\^6 colony-forming units (CFU) of freshly-harvested wild-type V. cholerae. At the onset of diarrhea, or at 48 hours after challenge, whichever occurred first, participants received oral iOWH032 500 mg tablets every 8 hours for 3 days. Participants received a 3-day course of antibiotics starting 4 days post-challenge, or sooner if the participant met the criterion for severe cholera diarrhea. arm 2: On Day 1, participants were challenged with 10\^6 CFU of freshly-harvested wild-type V. cholerae. At the onset of diarrhea, or at 48 hours after challenge, whichever occurred first, participants received oral matching iOWH032 placebo tablets every 8 hours for 3 days. Participants received a 3-day course of antibiotics starting 4 days post-challenge, or sooner if the participant met the criterion for severe cholera diarrhea.
Interventions: intervention 1: iOWH032 intervention 2: Placebo intervention 3: V. cholerae Challenge intervention 4: Antibiotics
Intervention descriptions: intervention 1: Anti-secretory synthetically manufactured small molecule designed to inhibit the cystic fibrosis transmembrane conductance regulator chloride channel. intervention 2: Oral tablets matching iOWH032 on taste, appearance, dissolution time with the same excipients but no active ingredients. intervention 3: Freshly-harvested, wild-type Vibrio (V.) cholerae El Tor Inaba strain N16961,10\^6 cfu suspended in 30 mL of sodium bicarbonate solution ingested orally. intervention 4: Antibiotic therapy may include:

* Ciprofloxacin 500 mg, twice daily;
* Azithromycin 500 mg, once daily (for those persons who are allergic to fluoroquinolones);
* Doxycycline 300 mg, once daily (for those persons who are allergic to fluoroquinolones)

Protocol documents:
Prot_000.pdf: 
PATH iOWH032 – v4.0 
13 March 2020 
Title 
A Phase 2a Randomized, Single-Center, Double-Blind, Placebo Controlled Study 
to Evaluate the Safety and Preliminary Efficacy of Oral iOWH032 Against Cholera 
Diarrhea in a Controlled Human Infection Model 
Protocol Number iOWH032 
DRG-032-PO-2-01-USA 
Name of Principal Investigator: 
 
National Clinical Trial (NCT) Identifier Number:   
NCT04150250 
Sponsor:  
PATH 
Funded by: 
 
 
Protocol Version Number: 
4.0 
REDACTED VERSION 
Version Date: 
13 March 2020 
Confidentiality Statement 
 
This document is confidential and is to be distributed for review only to investigators, potential investigators, consultants, 
study staff, applicable independent ethics committees or institutional review boards, and applicable regulatory 
authorities.  The contents of this document shall not be disclosed to others without written authorization from PATH (or 
others, as applicable), unless it is necessary to obtain informed consent from potential study participants. 


PATH iOWH032 – v4.0 
13 March 2020 
 
 
2 
 
TABLE OF CONTENTS 
 
 
 
Contents 
TABLE OF CONTENTS ............................................................................................................ 2 
LIST OF ABBREVIATIONS AND ACRONYMS ................................................................... 7 
STATEMENT OF COMPLIANCE ............................................................................................ 8 
PROTOCOL SUMMARY .......................................................................................................... 9 
1 
STUDY RATIONALE ................................................................................................. 13 
2 
KEY ROLES ................................................................................................................ 14 
3 
INTRODUCTION: BACKGROUND INFORMATION AND SCIENTIFIC 
RATIONALE ........................................................................................................................... 15 
3.1 
iOWH032 General Properties & Pharmacodynamics .................................................. 15 
3.2 
Summary of Nonclinical Studies of Study Drug .......................................................... 16 
3.3 
Summary of Clinical Studies of iOWH032 .................................................................. 16 
3.4 
Background Information on the Cholera Challenge Model ......................................... 18 
3.5 Potential Risks and Benefits ............................................................................................... 19 
3.5.1 Known Potential Risks ..................................................................................................... 20 
3.5.2 Known Potential Benefits ................................................................................................ 21 
3.6 
Overall Development Strategy ..................................................................................... 21 
4 
HYPOTHESIS, OBJECTIVES AND PURPOSE ........................................................ 21 
4.1 
Study Hypothesis / Hypotheses .................................................................................... 21 
4.2 
Study Objectives ........................................................................................................... 21 
5 
STUDY DESIGN AND ENDPOINTS ........................................................................ 22 
5.1 
Description of the Study Design .................................................................................. 22 
5.2 
Study Endpoints ........................................................................................................... 22 
6 
STUDY ENROLLMENT AND WITHDRAWAL ...................................................... 23 
6.1 
Participant Inclusion Criteria ........................................................................................ 23 
6.2 
Participant Exclusion Criteria ...................................................................................... 24 
6.3 
Strategies for Recruitment and Retention .................................................................... 25 
6.4 
Withdrawal from Study ................................................................................................ 25 
6.5 
Premature Termination or Suspension of Study ........................................................... 26 


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7 
STUDY AGENTS ........................................................................................................ 27 
7.1 
Study Agent and Control Description .......................................................................... 27 
7.2 
Challenge Strain Description ........................................................................................ 29 
7.3 
Study Agents Accountability Procedures ..................................................................... 30 
8 
STUDY PROCEDURES AND SCHEDULE .............................................................. 30 
8.1 
Study Procedures/Evaluations ...................................................................................... 30 
8.2 
Laboratory Procedures/Evaluations ............................................................................. 31 


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8.3 
Study Schedule ............................................................................................................. 32 
8.4 
Outpatient Clinic Visit 2 (Day 15 ±1) .......................................................................... 35 
8.5 
Outpatient Clinic Visit 3 (Day 29 ±2) .......................................................................... 35 
8.6 
Final Follow-up – Visit 4 (Day 180 ±14) ..................................................................... 35 
8.7 
Unscheduled Visit(s) .................................................................................................... 35 
8.8 
Concomitant Medications, Treatments, and Procedures .............................................. 36 
8.9 
Prohibited Medications, Treatments, and Procedures .................................................. 36 
9 
MANAGEMENT OF EXPECTED CHOLERA ILLNESS ......................................... 36 
9.1 
Clinical Evaluation ....................................................................................................... 36 
9.2 
Measurement of Diarrhea and Vomitus ....................................................................... 36 
9.3 
Management of Fluid Loss ........................................................................................... 36 
9.4 
Frequency of Vital Signs Assessment .......................................................................... 37 
9.5 
Fluid Therapy/Hydration .............................................................................................. 37 
9.6 
Indications for Antibiotics ............................................................................................ 37 
9.7 
Indications for Other Concomitant Medications .......................................................... 38 
10 
ASSESSMENT OF SAFETY ...................................................................................... 38 
10.1 
Specification of Safety Parameters ............................................................................... 38 
10.2 
Classification of an Adverse Event .............................................................................. 40 
10.3 
Time Period and Frequency for Event Assessment and Follow-up ............................. 41 
10.4 
Reporting Procedures ................................................................................................... 42 
10.5 
Study Halting Rules ...................................................................................................... 44 
10.6 
Safety Oversight ........................................................................................................... 44 
11 
CLINICAL MONITORING ......................................................................................... 45 
12 
STATISTICAL CONSIDERATIONS ......................................................................... 45 
12.1 Statistical and Analytical Plans ......................................................................................... 45 
12.2 Statistical Hypotheses ....................................................................................................... 45 


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12.3 
Timing of Analyses ...................................................................................................... 46 
12.4 
Analysis Datasets ......................................................................................................... 46 
12.5 
Description of Statistical Methods ............................................................................... 47 
12.6 
Sample Size .................................................................................................................. 51 
12.7 
Measures to Minimize Bias .......................................................................................... 53 
13 
SOURCE DOCUMENTS AND ACCESS TO SOURCE DATA/DOCUMENTS ..... 53 
14 
QUALITY ASSURANCE AND QUALITY CONTROL ........................................... 54 
15 
ETHICAL CONSIDERATIONS (AND INFORMED CONSENT)............................ 54 
15.1 
Ethical Standard ........................................................................................................... 54 
15.2 
Institutional Review Board ........................................................................................... 54 
15.3 
Informed Consent Process ............................................................................................ 55 
15.4 
Exclusion of Women, Minorities, and Children (Special Populations) ....................... 55 
15.5 
Participant and Data Confidentiality ............................................................................ 55 
15.6 
Future Use of Stored Samples ...................................................................................... 56 
16 
DATA HANDLING AND RECORDKEEPING ......................................................... 56 
16.1 
Data Collection and Data Management Responsibilities ............................................. 56 
16.2 
Study Records Retention .............................................................................................. 57 
16.3 
Protocol Deviations ...................................................................................................... 57 
16.4 
Publications and Data Sharing Policy .......................................................................... 58 
17 
FINANCING AND INSURANCE .............................................................................. 58 
18 
CONFLICT OF INTEREST POLICY ......................................................................... 58 
19 
REFERENCES ............................................................................................................. 60 
APPENDIX A: PROTOCOL REVISIONS ............................................................................. 62 


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APPENDIX B: SCREENING TESTS ..................................................................................... 63 
APPENDIX C: SCHEDULE OF STUDY VISITS AND EVALUATIONS ........................... 65 
APPENDIX D: CLINICAL SAFETY LABORATORY TOXICITY SCALES ..................... 67 
APPENDIX E: REACTOGENICITY AND SOLICITED ADVERSE EVENT TOXICITY 
SCALES ................................................................................................................................... 68 
 
 
 
 
 
 


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LIST OF ABBREVIATIONS AND ACRONYMS 
 
AE 
 
Adverse Events 
ALT 
 
Alanine Aminotransferase 
AST 
 
Aspartate Aminotransferase 
CFR 
 
Code of Federal Regulations 
CFU 
 
Colony-forming Unit 
CPK 
 
Creatine phosphokinase 
CRF 
 
Case Report Form 
CFTR  
Cystic Fibrosis Transmembrane Conductance Regulator 
CYP2C9 
Cytochrome P450 2C9 
  
 
ECG  
Electrocardiogram 
eCRF  
Electronic Case Report Form 
FDA  
Food and Drug Administration 
GCP 
 
Good Clinical Practices 
HIV 
 
Human Immunodeficiency Virus 
ICH 
 
International Council on Harmonisation 
IND 
 
Investigational New Drug 
IRB 
 
Institutional Review Board 
ITT 
 
Intent to Treat 
MedDRA 
Medical Dictionary for Regulatory Activities 
mg 
 
milligram 
mITT  
Modified Intent to Treat 
mL 
 
milliliter 
MOP  
Manual of Operating Procedures 
OHRP  
Office of Human Research Protections 
PI 
 
Principal Investigator 
SAE 
 
Serious Adverse Event 
SAD  
Single Ascending Dose  
SOP 
 
Standard Operating Procedure (s) 
SRC 
 
Safety Review Committee 
TA 
 
Test Article 
TEAE  
Treatment-emergent Adverse Events 
ULN  
Upper Limit of Normal 
WHO  
World Health Organization 
 
 
 


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STATEMENT OF COMPLIANCE 
The trial will be conducted in accordance with the ICH E6 and the Code of Federal 
Regulations on the Protection of Human Subjects (45 CFR Part 46). The Principal 
Investigator will assure that no deviation from, or changes to the protocol will take place 
without prior agreement from the sponsor and documented approval from the Institutional 
Review Board (IRB), except where necessary to eliminate an immediate hazard(s) to the trial 
participants. All personnel involved in the conduct of the study will have completed ICH 
GCP training, and that all key investigators will have completed HSP and RCR training.  
 
I agree to ensure that all staff members involved in the conduct of this study are informed 
about their obligations in meeting the above commitments. 
 
 
Principal Investigator:  
 
 
 
 
 
 
 
Print/Type Name 
 
 
Signed:  
___________________________  
 
Date: _______________ 
 
 
 
 
Signature 
  
 
 


PATH iOWH032 – v4.0 
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PROTOCOL SUMMARY 
 
Title 
A Phase 2a Randomized, Single-Center, Double-Blind, Placebo Controlled 
Study to Evaluate the Safety and Preliminary Efficacy of Oral iOWH032 
Against Cholera Diarrhea in a Controlled Human Infection Model 
Short Title 
iOWH032 Efficacy against Cholera
Protocol 
Number 
DRG-032-PO-2-01-USA
IND Number 
110,938 (iOWH032)
NCT Number 
NCT04150250 
Trial Phase 
Phase 2a 
Study 
Summary 
This is a single-center, randomized, double-blind, placebo-controlled trial to 
evaluate the therapeutic efficacy of oral doses of iOWH032 on diarrhea 
output and clinical symptoms after a cholera challenge.  There will be 24 
study participants challenged in the first cohort and an additional 24 study 
participants challenged in the second cohort, pending favorable results of an 
interim analysis. Participants will be randomized 1:1 to receive either 
iOWH032 500 mg every 8 hours for three days or matching placebo. Blinded 
therapeutic dosing will start at the onset of diarrhea or by 48 hours after 
ingesting the challenge inoculum of 106 cfu of V. cholerae El Tor Inaba strain 
N16961.  The observation and management of cholera diarrhea and 
symptomatology will occur on an inpatient isolation research ward over a 
duration of ~11 days, including a three-day course of antibiotics to treat all 
participants prior to discharge from the inpatient unit.  The final study follow-
up visit will be six months after the challenge via phone call.  
Study 
Hypotheses 
 
x 
iOWH032 will demonstrate reduction in fluid output against cholera 
diarrhea 
x 
iOWH032 will be safe and well-tolerated 
Objectives 
Primary Efficacy Objective:
x 
To measure the rate and extent of diarrhea following cholera challenge, 
in participants treated with iOWH032 compared to placebo 
Primary Safety Objective: 
x 
To evaluate safety and tolerability of oral doses of iOWH032 compared 
to placebo 
Secondary Efficacy Objectives:  
x 
To evaluate additional measures of efficacy against cholera illness 
following cholera challenge, among those randomized to be treated with 
iOWH032 versus placebo  
Secondary Safety Objectives: 
x 
To evaluate tolerability of oral doses of iOWH032 compared to placebo 
Exploratory Objectives: 
x 
To evaluate the pharmacokinetic (pK) properties of iOWH032  
x 
To evaluate time of cessation of cholera organism in stool after challenge  
x 
To evaluate a decrease for oral rehydration solution (ORS) and/or 
intravenous (IV) fluid replacement therapy need  


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Endpoint(s) 
 
Primary Efficacy Endpoint: 
x 
Diarrheal stool output rate, defined as the total volume of diarrheal stools 
(mL, Grade 3 and above) divided by the number of hours between 
initiation of study product dosing and initiation of antimicrobial therapy. 
Primary Safety Endpoint: 
x 
Frequency and incidence of serious adverse events (SAEs) throughout the 
study 
Secondary Efficacy Endpoints:  
x 
Proportion of participants with moderate to severe diarrhea following 
cholera challenge, defined as >3 liters and >5 liters of loose stools , 
respectively, within 48 hours following challenge 
x 
Attack rate of any diarrhea following cholera challenge, defined as the 
number of participants with either 2 or more loose stools (grades 3-5) 
totaling > 200 mL, or 1 loose (grade 3-5) stool > 300 mL, respectively, 
within 48 hours following challenge 
x 
Area under the curve (AUC) of diarrheal stool volume between challenge 
dose and initiation of antibiotics 
x 
Density of cholera organisms in post-challenge stool samples measured in 
stool samples via quantitative stool culture. 
x 
To evaluate cholera illness in each participant after cholera challenge, 
using the following objective parameters: 
1. Duration of diarrheal episode as defined by time to first 
formed stool 
2. Total number of loose (Grades 3-5) stools 
3. Occurrence of fever 
4. Occurrence of vomiting 
Secondary Safety Endpoints: 
x 
To measure the occurrence of solicited adverse effects during the three 
days of oral dosing, through 8 hours following the last dose, which are 
attributed to iOWH032 and placebo, to include:  
1. Nausea 
2. Abdominal discomfort and pain 
3. Occurrence of abdominal cramps 
4. Occurrence of headache 
5. Occurrence of malaise 
6. Anorexia 
7. Pollakiuria  
8. Micturition urgency 
9. Sinus tachycardia 
10. Increased alertness 
x 
To measure the frequency and incidence of unsolicited adverse effects up 
to 28 days after the last dose. 
 
Exploratory Endpoints:  
• 
To determine plasma levels of iOWH032 7±1 hours after the first and last 
dosing in all participants 


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x 
Time (hours) to cessation of detectable cholera in stool, defined as the 
time of the first sample negative via quantitative culture, after which all 
following samples are also negative for cholera. 
x 
ORS and/or IV fluid replacement measured in liters  
 
Description of 
Study Agent 
Study Therapeutic: iOWH032 is a synthetically manufactured small molecule 
designed to inhibit the cystic fibrosis transmembrane conductance regulator 
(CFTR) chloride channel. iOWH032 is a lipophilic product which is poorly 
soluble in aqueous solutions. iOWH032 is available as 250 mg tablets. 
Placebo: Matching placebo tablets. 
Challenge Agent: Freshly-harvested, wild-type V. cholerae El Tor Inaba strain 
N16961 will be the challenge agent. Strain N16961 will be delivered at 106 
colony forming units (cfu), suspended in 30 mL of sodium bicarbonate 
solution (~1.3% NaHCO3). 
Study and 
Participant 
Duration 
The entire study duration is anticipated to be approximately 8-9 months, 
inclusive of screening period up to 85 days prior to enrollment, at least 28 
days of scheduled follow-up after challenge and a six-month phone follow-up. 
Each participant is anticipated to be in the study for a period of 6 months 
following administration of the challenge dose.   
Study 
Population  
Healthy adults, 18-44 years old, inclusive. Participants with Blood type O 
(carrying a higher risk for Cholera Gravis vs. Non-O) will not be preferentially 
selected, although randomization to treatment will be stratified by blood type 
status (O vs Non-O).  
Number of 
Participants  
48 participants will be enrolled in the study, split into two cohorts of 24 each. 
24 total (12 in each cohort) will receive the test product (iOWH032) and 24 
total (12 in each cohort) will receive placebo in a randomized double-blind 
fashion.  Up to 5 volunteers will be admitted to the contained units as 
alternates to ensure a complete cohort, but discharged prior to challenge.  
Randomization Participants will be randomized in a 1:1 ratio in each of two sequential 
cohorts to receive either iOWH032 or placebo. The participants will be 
stratified based on blood type so that there are equivalent numbers of 
volunteers with blood type O in each study arm. A randomization list will be 
prepared allocating participant identification numbers to the study group. 
The randomization scheme will be provided to the unblinded research 
pharmacist(s).  
Bacteriological 
Assessments  
All loose stools (grade 3 and higher) will be weighed/measured for volume, 
and graded.  A maximum of two stool samples daily will be examined for the 
presence of V. cholerae prior to receipt of antibiotics. Following antibiotics, if 
a subject cannot produce stool, a rectal swab will be performed every 12 
hours in order to fulfillment of discharge criteria.  
 
  
Sample Size  
Study enrollment will proceed in two stages, with 24 participants randomized 
1:1 to iOWH032 vs. placebo within each stage. Following completion of the 
first stage, an interim analysis of the primary efficacy endpoint will be 
conducted and the 2nd cohort will be enrolled, provided the study does not 
demonstrate either overwhelming efficacy or evidence of futility based on the 
results of the first cohort. A simulation-based approach was used to select 


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boundaries for interim efficacy and futility, and to compute study power and 
maintain overall type I error at the nominal level. Using a stratified Wilcoxon 
rank-sum test (Van Elteren test) to compare diarrheal stool output rate 
between study arms in a group-sequential inferential framework, a total of 48 
participants split equally between study arms provides 90% power to observe 
a 50% reduction in the cumulative diarrheal stool volume, based on prior data 
of placebo-treated participants challenged with the same strain as used in this 
study.  
 
 


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1 
Study Rationale 
This clinical trial is designed to be a Phase 2a, which will document the preliminary efficacy of oral 
doses of iOWH032 in diminishing the severity of illness of cholera, using a well-established human 
challenge model. 
 
1.1  Schematic of Study Design 
 
In Cohort 1, 24 eligible healthy male and female participants will be randomized 1:1 to be assigned to 
the iOWH032 (active treatment) or placebo. Following a favorable assessment of iOWH032 in an 
interim analysis, Cohort 2 will include an additional 24 healthy participants randomized 1:1 to 
iOWH032 or placebo. All participants will be challenged with 106 cfu of V. cholerae El Tor Inaba strain 
N16961. Blinded treatment will begin at the onset of (any) diarrhea symptoms or 48 hours after 
challenge, whichever is sooner, and consists of three oral doses per day, over three days.  The dosage 
of iOWH032 is 500 mg (two 250 mg tablets) per dose.  
A 3-day course of antimicrobial therapy will be initiated at approximately 4 days post challenge or 
sooner if indicated.  The Principal Investigator will be able to initiate antibiotics upon severe cholera 
definition being satisfied. The blinded study treatment will continue during antimicrobial therapy. 
For participants that remain asymptomatic, the last dose of iOWH032 and first dose of antibiotic will 
be separated by 1-2 hours. Discharge from the Clinical Research Unit will be contingent upon each 
participant meeting all the following criteria:  
x 
3 consecutive stool cultures that are negative for growth of vibrios (with each culture 
separated by at least 12 hours) 
x 
The absence of moderate or higher grade objective reactogenicity (diarrhea, fever, and 
vomiting) for at least 12 hours prior to discharge 
x 
The completion of a 3-day course of antimicrobial (preferred agent, ciprofloxacin, 500 mg 
twice daily for 3 days) therapy 
No. 
Participants 
Treatment Group
Treatment duration
Challenge inoculum
Cohort 1
12 
iOWH032
3 days
N16961 at 106 cfu
12 
Placebo
3 days
N16961 at 106 cfu
Cohort 2
12 
iOWH032
3 days
N16961 at 106 cfu
12 
Placebo
3 days
N16961 at 106 cfu
 
 


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Principal Investigator  
 
 
 
Pharmaron CPC Inc. 
 
Co-Investigator 
 
Center for Vaccine Development 
University of Maryland School of Medicine 
 
 
 
University of Maryland School of Medicine 
 
Medical Officer 
 
PATH 
 
PATH Study Statistician  
 
PATH 
 
Project Coordinator 
 
PATH 
 
 
 
Clinical Site Monitor 
 
The Total Approach Inc. 
  
Drug Manufacturer Representative 
 
Syngene International  
 
Clinical Microbiology Laboratory 
 
Center for Vaccine Development and Global Health
Clinical Microbiology Laboratory 
 
Challenge Site 
Pharmaron CPC, Inc.
 
Data Management 
Pharmaron CPC, Inc. 
 
Ethics Committee 
Advarra IRB
6940 Columbia Gateway Dr. 
 #110, 
Columbia, MD 21046 
 
 
 
2 KEY ROLES 
 


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3 INTRODUCTION: BACKGROUND INFORMATION AND 
SCIENTIFIC RATIONALE 
Cholera is an acute gastrointestinal illness caused by the ingestion of food or water contaminated with 
the Gram-negative bacillus V. cholerae. Infection is associated with copious, toxin-mediated, watery 
diarrhea and can result in rapid and fatal dehydration if untreated. It remains a public health problem 
among underprivileged populations in many developing countries, and visitors to such areas. The 
cholera toxin (CT) of V. cholerae acts through the pathophysiologic mechanism of stimulation of the 
cystic fibrosis transmembrane conductance regulator (CFTR) chloride ion channel. The investigational 
new drug iOWH032 is being developed as an oral therapy for the treatment of acute secretory 
diarrhea in adults and children. While oral rehydration therapy (ORT) is a safe and cost-effective 
treatment for acute secretory diarrhea, it does not reduce diarrheal output, which may lead to poor 
outcome, compliance and acceptance of this therapy. An antisecretory agent that reduces 
dehydration, provides rapid symptomatic relief, and speeds recovery with a short course of therapy 
(e.g., two to three days) is expected to improve patient outcomes and compliance. While iOWH032 is 
intended as a treatment for acute secretory diarrhea in epidemic conditions of both adult and 
pediatric populations, iOWH032 will first be evaluated in adults in this Phase 2a study prior to a 
potential age-descending trial. 
 
3.1 iOWH032 General Properties & Pharmacodynamics 
iOWH032 is a synthetically manufactured small molecule designed to inhibit CFTR, a cyclic 
adenosine monophosphate (cAMP)-activated chloride ion (Cl−) channel expressed in epithelial cells 
of mammalian airways, intestine, pancreas, skin, and testis. iOWH032’s ability to inhibit the CFTR 
chloride channel was demonstrated in vitro using T84 colon carcinoma cells and Chinese hamster 
ovary (CHO) cells expressing human CFTR. The 50% inhibitory concentration in these assays ranged 
from 2-11 PM. iOWH032’s activity in vivo was demonstrated in two disease models: the mouse 
closed-loop model and cecectomized rat open-loop model. The CFTR chloride channel in the 
gastrointestinal (GI) tract has both intracellular and extracellular (luminal) inhibitor sites. It is 
hypothesized that the inhibitory activity, and hence the pharmacodynamic response of iOWH032, 
will most likely be driven by GI lumen concentrations rather than plasma concentrations. The 
activity at the extracellular site will depend directly on the concentration in solution within the GI 
tract. 
iOWH032 has a molecular weight of 545.18. It has a phenolic moiety and behaves as a weak acid 
(experimental pKa 7.84). It is a lipophilic molecule and is expected to be freely permeable across the 
lipid layer of biological membranes as demonstrated both by the parallel artificial membrane 
permeability assay and the use of Heterogeneous Human Epithelial Colorectal Adenocarcinoma cell 
line (Caco-2) cell line monolayers. It is also confirmed that iOWH032 was not a substrate for the 
efflux transporter, P-glycoprotein (efflux ratio < 2). iOWH032 does, however, show limited solubility 
(< 1 μM at pH 7.4), especially in acidic conditions. Its solubility increases in the presence of 
simulated fasted intestinal fluid (7.5 μM). 
 


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The plasma half-life of iOWH032 administered orally in rats and dogs was in the range of 1 to 3 
hours with maximum plasma concentrations (Cmax) observed at approximately 1 to 2 hours post 
dose. The absolute bioavailability of an oral dose of 5 mg/kg was 38.1% in rats, and bioavailability of 
an oral dose of 3 mg/kg was 81.2% in dogs, showing absorption and bioavailability from the oral 
route. In vitro studies showed iOWH032 to be > 99% protein-bound in human, rat, and dog plasma. 
The clearance of iOWH032 was low relative to hepatic blood flow, with values of 0.452 L/hr/kg in 
rats and 0.096 L/hr/kg in dogs. However, despite this low clearance in both species, the 
corresponding half-lives of iOWH032 were short, 0.4 hours and 3.18 hours in rats and dogs, 
respectively, most likely due to the low volume of distribution. 
3.2 Summary of Nonclinical Studies of Study Drug  
The toxicology program included genotoxicity studies, acute (single-dose) and repeat-dose studies 
up to 14 days in duration, and reproductive toxicology studies in rats and rabbits. Animals received 
up to 2,000 mg/kg/day and there were no major toxicity findings with potential clinical significance. 
iOWH032 was shown in an in vitro study to be a potent inhibitor of Cytochrome P450 2C9 (CYP2C9) 
(IC50 0.08 μM) and, to a lesser extent, CYP2C19 (IC50 24 μM). Concomitant administration of 
iOWH032 with drugs metabolized by CYP2C9 should be avoided. This includes warfarin, phenytoin, 
Nonsteroidal Anti-Inflammatory Drugs (NSAIDs), and other drugs described in the Appendix F. 
However, significant drug interactions in patients are not anticipated because of the short duration 
of treatment (≤ 3 days). 
In the in vitro human ether-à-go-go–related gene (hERG) assay, the IC20 for the inhibitory effect of 
iOWH032 on hERG potassium currents was 1.87 μM and the IC50 was estimated to be > 4.95 μM, 
approximately 150-fold higher than the unbound concentration (Cmax(unbound) = 33.6 nM) of iOWH032 
found in humans dosed with 500 mg every 8 hours. Cardiac safety was evaluated in telemetry- 
instrumented dogs in a cardiovascular safety pharmacology study and by monitoring ECGs during 
the repeat-dose toxicology study in dogs. No adverse effects of iOWH032 were observed on any 
cardiac parameter (blood pressure, heart rate, ECG including QTc) at doses up to 1000 mg/kg. In the 
clinical setting, protein binding is also expected to be high (99.79% in humans, based on in vitro 
studies) and, therefore, the free (unbound) concentration of iOWH032 in vivo is expected to be 
much lower than the concentration required to significantly inhibit the hERG potassium channel in 
vitro. 
3.3 Summary of Clinical Studies of iOWH032  
iOWH032 has been administered to 72 healthy adult volunteers in two Phase 1 studies conducted in 
the United States, and was found to be generally well-tolerated at single doses ranging from 30 mg 
to 1000 mg, and when administered for 3 days at doses ranging from 100 mg every 12 hours to 500 
mg every 8 hours.  A two-part bridging pharmacokinetic (DDP-CFT-PO-201) study was performed 
May-October 2013 in eight (8) healthy adult volunteers and twelve (12) adult male cholera patients 
in Bangladesh (NCT01823939).  
3.3.1 
iOWH032 Human Phamacokinetics Data  
In the single ascending dose (SAD) study, doses of iOWH032 administered under fasted conditions 
exhibited absorption that was dose-independent with a median time of maximum plasma 
concentration (Tmax) of 5 to 6.5 hours. Exposure to iOWH032 [Cmax and AUC0-inf] increased over the 
single-oral-dose range of 30 mg to 1000 mg in a less-than-dose-proportional manner. This was most 
pronounced at doses up to 300 mg; exposure at doses of 300 mg to 1000 mg appeared to increase 
in an approximately dose-proportional manner, but proportionality could not be confirmed 
statistically. Elimination half-life was similar across the tested dose range and was independent of 


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feeding status. However, there was a significant increase in iOWH032 exposure parameters [Cmax: 
3.85-fold and AUC0-inf: 3.67-fold higher] when 500 mg iOWH032 was administered with a high-fat 
breakfast. The highest exposure to iOWH032 in this study was observed when 500 mg iOWH032 was 
administered with a high-fat breakfast [geometric mean: AUC0-inf 124,000 ng·hr/mL; Cmax 7270 
ng/mL]. 
In the multiple ascending dose (MAD) study, the median Tmax ranged from 4 to 6 hours following 
single-dose administration on Day 1 and from 3.00 to 4.00 hours following multiple-dose 
administration on Day 4. Following Tmax on Day 4, the iOWH032 plasma concentrations declined in a 
mostly monophasic manner. The elimination half-life of iOHW032 (10.3 to 11.7 hours) was 
independent of the administered dose. After single doses, geometric mean Cmax values ranged from 
2000 ng/mL (100 mg every 12 hours) to 5210 ng/mL (500 mg every 12 hours) and geometric mean 
AUCtau values ranged from 14,900 ng·h/mL (100 mg every 12 hours) to 43,200 ng·h/mL (500 mg 
every 12 hours). After multiple dosing, geometric mean Cmax values ranged from 2520 ng/mL (100 
mg every 12 hours) to 8730 ng/mL (500 mg every 8 hours) and geometric mean AUCtau values 
ranged from 23,000 ng·h/mL (100 mg every 12 hours) to 72,000 ng·h/mL (500 mg every 12 hours). 
Regression analysis indicated that iOWH032 plasma exposure as measured by AUCtau indicated a 
less-than-proportional increase over the studied dose range. 
Human metabolism of iOWH032 was evaluated in vivo using liquid chromatography tandem mass 
spectrometry, demonstrating the major plasma and urinary metabolites to be a hydroxylated 
product with its glucuronide also being in urine but not in plasma. Thus, it would appear that in vivo, 
the major metabolite of iOWH032 circulating in human plasma is also the same as that circulating in 
rat plasma. 
3.3.2  
iOWH032 Human Safety Data  
In the SAD study, the most common adverse event (AE) was pollakiuria (extraordinary daytime 
urinary frequency). In the 30 mg dose group, 4 of 6 participants, all who received active drug and 
none who received placebo, reported pollakiuria beginning 3 to 6 hours following receipt of the 
single dose and persisting for 19 to 30 hours. Three of these same participants also reported 
micturition urgency, and two reported an increase in stool frequency (3 formed stools in one day 
rather than their usual 1 formed stool). All adverse events were rated as mild in severity and 
resolved spontaneously. Repeated urinalyses (including urine specific gravities), urine cultures, 
serum electrolytes, blood urea nitrogen (BUN), creatinine, and complete blood counts were all 
normal throughout the period of time the participants were symptomatic. Plasma concentrations of 
iOWH032 in these 4 participants ranged from approximately 400–900 ng/mL. No urinary symptoms 
were reported by participants enrolled in the 300, 500, or 1000 mg dose cohorts in the SAD portion 
of the study. One participant in the food effects portion of the study reported pollakiuria following 
his receipt of the 500 mg dose in both the fed and fasted portions of the study. This participant 
reported onset of symptoms beginning approximately 4 hours after dosing in each treatment period 
and on both occasions, reported being symptomatic for approximately 4 days. As observed in the 30 
mg dose cohort, repeated urinalyses (including urine specific gravities), urine cultures, serum 
electrolytes, BUN, creatinine, and CBCs were normal and the participant’s symptoms resolved 
spontaneously. This participant’s maximum iOWH032 plasma concentrations were 6492 ng/mL in 
the fed state and 3432 ng/mL in the fasting state. 
One 19-year-old female with no history of cardiovascular disease experienced isolated, 
asymptomatic premature ventricular contractions (PVCs) at 2 and 3 hours following a single dose of 
100 mg of iOWH032. ECGs that were obtained at screening and pre-dose were normal except for 
intermittent sinus tachycardia. On telemetry during the next 24 hours, she persisted in having 


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isolated asymptomatic PVCs without any accompanying symptoms of palpitations, chest pain, or 
dizziness. A thorough cardiac evaluation by a board-certified cardiologist revealed a normal cardiac 
exam, normal stress echocardiogram, normal ECG, and no evidence of structural cardiac disease. 
In the MAD study, the most frequently occurring treatment-emergent adverse events (TEAEs) 
reported in 2 or more participants overall were application site irritation (at the site of 
electrocardiogram patches) (11/40, 27.5%), sinus tachycardia (3/40, 7.5%), catheter site pain (2/40, 
5.0%), decreased appetite (2/40, 5.0%), and headache (2/40, 5.0%). Treatment-emergent sinus 
tachycardia and decreased appetite were not reported in participants who received placebo. 
Three mild TEAEs of sinus tachycardia were reported by a single participant in each of the following 
3-dose cohorts: 300 mg iOWH032 every 8 hours, 500 mg iOWH032 every 12 hours, and 500 mg 
iOWH032 every 8 hours. No clinically significant changes in blood pressure or other ECG parameters 
accompanied the tachycardia. Two of these participants had the maximum iOWH032 exposures in 
their dose cohorts and had close to the maximum overall exposure of all participants during the 
time of their sinus tachycardia; 1 of the 2 participants experienced an accompanying TEAE of 
increased alertness and the other participant reported no accompanying symptoms. The third 
participant who had a TEAE of sinus tachycardia reported an accompanying symptom of a 
“stimulated feeling” and had iOWH032 exposures that approximated the median for his dose cohort 
and also for all participants. One participant reported a TEAE of palpitations that was 
unaccompanied by tachycardia. A slight upward trend in average pulse rate change from baseline 
was observed with increasing iOWH032 plasma concentration, but it was not statistically significant. 
Mean systolic blood pressure decreased by approximately 10 mmHg in the 300-mg and 500-mg 
iOWH032 every-8-hour dose cohorts compared to the pooled placebo group, beginning 
approximately 1 hour following receipt of Dose 1 and persisting until approximately 112 hours after 
Dose 1 (approximately 40 hours after the receipt of Dose 10 of iOWH032). None of the individual 
decreases were considered clinically important decreases in systolic blood pressure and thus were 
not reported as adverse events. 
In the PK bridging study (Bangladesh, DDP-CFT-PO-201), one SAE was reported in a patient with 
cholera. This SAE of elevated serum creatine kinase/creatine phosphokinase was deemed unlikely 
related to the drug, as there were no clinically relevant features or changes in ECG parameters that 
were found to be associated.  No treatment was provided to the participant in response to the 
event. No action was taken with regards to the study drug due to the event.  
3.4 Background Information on the Cholera Challenge Model 
Cholera continues to be a major public health problem in nearly all developing countries including 
the Western hemisphere, afflicting both children and adults, and is an endemic disease in over 100 
countries of the world. There are over 200 serogroups of cholera, based on the polysaccharide O-
antigen. Epidemic cholera is associated with the O1 and, more recently, O139 serogroups. The O1 
serogroup is further classified by biotype, classical or El Tor, and within this biotype by serotype, 
Ogawa or Inaba. Carefully conducted human challenge studies have been used to demonstrate the 
efficacy of cholera vaccine candidates. Pioneered at the University of Maryland, Baltimore (UMB), 
Center for Vaccine Development (CVD), cholera challenge models have been safely and successfully 
used for over 40 years. Investigators at CVD developed a challenge model using freshly harvested V. 
cholerae El Tor Inaba strain N16961.  Previous studies demonstrated a clear dose-response (Table 1) 
and indicated that a 106 cfu challenge dose of freshly harvested V. cholerae strain N16961 produced 
consistent disease (Table 2). 


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Table 1: Clinical and bacteriological responses of healthy volunteers to various doses of freshly harvested V. 
cholerae El Tor Inaba strain N16961 
Inoculum 
Attack 
Rate for 
Diarrhea 
Mean 
Incubation 
(Hours) 
Mean 
Diarrheal 
Stool 
Volume 
(Liters)
Mean 
Number 
Diarrheal 
Stools 
(per day)
Vomiting
Fever
Positive 
Stool 
Culture
103 
4/6 
33 
0.9
5.1
0/6
0/6
6/6
104 
4/5 
36 
1.1
6.5
0/5
0/5
4/5
105 
3/5 
18 
3.1
15
0/5
0/5
4/5
106 
9/10 
25 
3.2
12.9
2/10
2/10
10/10
 
Table 2: Diarrheal attack rates with freshly harvested V. 
cholerae El Tor Inaba strain N16961, across multiple 
challenge studies 
CVD Protocol
Inoculum
Attack Rate for 
Diarrhea
3002A 
0.62 x106 
5/5 
3005B 
1.0 x106 
4/5 
5009 
1.9 x106 
6/8 
5011 
2.0 x106 
5/7 
5013 
1.4 x106 
7/8 
5025 
2.2 x106 
9/9 
6001 
2.3 x106 
6/7 
6005 
1.98 x106 
10/11 
6011 
1.74 x106 
6/6 
7002 
1.74 x106 
7/7 
7004 
1.79 x106 
6/8 
9003 
1.0 x106 
7/8 
11002 
1.07 x106 
13/13 
TOTAL 
91/102 (89%) 
It is intended that 106 cfu challenge doses of freshly harvested V. cholerae strain N16961 will be 
administered to study participants in this clinical trial, to allow for a preliminary assessment of the 
efficacy of iOWH032. 
3.5 Potential Risks and Benefits 
No benefits are expected to participants for their participation in this research study.  The study 
screening visit will be free of charge, but that if any clinical abnormalities are noted during that visit 
that participants will be referred to their own physician for further follow-up. 
There is the potential for some therapeutic efficacy against acute secretory diarrhea with oral doses 
of iOWH032, but this has not been proven.  Participants that undergo challenge with cholera should 
expect to have diarrhea, in some cases the diarrhea can be severe. Cholera illness may also be 
associated with nausea, vomiting, abdominal cramping or discomfort, loss of appetite, muscle aches, 
and tiredness. The electrolyte losses with the secretory diarrhea may result in cardiac conduction 
abnormalities, if uncorrected. Intravascular volume losses with diarrhea can be associated with 
postural hypotension, shock, and death, if uncorrected. In this study, participants will be closely 
monitored to detect and prevent such effects, with rescue treatment readily provided (See section 
9). 


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3.5.1 Known Potential Risks 
Oral doses of iOWH032. Doses of 500 mg administered every 8 hours for 10 consecutive doses (3 
days plus a single dose on Day 4) have generally been well tolerated in the limited studies that have 
been performed. Potential known AEs of iOWH032 are pollakiuria, micturition urgency, sinus 
tachycardia and increased alertness. There may be other unknown risks, discomforts, or side effects 
from iOWH032.  Any significant adverse events, which are deemed possibly related to iOWH032, will 
be disclosed, as appropriate. 
Challenge with V. cholerae strain N16961. The intended dose of 106 cfu is expected to elicit acute 
watery diarrhea within 18-48 hours of ingestion; about 20% are expected to have severe cholera. 
Persons that are blood group O are known to be more likely to have severe cholera.  Nausea or 
vomiting, abdominal cramping or discomfort, and loss of appetite are also common symptoms that 
accompany cholera. Fever is a rarer symptom with cholera. The signs of severe dehydration can 
involve dry mouth, decreased urine, thirst, cold clammy skin, hypotension, lethargy, stupor, and 
muscle cramping. The complications of too much fluid lost through excessive diarrhea include 
hypoglycemia (low blood sugar), acidosis, kidney failure, pulmonary edema (fluid in lungs), 
arrhythmia (heart rhythm abnormalities), coma, and death. Anticipated dehydration will be 
managed with aggressive fluid rehydration (oral and/or intravenous) with potassium repletion. Early 
antibiotic therapy will be provided to participants that achieve severe cholera illness; otherwise all 
participants will be treated with antibiotics at approximately 4 days post-challenge. It is expected 
that shedding will occur in the stool; however, transmission from person to person is mitigated by 
housing the participants on the Clinical Research Unit until they meet the protocol discharge criteria. 
Participants will be counselled about use of standard hygiene practices (i.e., hand washing with soap 
and water after defecation), to decrease the likelihood of transmission of the strain person to 
person.  
Risk of Allergic Reaction. There is a very rare risk for an allergic reaction to the study drug iOWH032, 
the placebo, the challenge inoculum, or the antibiotic. Allergic reactions generally can range from 
mild (for example, skin rash, itching, swelling, or numbness) to severe (for example, low blood 
pressure, difficulty breathing, shock, heart arrest, or death). The symptoms of a mild allergic 
reaction typically go away without treatment, but some cases may require treatment with 
antihistamines or steroids (medications used to treat allergic reactions). In the case of more severe 
allergic reactions such as shock, immediate and intensive medical treatment is necessary. Although 
the risk of an allergic reaction is very small, epinephrine, antihistamines, and other equipment will 
be available to treat anaphylaxis or immediate hypersensitivity reactions. 
Risk with Pregnancy. The study drug iOWH032 has not been evaluated in pregnant women. 
Therefore, the risk to the unborn fetus or pregnant mother is unknown. Cholera infection in a 
pregnant woman can harm the fetus. All female study participants will be tested for pregnancy, 
during screening and one day before the challenge. To be eligible, female study participants must be 
using a highly effective form of contraception for the duration of the trial and the four weeks before 
and after the challenge.  If a pregnancy occurs between challenge and 28 days post-challenge, it will 
be reported immediately (within 24 hours of identification) to the sponsor’s medical officer. A 
pregnancy is reported as an AE or SAE only when there is suspicion that the investigational product 
may have interfered with the effectiveness of contraception or there was a serious complication in 
the pregnancy including a spontaneous abortion or an elective termination for medical rationale, or 
a birth defect in the baby. 
Risk with Blood Draws. Blood will be drawn at several times during the study and may also be 
performed to help manage the diarrheal illness. The drawing of blood may cause pain, bruising, 
feeling faint, fainting, needle site infections, swelling, and rarely other infections.  Bruising at the site 


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of blood drawing can be prevented by applying pressure for several minutes.  To reduce the risk of 
infection, the skin site is prepared with an alcohol wipe and sterile technique is used.  
Rectal swab (if a stool sample is not provided). Rectal swabs may be performed as part of the 
requirement for discharge criteria (i.e., three sequential negative stool cultures separated by 12 
hours). The rectal swab may cause the sensation of rectal pressure, but is not painful. 
Risks to Confidentiality. Personal health information will be collected as a part of this study and 
efforts will be made to maintain confidentiality. There is a small risk of loss of confidentiality that an 
unauthorized person may gain access to viewing the research records. In order to maintain 
confidentiality, all study records will be stored in a secure location, such as a locked office and/or 
locked cabinet. Electronic data will be password-protected.  Study records and specimens obtained 
will be coded.  Research records will only be shared with authorized personnel and only in 
connection with carrying out the obligations relating to the study. Every effort will be made to keep 
the records as confidential as possible, within the limits of the law. 
3.5.2 Known Potential Benefits  
This is a healthy volunteer study which does not provide any guarantee of benefit to the 
participants. The benefit is largely the scientific knowledge to be gained from the study. 
3.6  Overall Development Strategy  
To predict the usefulness of iOWH032 as a public health tool in the treatment of cholera, PATH 
proposes to first determine the degree of protection provided against moderate to severe diarrheal 
disease with V. cholerae using a human challenge study as a Phase 2a trial, before proceeding to 
field studies.  While a cholera challenge study will not answer all of the questions regarding the use 
of an antisecretory drug in the field, it will provide an answer on the degree of reduction of diarrheal 
disease under these optimal conditions.  If it is not efficacious in the challenge model it is unlikely to 
be effective in the field.  Therefore, a positive result in the challenge model would justify further 
field efficacy trials for fluid reduction in cholera. 
 
If efficacy is found in the challenge study, the next step is to perform field trials in areas where 
cholera occurs. Our goal is to achieve US approval by submitting a New Drug Application to the FDA, 
followed by prequalification by the World Health Organization (WHO) to support product acquisition 
by UNICEF and other humanitarian agencies for its distribution to low- and middle- income 
countries.   
4 HYPOTHESIS, OBJECTIVES AND PURPOSE 
4.1 Study Hypothesis / Hypotheses 
x iOWH032 will demonstrate reduction in fluid output against cholera diarrhea  
x iOWH032 will be safe and well-tolerated 
 
4.2  Study Objectives 
4.2.1 Primary Efficacy Objective 
x 
To measure the rate and extent of diarrhea following cholera challenge, in participants treated 
with iOWH032 compared to placebo 
4.2.2 Primary Safety Objective: 
x 
To evaluate safety of oral doses of iOWH032 compared to placebo 


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4.2.3 Secondary Efficacy Objectives: 
x 
To evaluate additional measures of efficacy against cholera illness following cholera challenge, 
among those randomized to be treated with iOWH032 versus placebo  
4.2.4 Secondary Safety Objective: 
x 
To evaluate tolerability of oral doses of iOWH032 compared to placebo 
4.2.5 Exploratory Objectives: 
x 
To evaluate the pharmacokinetic (pK) properties of iOWH032 
x 
To evaluate time of cessation of cholera organism in stool after challenge  
x 
To evaluate a decrease for ORS and/or IV fluid replacement therapy need 
5 STUDY DESIGN AND ENDPOINTS 
 
5.1 Description of the Study Design 
 
This is a single-center, randomized, double-blind, placebo-controlled challenge and therapeutic trial 
assessing diarrheal output and clinical symptoms when given either placebo or oral iOWH032 500 
mg (two 250 mg tablets) every 8 hours for three days starting at the onset of any diarrhea (upon first 
Grade 3-5 stool, regardless of volume) stool or 48 hours after ingesting an inoculum of V. cholerae 
N16961, whichever is sooner, and consists of three oral doses per day, over three days.     
 
Test Article (TA) 
N 
Dose 
iOWH032 
24 
500 mg (two 250 mg tablets) every 8 hours for 3 days 
with meals 
Placebo 
24 
Two placebo tablets every 8 hours for 3 days with meals
Challenge 
48 
106 CFU Vibrio cholerae N16961, once on challenge day 
 
 
5.2  Study Endpoints 
5.2.1 Primary Efficacy Endpoints 
x 
Diarrheal stool output rate, defined as the total volume of diarrheal stools (mL, Grade 3 and 
above) divided by the number of hours between initiation of study product dosing and 
initiation of antimicrobial therapy  
5.2.1 Primary Safety Endpoint: 
x 
Frequency and incidence of serious adverse events (SAEs) throughout the study 
5.2.2 Secondary Efficacy Endpoints: 
x 
Proportion of participants with moderate to severe diarrhea following cholera challenge, 
defined as >3 liters and >5 liters of loose stools, respectively, within 48 hours following 
challenge  
x 
Attack rate of any diarrhea following cholera challenge, defined as the number of participants 
with either 2 or more loose stools (grades 3-5) totaling > 200 mL or 1 loose (grade 3-5) stool > 
300 mL, respectively, within 48 hours following challenge 


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x 
Area under the curve (AUC) of diarrheal stool volume between challenge dose and initiation of 
antibiotics 
x 
Density of cholera organisms in stool samples measured in  stool samples via quantitative stool 
culture 
x 
To evaluate cholera illness in each participant after cholera challenge, using the following 
objective parameters: 
1. Duration of diarrheal episode as defined by time to first formed stool 
2. Total number of loose (Grades 3-5) stools 
3. Occurrence of fever 
4. Occurrence of vomiting 
5.2.3 Secondary Safety Endpoints: 
x 
To measure the occurrence of solicited adverse effects during the three days of oral dosing, 
through 8 hours following the last dose, which are attributed to iOWH032 and placebo, to 
include:  
1. Nausea 
2. Abdominal discomfort and pain 
3. Occurrence of abdominal cramps 
4. Occurrence of headache 
5. Occurrence of malaise 
6. Anorexia 
7. Pollakiuria  
8. Micturition urgency 
9. Sinus tachycardia 
10. Increased alertness 
x 
To measure the frequency and incidence of unsolicited adverse effects up to 28 days after the 
last dose. 
5.2.4  Exploratory Endpoints  
x 
To determine plasma levels of iOWH032 7±1 hours after the first and last dosing in all 
participants 
x 
Time (hours) to cessation of detectable cholera in stool, defined as the time of the first sample 
negative via quantitative stool culture, after which all following samples are also negative for 
cholera. 
x 
ORS and/or IV fluid replacement in measured liters  
6 STUDY ENROLLMENT AND WITHDRAWAL 
 
6.1 Participant Inclusion Criteria 
 
1. Willing and able to understand and provide written informed consent 
2. Healthy male and female adults, age 18 to 44 years (inclusive), without clinically significant 
medical history, physical or clinical laboratory abnormalities (as per protocol-defined 
acceptable ranges), and protocol-defined abnormal electrocardiogram results at screening 
3. All women must have a negative serum pregnancy test at screening and one day prior to 
challenge.  
4. Agreement by participants to use an adequate method of contraception§ during the study 
and for 4 weeks before and after the challenge. 


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5. Able to pass a written examination (comprehension assessment test) with a score of ≥70%, 
in order to demonstrate their comprehension of this study. If a participant scores at least 
50%, then they will be given one more opportunity to re-test after further re-education. 
6. Willing and able to comply with the study requirements and procedures. 
§ Adequate contraception is defined as a contraceptive method with failure rate of less than 1% 
per year when used consistently and correctly and when applicable, in accordance with the 
product label; includes, but is not limited to, barrier with additional spermicidal foam or jelly, 
intrauterine device, hormonal contraception (started at least 4 weeks prior to study 
enrollment), or women who have intercourse limited to men who underwent vasectomy. 
6.2 Participant Exclusion Criteria 
1. Clinically significant history of immunodeficiency, cardiovascular disease, respiratory 
disease, endocrine disorder, liver disease, renal disease, gastrointestinal disease, anal or 
rectal disorders, neurologic disease,  
2. Current nicotine use or drug, alcohol abuse within the past 6 months 
3. Recipient of bone marrow or solid organ transplant 
4. Use of systemic chemotherapy in the past 5 years 
5. Has a malignancy (excluding localized non-melanoma skin cancers) or lymphoproliferative 
disorders diagnosed or treated within the past 5 years 
6. Received or plans to receive systemic immunosuppressive therapy, radiation therapy, 
parenteral or high-dosage inhaled steroids (> 800 Pg/day of beclomethasone dipropionate 
or equivalent) within 6 months prior to the enrollment through 28 days after challenge 
7. Have a history of hospitalization for psychiatric illness, suicide attempt, or confinement for 
danger to self or others, within the past 10 years. Participants with a psychiatric disorder 
(not meeting exclusion criteria, e.g., attention-deficit hyperactivity disorder) that is 
controlled for a minimum of 3 months and the investigator has determined that the 
participant’s mental status will not compromise the participant’s ability to comply with 
protocol requirements may be enrolled 
8. Have an elevated blood pressure, systolic ≥150 mmHg or diastolic ≥90 mmHg, before 
challenge 
9. Taking any of the drugs listed in Appendix F that are metabolized by CYP2C9 or any of the 
following psychiatric medications: aripiprazole, carbamazepine, chlorpromazine, 
chlorprothixene, clozapine, divalproex sodium, fluphenazine, haloperidol, lithium carbonate, 
lithium citrate, loxapine, mesoridazine, molindone, olanzapine, perphenazine, pimozide, 
quetiapine, risperidone, thioridazine, thiothixene, trifluoperazine, triflupromazine, or 
ziprasidone 
10. History of Guillain-Barré Syndrome  
11. Too low or too high a BMI (BMI < 18.5 or > 39) 
12. Has an abnormal stool pattern defined as fewer than 3 stools per week or more than 2 
stools per day within the past 6 months, and any loose stools (grade 3 or higher) during the 
1–2 day acclimation period before challenge 
13. Has regularly used laxatives in the past 6 months 
14. Has a history of eating disorders (e.g. anorexia or bulimia) within the past 10 years 
15. Known allergy or previous severe adverse effect to all of the following antibiotics:  
ciprofloxacin (or quinolones), azithromycin and doxycycline.  
16. Previously received a licensed or investigational cholera vaccine, within 10 years 
17. History of cholera or enterotoxigenic Escherichia coli (ETEC) infection (lab-confirmed natural 
infection or experimental challenge), within 10 years 


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18. Travel to a cholera-endemic area in the past 5 years 
19. Pregnant or nursing 
20. Positive serology for human immunodeficiency virus (HIV), hepatitis B antigen, or hepatitis C 
antibody 
21. Protocol-defined (appendix B) clinically abnormal 12-lead ECG at screening in the judgment 
of the Investigator, or based on the formal 12-lead ECG reading by a cardiologist; history of 
any cardiac abnormalities, including conduction abnormalities such as Wolff-Parkinson-
White, dysrhythmias, or coronary artery disease 
22. Presence of a clinically significant abnormality on physical examination, including (but not 
limited to): pathologic heart murmur, lymphadenopathy, hepatosplenomegaly, large 
abdominal scar of unclear origin 
23. Has poor venous access, defined as the inability to obtain screening blood tests after three 
attempts 
24. Currently on, or plans to be on, antibiotics (e.g., doxycycline) within 14 days prior to 
challenge and through 28 days after challenge 
25. Presence of an acute illness or fever (>100.4°F) within 72 hours of admission to the inpatient 
Clinical Research Unit 
26. Taking any prescription or over-the-counter medications that contain aspirin, non-steroidal 
anti-inflammatory drugs (NSAIDs), antacids, proton pump inhibitors (PPIs), anti-diarrheals, 
etc. within 72 hours prior to challenge 
27. Received an investigational product within 30 days prior to randomization ( for the 
monoclonal antibodies- 90 days prior to randomization) or planning to participate in 
another research study involving investigational product during the conduct of this study 
28. Participants must not have donated blood in 8 weeks prior to study entry and agreed to not 
donate blood during and for 4 weeks following their active participation in this study 
29. Lack of ability to fully understand the informed consent 
30. Any other condition(s) that in the opinion of the investigator would jeopardize the safety or 
rights of a participant participating in the trial or would render the participant unable to 
comply with the protocol 
 
6.3 Strategies for Recruitment and Retention 
 
Volunteers will be recruited from the greater Baltimore area.  They will be recruited using standard 
procedures for Pharmaron clinical trials. Volunteers for inpatient challenge studies are carefully 
screened on the details of the protocol, their time commitment, and the nature of the confinement 
on a quarantine ward. At the end of screening volunteers will take a test to make sure that they 
understand the study.  They are also screened for general health.  Volunteers receive a stipend for 
each visit and day in confinement that has been approved by the Advarra IRB.  
 
6.4  Withdrawal from Study 
6.4.1 
Reasons for Withdrawal or Termination 
Participation in the study is strictly voluntary.  Participants have the right to withdraw from the 
study at any time and for any reason, without penalty.   
The Principal Investigator (PI) and/or designee may withdraw a participant from continuing in the 
study for the following reasons: 
• 
Pregnancy 


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• 
Significant study intervention non-compliance  
• 
If any clinical adverse event (AE), laboratory abnormality, or other medical condition or 
situation occurs such that continued participation in the study would not be in the best 
interest of the participant 
• 
Disease progression which requires discontinuation of the study intervention 
• 
If the participant meets an exclusion criterion (either newly developed or not previously 
recognized) that precludes further study participation 
• 
It is considered to be in the participant’s best interest, or if the participant is not willing or 
able to comply with the study requirements.   
6.4.2 Handling of Participant Withdrawals or Termination 
Every effort will be made to undertake protocol-specified safety follow-up procedures to capture 
AEs, serious adverse events (SAEs), and unanticipated problems (UPs). In the event of withdrawal 
from study, reasonable efforts should be made to conduct the following procedures: 
x 
Update any ongoing AE/SAEs that remain ongoing at time of participant’s last visit prior to 
withdrawal  
x 
Query about AEs, SAEs and concomitant medications if the interval between the 
participant’s last visit and the time of withdrawal is within the protocol defined reporting 
period 
x 
Physical examination 
x 
Blood for safety laboratory testing if withdrawal occurs before Visit 2 
x 
Update contact information 
The reason for participant discontinuation or withdrawal from the study will be recorded on the 
Case Report Form (CRF). Participants who sign the informed consent form and are randomized but 
do not receive the challenge may be replaced.  Participants who sign the informed consent form, 
and are randomized and receive the challenge, and subsequently withdraw, or are withdrawn or 
discontinued from the study, will not be replaced. 
 
6.5 Premature Termination or Suspension of Study  
Although the study Sponsor has every intention of completing this study, it may be temporarily 
suspended or prematurely terminated if there is sufficient reasonable cause. The Sponsor reserves 
the right to terminate the study at any time for clinical or administrative reasons. Written 
notification, documenting the reason for study suspension or termination, will be provided by the 
suspending or terminating party to PI and the FDA. If the study is prematurely terminated or 
suspended, the PI and/or designee will promptly inform the IRB and will provide the reason(s) for 
the termination or suspension.  
Circumstances that may warrant termination or suspension include, but are not limited to:  
• 
Determination of unexpected, significant, or unacceptable risk to participants  
• 
Demonstration of efficacy that would warrant stopping  
• 
Insufficient compliance to protocol requirements  
• 
Data that are not sufficiently complete and/or evaluable  
• 
Determination of futility  
Study may resume once concerns about safety, protocol compliance, data quality are addressed and 
satisfy the sponsor, IRB and/or FDA. 


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7 
STUDY AGENTS 
 
7.1 Study Agent and Control Description  
7.1.1 Product Description 
The iOWH032 drug product is a chemically and physically stable immediate-release oral tablet.  The 
key characteristics of iOWH032 tablets include fast disintegration time (less than five minutes), 
similar to micro-suspension (which was used for phase 1 SAD and MAD studies), and good chemical 
and physical stability under long-term storage conditions. The placebo will be matched in terms of 
visual appearance, disintegration time, and taste. 
7.1.2 Manufacturer   
The iOWH032 drug substance for this study was manufactured by Albany Molecular Research, Inc., 
Albany, New York, United States (Batch Number 6798).  
 
The iOWH032 drug product and placebo for this study will be manufactured by Syngene 
International Ltd., Bengaluru, India (Batch Number TBD) 
7.1.3 Acquisition 
iOWH032 drug product and placebo will be shipped to the site from Syngene International Ltd., 
Bengaluru, India.   
7.1.4 Formulation, Appearance, Packaging, and Labeling   
Each 250 mg iOWH032 tablet contains 40% of iOWH032 (w/w) and 60% excipients (w/w) including 
Mannitol 60®, Avicel® PH-101, Kollion CL-F®, Kollidon30®, Aerosil® 200 Pharma, and magnesium 
stearate. The matching placebo is made of the same excipients, but without the active drug 
substance. 
 
Uncoated iOWH032 tablets and matching placebos have a white, circular, biconvex appearance and 
their manufacturing process involves blending, wet granulation, drying, milling, and tableting. 
 
All packaging and labeling operations will be performed according to current Good Manufacturing 
Practices (cGMPs) and Good Clinical Practices (GCPs) and local requirements.   
 
All drug packages should be inspected on receipt at the study site for signs of damage. Evidence of 
damaged study drug is to be reported to the sponsor and stored under the labeled conditions until 
further instructions have been provided. 
7.1.5 Product Storage and Stability 
The study drugs must be kept in a securely locked, substantially constructed enclosure to which 
access is limited (e.g., locked cabinet). The investigator will take adequate precautions, including 
locked storage, to prevent theft or diversion of the study drug, consistent with International Council 
on Harmonisation-Good Clinical Practice (ICH-GCP). Within the locked storage area, study drug will 
be stored in accordance with the conditions specified on the drug labels (i.e. stored in tightly closed 
bottles at or below 30°C without direct exposure to sunlight) until used.  
 


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On receipt of the study drug, the investigator or designee will perform an inventory of the shipment, 
comparing the shipment inventory to study drug actually received, and complete and sign an 
inventory log. The investigator or designee must count and verify that the shipment contains all of 
the items appearing on the shipment inventory. The investigator must immediately notify PATH or 
the drug distribution contractor of any damaged or unusable study drug that the site receives, and 
document any damaged or unusable study drug in the inventory log.  
 
The investigator or designee will retain a copy of the shipment inventory received with the drug 
supply in the study file, send a copy to the PATH-designated representative, and forward the original 
to the drug distribution center.  
 
Previous lots of iOWH032 have demonstrated good physical and chemical stability under a variety of 
storage conditions (25°C/60% RH, 30°C/75% RH, 40°C/75% RH) in studies lasting 18 months. The 
current lot will be under stability for 18 months at 25°C/60% RH. 
7.1.6 Preparation 
Dose preparation, as described in detail in the Pharmacy Manual, will be carried out by a qualified 
unblinded research pharmacist.  
7.1.7 Dosing and Administration   
All participants will receive 500 mg iOWH032 (two 250 mg tablets) or matching placebo three times 
a day with meals, approximately 8 hours part, for 3 consecutive days according to randomized 
treatment assignment. The treatment will begin at the onset of diarrhea symptoms (first Grade 3 or 
over stool) or 48 hours after challenge, whichever is sooner. 
7.1.8 Route of Administration  
iOWH032 and placebo tablets will be administered orally.   
7.1.9 Starting Dose 
All doses are set at 500 mg (two 250 mg tablets) for iOWH032 or two matching placebo tablets. 
7.1.10 Dose Adjustments/Modifications/Delays 
No dose adjustments are allowed.  A delay in dosing for safety reasons is allowed, in case of delayed 
dosing those participants will be removed from the pK population.  PATH Medical Officer should be 
notified if the investigator delays dosing due to safety reasons.  PATH Medical Officer, in 
collaboration with the investigator, will determine if dosing can be reinitiated. The date and reason 
that dosing was delayed must be recorded in the source documentation and case report form, as 
applicable. 
7.1.11 Duration of Therapy 
Study drug will be administered three times a day, approximately 8 hours part, for 3 consecutive 
days.   
7.1.12 Tracking of Dose 
Each dosing will be directly observed by study staff. Appropriate staff will record each study drug 
administration (date and stop/start times) in the participant’s chart (source document) and 
electronic case report form (eCRF).  


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7.2  Challenge Strain Description 
7.2.1 Product Description 
Freshly-harvested, wild-type V. cholerae El Tor Inaba strain N16961 will be the challenge agent. 
Strain N16961 will be delivered at 106 cfu, suspended in 30 mL of sodium bicarbonate solution 
(~1.3% NaHCO3). 
7.2.2 Manufacturer   
Investigators at CVD developed the cholera challenge model using freshly harvested V. cholerae El 
Tor Inaba strain N16961. For this study, CVD will develop the master cell bank to be administered to 
participants. 
7.2.3 Acquisition 
Freshly-harvested, wild-type V. cholerae El Tor Inaba strain N16961 will be prepared by University of 
Maryland, Baltimore (UMB), Center for Vaccine Development (CVD). Vials of strain N16961 are 
stored in the CVD Clinical Microbiology Lab at -70°C (-60°C or colder). 
7.2.4 Formulation, Appearance, Packaging, and Labeling   
Final containers hold 1 mL strain N16961 with ~109 cfu/mL in cryovials with gasketed screw-caps. 
The following information is contained on the label of each vial: “New Drug Limited by Federal (US) 
Law to Investigational Use”, Lot #xxx, Manufacture date xx/xxx/xxxx.” 
7.2.5 Product Storage and Stability 
It is anticipated that the cell bank will be stable for years if maintained frozen at -70°C (-60°C or 
colder). 
7.2.6 Preparation 
Two days before the challenge date, inoculum preparation will begin from a WCB vial of frozen V. 
cholerae strain N16961.The WCB strain will be plated onto Hy-Soy Agar plates. After incubation at 
37°C overnight, one colony will be selected and will be plated onto Hy-Soy Agar plate and 
Thiosulfate-citrate-bile salts-sucrose agar plate (TCBS). After incubation at 37°C overnight, colonies 
that exhibit characteristic Vibrio appearance will be picked for heavy growth of organisms as well as 
for identity tests (morphology, Gram’s staining and agglutination with Vibrio antiserum). From the 
heavily seeded Hy-Soy Agar plates, growth will be harvested into sterile phosphate buffered saline 
(PBS). The bacterial suspension will be diluted with sterile PBS to produce a turbidity measured by 
optical density (OD) 660 nm to correspond to the desired bacterial count of a target of 106 CFU/mL. 
The final stock dilution of challenge inoculum will be in a tube labeled with:  organism identity, 
concentration, date, time, and “Caution: New Drug (limited to investigational use)”. This tube will be 
kept at room temperature and transported to the Research Isolation Ward. Inoculum colony counts 
will be performed before and after challenge to document the final concentration of inoculum.  
7.2.7 Dosing and Administration   
Fasting eligible participants will drink 120 mL of sodium bicarbonate solution (~1.3% NaHCO3); 
approximately 1 minute later, participants will ingest 106 cfu of the challenge strain suspended in 30 
mL of sodium bicarbonate solution (~1.3% NaHCO3).  Participants will have nothing by mouth, 
except water, for 90 minutes before or following ingestion of the challenge inoculum. 


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7.2.8 Route of Administration  
The challenge strain will be administered orally. 
7.2.9 Starting Dose 
The challenge dose is a single inoculum of 106 cfu of V. cholerae El Tor Inaba strain N16961. 
7.2.10 Dose Adjustments/Modifications/Delays 
No dose adjustments are allowed. Challenge will not be re-administered if a subject who vomits the 
challenge dose shortly after dosing.  
7.2.11 Duration of Therapy 
The challenge inoculum will be administered in a single dose only. 
7.2.12 Tracking of Dose 
The challenge inoculum dosing will be directly observed by study staff. Appropriate staff will record 
each study drug administration (date and stop/start times) in the participant’s chart (source 
document) and eCRF. 
 
7.3 Study Agents Accountability Procedures 
The site pharmacist will maintain complete records of all study drug received from the Sponsor and 
will be responsible for maintaining an accurate record of the randomization codes, inventory, and an 
accountability record of iOWH032, challenge material and placebo for this study.  The site 
pharmacist will also be responsible for ensuring the security of these documents.   At the end of the 
study, the site will receive instruction from the Sponsor regarding the final disposition of any 
remaining study drug.   
8 
STUDY PROCEDURES AND SCHEDULE 
 
8.1 Study Procedures/Evaluations 
8.1.1 Study Specific Procedures 
Study procedures and evaluations done as part of the study include: 
x 
Medical history: a comprehensive medical history will be collected by interview with the 
participant including participation in clinical trials, surgery, previous hospitalization, allergy 
to food/drugs, and history of any chronic or recurrent medical conditions.  
x 
Interval medical history: inquiry regarding changes since the last medical history discussion 
including signs and symptoms. 
x 
Medication history from the past 4-weeks and current medications taken, including 
prescription and over-the-counter medications. 
x 
Physical examination: full physical examination will include assessment of vital signs, head, 
eyes, ears, nose, oropharynx, neck, chest (auscultation), lymph nodes (neck, supraclavicular, 
axillary, inguinal), abdomen (auscultation and palpation), musculoskeletal, skin, and 
neurological.  
x 
ECG and pregnancy testing 
x 
Safety Laboratory: Blood, stool, and urine specimens collected and evaluated 
x 
Blood sampling for study drug pK analysis 
x 
Cholera Challenge, iOWH032 and placebo administration and antibiotic treatment   


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8.1.2 Standard of Care Study Procedures 
As all participants in this trial are healthy volunteers, there are no standard of care procedures that 
would have been conducted if the individuals were not enrolled into the trial.  
 
Standard of care procedures for the treatment of cholera include management of hydration levels, 
electrolyte balance, and also prescription with a course of antimicrobials. Details of these 
procedures can be found in Section 9.  
 
8.2 Laboratory Procedures/Evaluations 
8.2.1 Clinical Laboratory Evaluations 
Protocol-mandated clinical screening and safety laboratory tests will be conducted in real time by 
laboratories that are properly accredited. These tests include: 
x 
Biochemistry: Sodium, Potassium, creatinine, alkaline Phosphatase, total bilirubin, alanine 
aminotransferase (ALT), albumin, aspartate aminotransferase (AST), Creatinine 
Phosphokinase (CPK), Glucose 
x 
Hematology: hemoglobin, white blood cells with differential count, platelet count 
x 
Serology: HbSAg, anti-HCV, and HIV 
x 
Blood typing 
x 
Pregnancy test: Serum β-HCG 
Screening labs may be repeated once if certain values are outside the acceptable ranges and if the 
value does not increase the risk to the subject in the opinion of the clinical investigator. If a subject 
with repeated Grade 1 abnormal lab values judged not clinically significant by the PI, with the 
concurrence of the Sponsor’s Medical Officer, she/he will remain eligible for enrollment into the 
study.  
 
Additional blood specimens (2 mL) will be collected for iOWH032 pK analysis Day 1 and Day 3 of the 
study.   
8.2.2 Stool Specimen Preparation, Handling, and Storage 
Stool Culture: All loose stools will be collected, graded, and weighed while volunteers are in the 
Clinical Research Unit. Quantitative cultures will be performed on the first two stool samples of each 
24-hour period prior to the initiation of antibiotics. Stool specimens will be inoculated onto 
thiosulfate citrate bile salts sucrose (TCBS) agar plates either directly or after overnight incubation 
enrichment in alkaline peptone water before plating onto TCBS agar. Up to 2 stools daily will be 
cultured to determine the number of organisms per gram of stool. A rectal swab will be obtained if 
no stool was passed. Suspicious colonies will be agglutinated with polyvalent anti-O1 antisera.6 
Subsequent to the initiation of antibiotics, one stool sample for each 12-hour period (or a rectal 
swab if subject cannot produce stool) will be submitted for qualitative culture only. Three sequential 
negative stool cultures are required for discharge from the Clinical Research Unit.  
8.2.3 Specimen Shipment 
All biological specimens will be transported using packaging mandated by CFR 42 Part 72.  All 
dangerous goods materials, including diagnostic specimens and infectious substances, must be 
transported according to instructions detailed in the International Air Transport Association 
Dangerous Goods Regulations. Biohazardous waste will be contained according to institutional, 
transportation/carrier, and all other applicable regulations.   


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8.3 Study Schedule 
8.3.1 Screening (Outpatient Clinic Visit 1) 
Potential volunteers may be screened for eligibility up to 85 days prior to enrollment. Interested 
potential participants will be recruited through standard procedures at Pharmaron and brought to 
the study site for screening and consenting. 
Pharmaron research staff will provide a detailed description of all aspects of the study, including the 
rationale and background, the public health significance, the procedures and schedule of visits, and 
a detailed discussion of the risks and the presence or absence of benefit to them, as appropriate.  
After the informed consent form is signed, the participant is interviewed one-on-one by a member 
of the study team to discuss the study. A brief written examination is administered to assess the 
volunteer’s comprehension of the study (i.e., Comprehension Assessment Tool). If this quiz is passed 
(≥70% correct answers), the research staff complete the medical history and concomitant 
medication forms, draw blood for eligibility testing, and obtain an electrocardiogram (ECG). The 
participant also meets the principal investigator, or designee, and has a physical examination to 
complete the eligibility screening. If the participant has passed all the eligibility criteria, he/she is 
then invited to proceed in the study and is given an appointment for the next visit.  The screening 
procedures may be completed in a single day or on multiple days, as long as the screening 
procedure dates do not exceed 85 days prior to challenge. 
It is recognized that volunteer studies must be carried out in an environment where no coercion is 
applied, and where volunteers can be adequately informed of the purpose, nature, procedures, risks 
and hazards of the study. To assess and document comprehension of the material presented, each 
participant must pass a written quiz (score 70% or higher) containing approximately 20 multiple 
choice and true/false questions covering all aspects of the study including the purpose, procedures, 
risks, benefits and pertinent microbiology. Incorrect answers will be reviewed with the participant.   
If the participant scores at least 50% the quiz may be retaken once, after further education and a 
review of the consent form. The Comprehension Assessment Tool is dated and signed by the 
participant and by a research staff member and made part of the permanent record.  
Another important feature of the consent process is the repeated demonstration of both initiative 
and reliability by the prospective participant. There are multiple opportunities for the participant to 
decline to proceed further in the process. This deliberate education and screening process 
contributes to the informed nature of the participant’s consent. The process also increases the 
likelihood that the participant will be committed to completing the entire inpatient containment 
portion of this study. Prospective participants will be carefully screened to ensure that they are in 
good physical and mental health. 
The screening procedures include:  
• 
Signed, witnessed informed consent 
• 
Administration of the study Comprehension Assessment Tool  
• 
Obtain vital signs (oral temperature, blood pressure, pulse, height, and weight) 
• 
Collection of medical history 
• 
Collection of concomitant medication history 
• 
Perform physical examination, to be performed by a study PI or designee 
• 
Perform 12-lead electrocardiogram (ECG); the interpretation of screening ECG eligibility is 
defined in Appendix B. 


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• 
Obtain the following screening laboratory studies (~18 mL blood); the acceptable values for 
the screening laboratory tests are defined in Appendix B: 
o 
Complete Blood Count (CBC) with differential and platelet count for the evaluation of 
WBC, ANC, Hemoglobin, and Platelets 
o 
Sodium, Potassium, Creatinine, Albumin, AST (SGOT), ALT (SGPT), Alkaline Phosphatase, 
and Total Bilirubin, CPK, Glucose 
o 
Blood Type (e.g., A, B, or O) 
o 
Urinalysis for the evaluation of glucose and protein 
o 
Pregnancy test (serum β-HCG) 
o 
HIV antibody 
o 
Hepatitis B surface antigen 
o 
Hepatitis C antibody 
Note: Since intravenous fluids is a part of the planned management of severe diarrhea, if more than 
3 attempts at venipuncture are required for obtaining screening labs, then we will consider that 
person ineligible on the basis of poor venous access. 
8.3.2 Inpatient Containment Period 
Study participants determined to be eligible will be scheduled for admission to the inpatient 
containment unit (Clinical Research Unit). The anticipated duration of the inpatient stay is 11 days, 
participant to completion of discharge criteria, described below. 
8.3.3 Acclimatization (1 day prior to challenge, Day -1) 
Study participants will start their inpatient stay 1 day prior to challenge for acclimatization, during 
which we educate and familiarize each participant with the protocol-required procedures (e.g., stool 
handling), hygiene practices, and the “Rules and Procedures” to be followed while in the inpatient 
unit.  In addition, during the acclimatization period, we monitor behavior, person-to-person 
interactions, mood, etc. to assess each study participant for any behavior or attitudes, which might 
not be appropriate for an inpatient containment study (i.e., combativeness, anti-social behavior, 
anger outbursts, destruction of property, etc.).  Any evidence that a participant may demonstrate 
which might pose a safety risk to themselves, other participants, or staff could be cause for 
ineligibility for challenge and the remainder of the inpatient stay. Refusal to comply with protocol-
required procedures, adherence to hygiene practices, or repetitive breaking of the “Rules and 
Procedures” could also constitute ineligibility.  This observation during the acclimatization period 
may be considered an imperfect and rather subjective method, but we have not identified any other 
good alternate objective measures that substitute for this direct observation procedure.  Any 
participant who is deemed ineligible will be discharged, prior to challenge.  
Baseline Clinical Safety Laboratory (~10 mL) will be obtained prior to challenge to include: 
x 
Complete Blood Count (CBC) with differential and platelet count for the evaluation of WBC, 
ANC, Hemoglobin, and Platelets 
x 
Sodium, Potassium, Creatinine, AST (SGOT), ALT (SGPT), Albumin, Alkaline Phosphatase, and 
Total Bilirubin, CPK, Glucose 
A serum pregnancy test (must be negative) will be performed for all women. A pre-challenge stool 
sample will be collected as a baseline, however if a subject fails to produce a screening stool sample 
it will not be considered a protocol deviation. 
All participants that continue to demonstrate eligibility and provide continuing consent will fast 
(defined by nothing by mouth except for water) for 90 minutes before ingestion of the challenge 
inoculum. 


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8.3.4 Challenge Day (Day 1) 
On the morning of challenge, fasting participants will have baseline vitals (oral temperature, pulse, 
and blood pressure) recorded and a final eligibility confirmation will be completed prior to oral 
ingestion of the challenge inoculum.   
Fasting eligible participants will drink 120 mL of sodium bicarbonate solution (~1.3% NaHCO3); 
approximately 1 minute later, participants will ingest 106 cfu of the challenge strain suspended in 30 
mL of sodium bicarbonate solution (~1.3% NaHCO3).  Participants will have nothing by mouth, 
except water, for 90 minutes before and following ingestion of the challenge inoculum. 
All back-up study participants that are not challenged will be discharged from the inpatient ward 
upon the completion of the target number of challenges.  Blinded randomization will occur for all 
participants that successfully complete the ingestion of challenge.  
8.3.5 Post-Challenge Observation Period (Day 1 through discharge) 
At the onset of symptoms or by 48 hours after ingesting challenge inoculum of 106 cfu of V. cholerae 
El Tor Inaba strain N16961, blinded study drug will be administered for 3 days every 8 hours to all 
study participants. After each dose of blinded study drug, the following tolerability will be assessed: 
nausea, abdominal discomfort and pain, occurrence of abdominal cramps, occurrence of headache, 
and occurrence of malaise, anorexia, pollakiuria, micturition urgency, sinus tachycardia, increased 
alertness ; an attribution must be determined as related to study drug, cholera, or an alternate 
etiology (unrelated to study). 
Following ingestion of the challenge inoculum, the following procedures will be performed: 
• All stools will be graded for consistency (Grade 1-5) and any diarrheal stool (Grade 3 or 
higher) will be weighed.  We assume a 1:1 weight per volume conversion for diarrheal stools.  
• Any vomitus will also be collected and weighed.   
• Vital signs will be measured at least 3 times daily (approximately every 8 hours). On Day 1 
including the pre-challenge vitals assessment, there will be 4 vital signs assessments (1x pre-
challenge and 3x post-challenge). Vital signs will also be measured every 4 hours when a 
participant has a fever ≥ 39˚C (102.1˚F) and diarrhea, until the participant has 2 consecutive 
temperatures of ≤ 38˚C (100.4˚F) and cessation of diarrhea. (See Section 9 for detailed 
procedures for the management of illness) 
• During the first 7 days following challenge, the highest grade of an adverse symptom over 
that day will be assessed.  The documentation of the previous day’s highest grade of an 
adverse symptom (prior 24 hours) will include an attribution: study drug, cholera, or an 
alternate etiology (unrelated to study). The anticipated subjective adverse symptoms include 
abdominal discomfort and pain, nausea, abdominal cramps, malaise, headache, and 
anorexia. These adverse symptoms will be graded according to the scales shown in Appendix 
E. 
• For each of the first 7 days, the maximum temperature, total diarrheal stool volume, number 
of diarrheal stools, total vomitus volume, and number of vomiting episodes will be 
calculated. 
x 
Serum samples will be collected 7±1 hour after the first and the last dose of the iOWH032 for 
pK analysis 
The management of clinical illness is described in Section 9.0 and Manual of Operating Procedures 
(MOP); including: criteria for oral and/or intravenous rehydration, necessity of increased frequency 
of vital signs monitoring, unscheduled physical examinations, obtaining clinical safety labs, and the 
initiation of antibiotic or anti-pyretic therapy. 


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8.3.6 
Discharge Criteria 
Participants will not be allowed to leave the Clinical Research Unit until they demonstrate that they 
are no longer infectious and cannot present a hazard to the community. Therefore, each participant 
must meet all the following criteria to be discharged: 
• The participant has at least three negative sequential stool cultures (separated by ~12 hours 
apart each) from which V. cholerae does not grow 
• The absence of moderate or higher grade objective reactogenicity (diarrhea, fever, and 
vomiting) for at least 12 hours prior to discharge 
• The participant has completed a 3-day course of antibiotic therapy, which may include  
o Ciprofloxacin 500 mg, twice daily 
o Azithromycin 500 mg, once daily (for those persons who are allergic to 
fluoroquinolones) 
o Doxycycline 300 mg, once daily (for those persons who are allergic to 
fluoroquinolones) 
8.3.7 
Early Termination/Early Discharge 
Participants that desire to withdraw from the study or are withdrawn by the PI (e.g., due to 
continuing non-compliance or safety concerns) during the inpatient period will be given instructions 
on appropriate follow up. If a participant desires to leave the inpatient containment unit early, we 
will need to ensure that the participant is not infectious to the community.  Therefore, we plan to 
administer either Ciprofloxacin 1000 mg or Azithromycin 1000 mg, as a directly observed oral dose 
prior to discharge—selection of antimicrobial will be based on potential allergies to medications and 
the subject must have a negative stool culture before being allowed to leave.  We will provide 
medication and give instructions for 2 additional days of the respective antimicrobial, for self-
administration.  A document of early termination/withdrawal will be completed by the study staff 
and co-signed by the participant.  The participant will not be compensated for any inpatient days 
that are not completed, including the day of early termination. 
8.4 Outpatient Clinic Visit 2 (Day 15 ±1) 
Participants will be scheduled for an outpatient visit shortly after discharge, approximately 2 weeks 
after challenge. During this visit we will assess the emergence of adverse events and for any changes 
to medical history and concomitant medications. 10 mL blood will be drawn for safety labs. 
8.5 Outpatient Clinic Visit 3 (Day 29 ±2) 
Participants will be scheduled for an outpatient visit approximately 4 weeks after challenge. During 
this visit we will assess the emergence of adverse events and for any changes to medical history and 
concomitant medications. This is the last clinic visit scheduled. 
8.6 Final Follow-up – Visit 4 (Day 180 ±14) 
A final follow up visit will be completed by phone.  This visit will only be for the collection of serious 
adverse events. 
8.7 Unscheduled Visit(s) 
Participants that experience any serious or severe adverse effects or experience an event of concern 
can be scheduled for an outpatient visit to have further evaluation. If an unscheduled visit occurs, a 
member of the clinical study team (PI, sub-investigator, nurse coordinator, or clinical nurse) will 
interview and evaluate the participant to determine the cause of the visit and provide care as 
needed. 


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8.8 Concomitant Medications, Treatments, and Procedures 
Drugs used during the study will be reported in the Concomitant Medication CRF, including both 
prescription, non-prescription, and over-the-counter medication. 
 
8.9 Prohibited Medications, Treatments, and Procedures 
Treatment with drug listed in Appendix F and antimicrobial agents other than the ones that will be 
used for the treatment of Cholera diarrhea will not be permitted during the inpatient period unless 
discussed and approved by the PATH Medical Officer and PI. 
9 
MANAGEMENT OF EXPECTED CHOLERA ILLNESS 
9.1 Clinical Evaluation 
Subsequent to cholera challenge, participants will remain on the ward for approximately 10-11 days.  
Vital signs and oral temperature will be measured at least every 8 ±1 hours by staff nurses who 
remain on the ward 24h a day. Participants will be interviewed daily by a study physician to 
determine the occurrence of illness signs and symptoms (e.g., anorexia, malaise, abdominal cramps, 
and headache) which occurred during the previous study day; these data will be recorded on a 
standardized form and graded in severity according to Appendix E.  A focused physical examination 
may be performed at the discretion of the physician according to the nature of a participant’s 
complaint. 
9.2 Measurement of Diarrhea and Vomitus 
Since diarrhea is anticipated to be a common occurrence, all participants will be expected to collect 
every stool that is passed, from the time of challenge until discharge.  Participants will be instructed 
to use a plastic stool collection basin, commonly called a “hat”.  All stools will be graded by the study 
staff.  The grading of stool is based on consistency and the definition of diarrhea is a grade 3 or 
higher stool, as follows: 
x 
Grade 1 – well formed (normal stool, does not take the shape of the container) 
x 
Grade 2 – soft (normal stool, does not take the shape of the container) 
x 
Grade 3 – thick liquid (diarrhea, takes the shape of the container readily) 
x 
Grade 4 – opaque watery diarrhea 
x 
Grade 5 – rice water diarrhea (clear watery) 
Any Grade 3 or higher stool (diarrheal stool) must be weighed, to estimate the volume of fluid loss 
(assume ~1 g diarrheal stool = 1 mL of fluid lost).   
Similarly, any episodes of vomiting should be collected in either a stool “hat” or a plastic “kidney” 
basin, provided.  If a vomiting or diarrhea episode is not able to be collected in a basin (e.g., the 
participant has an “accident” while sleeping or before they are able to reach a toilet), then the 
volume of the output will be estimated. 
9.3 Management of Fluid Loss 
Oral Rehydration Solution (ORS) will be offered as the primary means of management of fluid loss.  
Participants who develop diarrheal stools (Grade 3 or higher) during the inpatient observation will 
be required to ingest standard WHO Oral Rehydration Solution (ORS) at 1.5 times the stool volume. 
Vomitus will be replaced with ORS in equal amounts, 1:1 ratio. At the discretion of the investigator, 
additional ORS may be administered. ORS will be prepared according to the manufacturer’s package 
insert (Jianas Bros ORS).  Unused ORS should be discarded 24 hours after preparation. 


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For the duration of diarrhea, the participant will be requested to provide a urine specimen from 
every void that they experience; the specimen will be tested for urine specific gravity.  In the event 
that intravenous fluids are required, serum electrolytes (Sodium, Potassium, Chloride, Bicarbonate), 
BUN, and creatinine will be measured.   
A physician is available by telephone or beeper at all times. Nurses will notify the on-call physician if 
any of the following occurs in a participant who is experiencing diarrhea and/or vomiting: 
x 
Syncope 
x 
Complaint of dizziness or lightheadedness or established orthostatic hypotension 
x 
Urine specific gravity > 1.025 
x 
> 500 mL behind in ORS replacement 
x 
Vomiting of ≥ 500 mL once or total volume within the past 4 hours 
x 
High fever ≥ 39qC (102.1qF) 
x 
Severe headache, severe malaise, or severe abdominal pain 
x 
Participant has a complaint for which he/she requests treatment 
x 
Any other clinical situation that concerns the nurse 
 
If a participant develops severe watery diarrhea or persistent vomiting and cannot maintain full 
hydration by oral means, according to the criteria on section 9.5, IV fluid replacement will be 
administered.   
9.4 Frequency of Vital Signs Assessment 
Vital signs (blood pressure, pulse, and oral temperature) will be measured approximately every 8±1 
hours, unless more frequent monitoring is needed. Once a participant has passed a diarrheal stool 
(Grade 3 or higher), vital signs will be measured every 4 hours until the participant passes a Grade 1 
or 2 stool or 24 hours have passed since the last Grade 3 – 5 stool, whichever comes first.   
Vital signs will also be measured every 4 hours when a participant has a fever ≥ 39qC (102.1qF), until 
the participant has 2 consecutive temperatures of ≤ 38qC (100.4qF).  
Any participant that complains of dizziness or lightheadedness upon standing will have orthostatic 
blood pressures assessed—BP after supine for ~5 minutes and BP after standing ~2-3 minutes. 
Orthostatic hypotension is defined as a drop in systolic BP > 20 mmHg or in diastolic BP > 10 mmHg. 
9.5 Fluid Therapy/Hydration 
Intravenous fluids (Lactated Ringers solution) will be administered to participants with diarrhea who 
meet any of the following criteria: 
x 
Orthostatic hypotension 
x 
Urine specific gravity > 1.025 
x 
No urine output for ≥ 8 hours 
x 
> 1000 mL deficit in ORS replacement 
x 
At the investigator’s discretion, based on clinical evaluation or on a participant’s difficulty in 
keeping up with ongoing diarrheal losses by oral rehydration alone 
Intravenous therapy will continue until the above criteria are no longer satisfied, the participant is 
able to take fluid by mouth, and a study physician determines that it is no longer required. 
9.6 Indications for Antibiotics 
Antimicrobials will be administered immediately if a participant meets the criteria for severe cholera 
diarrhea, defined as ≥5 L (5 kg) of cumulative diarrheal stool.  All other participants who have not 


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met the criteria for severe cholera diarrhea will receive antibiotics at approximately 4 days after the 
challenge. Antimicrobials will continue for the completion of a 3-day course. In rare and unexpected 
instances, the study investigator will be allowed to administer antimicrobials for alternate reasons—
e.g., if in his/her judgment it is necessary to ensure the safety of the participant.  
The administration of a defined course of antibiotics (which starts, at the latest, on the fourth day 
post-challenge) results in the rapid elimination of susceptible cholera organisms from the stool, such 
that it is extremely unlikely that the discharge criterion (for three sequential negative stool cultures, 
~12 hours apart) will fail to be achieved by the tenth day post-challenge.  
If cholera organisms continue to be grown from stool cultures after the completion of the 3-day 
course of antibiotics, then another course of antibiotics can be entertained.  Study participants will 
continue to be required to reside in the Clinical Research Unit until all the discharge criteria are met.  
Participants will be compensated for the additional days on the Clinical Research Unit.  This event is 
considered to be a very rare likelihood since failure to eradicate the cholera challenge strain never 
occurred during nearly 40 years of experience using the N16961 strain. 
9.7 Indications for Other Concomitant Medications 
Any concomitant medication, prescription or over-the-counter, will be evaluated for continuation 
during the inpatient setting.  Any such medication will need to be discussed with and approved by 
the investigator prior to admission.  The supply of such medication will be the responsibility of the 
participant and will be handed over to the research staff upon admission; daily administration will 
be recorded.  Any un-declared prescription or over-the-counter medications that are discovered 
during the inpatient stay will constitute a violation of the ward “Rules and Procedures.”  
Other medications may be administered during the study period as follows: 
x 
Smoking is not allowed on the ward, but participants will be able to request a nicotine 
patch.  
x 
An anti-pyretic and analgesic (i.e. acetaminophen) may be prescribed for severe headaches, 
other pains, or fevers (e.g., sustained temperatures of ≥ 39qC).   
x 
At the investigator’s discretion (e.g., upon review of serum electrolyte results during severe 
diarrhea), oral potassium may be administered for repletion of electrolyte losses. 
x 
Other medications, which are deemed necessary for the safety and welfare of the 
participant  
The prescription of any medication must be ordered and signed by the investigator and each 
administration recorded. 
10 ASSESSMENT OF SAFETY 
The assessment of the safety of the study drug and challenge agent will be through the detection 
and documentation of adverse effects, both solicited adverse events (AEs) and unsolicited AEs, 
and/or clinically significant laboratory abnormalities, through 28 days post-challenge. Only the 
occurrence of serious adverse events (SAEs) will be reported between 28 days and 180 days post-
challenge. 
10.1 Specification of Safety Parameters 
10.1.1 Definition of Adverse Event (AE) 
An adverse event is any untoward medical occurrence in a participant after administration of the 
investigational products and that does not necessarily have a causal relationship with the 


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investigational products.  An AE can therefore be any unfavorable and unintended sign (including 
abnormal laboratory findings), symptoms, physical examinations, or disease temporally associated 
with the use of the investigational products, whether or not related to the investigational products.  
This definition includes exacerbations of pre-existing conditions.  Stable pre-existing conditions 
which do not change in nature or severity during the study are not considered AEs; however, these 
should be reported as part of the medical history. In certain instances, it may be not be possible to 
distinguish between relatedness due to study drug or cholera; in those cases an AE may be noted as 
related to both. 
Any AEs that are reported after signing the study ICF up to the Day -1 inpatient ward admission will 
be reported as a part of the medical history. 
Solicited AEs are pre-specified AEs that could potentially be in association with the study drug or 
cholera infection. Investigators will attempt to assign causality of solicited AEs to either the study 
drug, cholera infection, or an alternate etiology. A solicited AE is a predetermined event, identified 
in the Investigator’s Brochure, which may reflect safety concerns related to the investigational 
product. 
Unsolicited AEs are any AEs reported spontaneously by the participant, observed by the study 
personnel during study visits or those identified during review of medical records or source 
documents. Investigators will attempt to assign causality of unsolicited AEs to either the study drug, 
cholera infection, or an alternate etiology. 
10.1.2 Definition of Serious Adverse Event (SAE) 
A serious adverse event (SAE) is any adverse event that results in any of the following outcomes: 
1. Death 
2. A life-threatening event. Life-threatening events mean that the study participant was, in 
the opinion of the site PI or Sponsor, 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  
4. Results in persistent or significant incapacity or substantial disruption of the ability to 
conduct normal life functions  
5. Congenital abnormality or birth defect 
6. Important medical event that may not result in one of the above outcomes but may 
jeopardize the health of the study participant and/or requires medical or surgical 
intervention to prevent one of the outcomes listed in the above definition of serious 
adverse event. Examples of such medical events include allergic bronchospasm requiring 
intensive treatment in an emergency room or at home, blood dyscrasias or convulsions 
that do not result in inpatient hospitalization, or the development of drug dependency 
or drug abuse.  
All SAEs will be: 
• recorded in the research record  
• reported to the local IRB, per local IRB guidelines 
• reviewed and evaluated by a study clinician, the PI and Independent Medical Monitor  
• followed through resolution by a study clinician 
All SAEs, whether related or unrelated, will be reported to the PATH medical officer within 24 hours 
of site awareness.  


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10.1.3 Definition of Unanticipated Problems (UP) 
The Office of Human Research Protections (OHRP) considers unanticipated problems involving risks 
to participants or others to include, any incident, experience, or outcome that meets all of the 
following criteria: 
x 
Unexpected in terms of nature, severity, or frequency given (a) the research procedures that 
are described in the protocol-related documents, such as the IRB-approved research 
protocol and informed consent document; and (b) the characteristics of the participant 
population being studied; 
x 
Related or possibly related to participation in the research (“possibly related” means there is 
a reasonable possibility that the incident, experience, or outcome may have been caused by 
the procedures involved in the research); and 
x 
Suggests that the research places participants or others at a greater risk of harm (including 
physical, psychological, economic, or social harm) than was previously known or recognized. 
 
This study will use the OHRP definition of UP. 
 
10.2  Classification of an Adverse Event 
10.2.1 Severity of Event 
Severity of Adverse Events:  All AEs will be assessed by the clinician using a protocol-defined grading 
system in Appendix E.  For events not included in the protocol-defined grading system, the following 
guidelines will be used to quantify severity:  
Mild:  events require minimal or no treatment and do not interfere with the participant’s 
daily activities. 
Moderate:  events result in a low level of inconvenience or concern with the therapeutic 
measures. Moderate events may cause some interference with functioning. 
Severe:  events interrupt a participant’s usual daily activity and may require systemic drug 
therapy or other treatment. Severe events are usually incapacitating. 
Life threatening:  any adverse drug experience that places the participant, in the view of the 
investigator, at immediate risk of death from the reaction as it occurred, i.e., it does 
not include a reaction that had it occurred in a more severe form, might have caused 
death. 
Changes in the severity of an AE should be documented to allow an assessment of the duration of 
the event at each level of intensity to be performed.  Adverse events characterized as intermittent 
require documentation of onset and duration of each episode. 
10.2.2 Relationship to Study Agent or Cholera Infection 
Relationship (causality or attribution) of all AEs to the study drug, known cholera infection, or to an 
alternate etiology (unrelated to the study) is part of the documentation process, but it is not a factor 
in determining what is (or is not) reported in the study.  If there is any doubt as to whether a clinical 
observation is an AE, the event should be reported.  
The PI must assign a relationship of each AE to the receipt of the investigational product. The 
investigator will use clinical judgment in conjunction with the assessment of a plausible biologic 
mechanism, a temporal relationship between the onset of the event in relation to receipt of the 


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investigational product, and identification of possible alternate etiologies including underlying 
disease, concurrent illness, or concomitant medications. 
All AEs must have their relationship assessed using one of three terms:  related to study drug, 
related to cholera, or not related (an alternate etiology can be identified and is likely or feasible).  To 
help assess, the following guidelines are used. 
Related to study drug (i.e., iOWH032 or placebo) – There is a reasonable possibility (perhaps 
due to the timing of onset of the symptoms) that the study drug caused the adverse 
event. Reasonable possibility means that there is evidence to suggest a causal 
relationship between the study drug and the adverse event.  
Related to cholera - There is a reasonable possibility that the cholera infection caused the 
adverse event. Reasonable possibility means that there is evidence to suggest a 
causal relationship between the cholera illness and the adverse event. 
Not Related – There is not a reasonable possibility that the event is related to either the 
study drug (i.e., iOWH032 or placebo) or cholera and there is a reasonable alternate 
etiology. Thus, allergic reactions to the protocol-required antimicrobial will be 
classified as “not related” and the alternate etiology will be designated as a reaction 
to the antimicrobial. 
10.2.3 Expectedness 
The study investigator will be responsible for determining whether an AE is expected or unexpected. 
An AE will be considered unexpected if the nature, severity, or frequency of the event is not 
consistent with the risk information previously described for the study agent in the Investigator’s 
Brochure. 
 
10.3 Time Period and Frequency for Event Assessment and Follow-up 
The occurrence of an AE or SAE may come to the attention of study personnel during study visits 
or upon review by a study monitor. All AEs including local and systemic reactions not meeting the 
criteria for SAEs will be captured on the appropriate CRF. Information to be collected includes 
event description, time of onset, clinician’s assessment of severity, relationship to study product, 
and time of resolution/stabilization of the event. All AEs occurring through 28 days post-
challenge, must be documented appropriately regardless of relationship. All AEs will be followed 
to adequate resolution.  
 
Any medical condition that is present at the time that the participant is screened will be 
considered as baseline and not reported as an AE. However, if the study participant’s condition 
deteriorates at any time during the study, it will be recorded as an AE. UPs will be recorded in the 
data collection system throughout the study.  
Changes in the severity of an AE will be documented to allow an assessment of the duration of the 
event at each level of severity to be performed. AEs characterized as intermittent require 
documentation of onset and duration of each episode.  
The PI will record all reportable events with start dates occurring any time after the challenge is 
obtained until 28 (for unsolicited AEs) or 180 days (for SAEs) after the last day of study participation. 
At each study visit, the investigator will inquire about the occurrence of AE/SAEs since the last visit. 
Events will be followed for outcome information until resolution or stabilization. 
 


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10.4  Reporting Procedures 
10.4.1 Adverse Event Reporting 
To improve the quality and precision of AE data, the investigator should observe the following 
guidelines: 
• AEs must be graded, assessed for severity and causality, and reviewed by a site investigator. 
• Whenever possible, use recognized medical terms when reporting AEs and avoid the use of 
colloquialisms or abbreviations. 
• If known, report the diagnosis (i.e., syndrome or disease) rather than component symptoms, 
signs or laboratory values (e.g., report congestive heart failure rather than dyspnea, rales, and 
cyanosis); however, symptoms or signs that are considered unrelated to an observed syndrome 
or disease should be reported as individual AEs (e.g., if congestive heart failure and severe 
headache are observed at the same time, each event should be recorded as an individual AE). 
• AEs occurring secondary to other events (e.g., sequelae) should be identified by the primary 
cause. A ‘primary’ AE, if clearly identifiable, generally represents the most accurate clinical term 
to report. For example: orthostatic hypotension → fainting and fall to floor → head trauma → 
neck pain; the primary AE is orthostatic hypotension, which is what should be reported. If a 
primary SAE is reported, events occurring secondary to the primary event should be described in 
the narrative description of the case. 
• Death is an outcome of an event. The event that resulted in the death should be reported as 
the SAE. 
• For hospitalizations for surgical or diagnostic procedures, the illness leading to the surgical 
or diagnostic procedure should be recorded as the SAE, not the procedure itself. The procedure 
should be captured in the case narrative as part of the action taken in response to the illness. 
• Elective surgical or diagnostic procedures with or without hospitalizations (e.g., circumcision 
or elective abortion of a pregnancy) will not be recorded as an AE. The procedure should be 
captured in the case narrative as part of medical history. 
10.4.2 Serious Adverse Event Reporting 
The study clinician will complete a SAE Form within the following timelines:  
x 
All SAEs, whether related or unrelated, will be recorded on the SAE Form and submitted to 
the PATH Medical Officer within 24 hours of site awareness. See Section 1, Key Roles for 
contact information.  
 
Even if the investigator does not have complete information regarding an SAE, the SAE should still 
be reported within 24 hours and once additional relevant information is received, the report should 
be updated within 24 hours. The investigator should always provide an assessment of causality at 
the time of the initial report, even though this assessment may subsequently change based on the 
follow-up information received. After receipt of the initial report, PATH Medical Officer will contact 
the investigator if it is necessary to obtain further information for assessment of the event. 
 
All SAEs will be followed until satisfactory resolution or until the site investigator deems the event to 
be chronic or the adherence to be stable. Other supporting documentation of the event may be 
requested by PATH and should be provided as soon as possible. PATH will be responsible for 
notifying FDA of any unexpected fatal or life-threatening suspected adverse reaction as soon as 
possible but in no case later than 7 calendar days after the PATH’s initial receipt of the information. 


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10.4.3 Unanticipated Problem Reporting 
Incidents or events that meet the OHRP criteria for UPs require the creation and completion of 
an UP report form. It is the site investigator’s responsibility to report UPs to Advarra and to 
PATH. The UP report will include the following information:  
x 
Protocol identifying information: protocol title and number, PI’s name, and the Advarra 
Protocol number; 
x 
A detailed description of the event, incident, experience, or outcome; 
x 
An explanation of the basis for determining that the event, incident, experience, or outcome 
represents an UP; 
x 
A description of any changes to the protocol or other corrective actions that have been 
taken or are proposed in response to the UP. 
 
To satisfy the requirement for prompt reporting, UPs will be reported using the following timeline:  
x 
UPs that are SAEs will be reported to the IRB and to PATH within 24 hours of the investigator 
becoming aware of the event. 
x 
Any other UP will be reported to the IRB and to the study sponsor within 72 hours of the 
investigator becoming aware of the problem. 
x 
All UPs should be reported to appropriate institutional officials (as required by an 
institution’s written reporting procedures), the supporting agency head (or designee), and 
OHRP within 96 hours of the IRB’s receipt of the report of the problem from the 
investigator. 
10.4.4 Reporting of Pregnancy 
iOWH032 has not been shown to be safe during pregnancy, so pregnant women may not participate 
in the study and women with child-bearing potential must agree to use effective methods of birth 
control 4 weeks prior to challenge and continue throughout the study. Although every effort will be 
made to counsel women about the pregnancy precautions related to study participation, if a woman 
becomes pregnant during the treatment phase of the trial, efforts will be made to obtain permission 
to continue to follow the participant through the pregnancy and to document the outcome of the 
pregnancy. The pregnancy and its outcome will be documented, even if birth occurs after the 
scheduled end of the study for the participant. A pregnancy in a participant is not in and of itself an 
AE. A pregnancy is reported as an AE or SAE only when there is suspicion that the investigational 
product may have interfered with the effectiveness of contraception or there was a serious 
complication in the pregnancy including a spontaneous abortion or an elective termination for 
medical rationale. The participant will be asked to provide serial follow-ups on the status of her 
pregnancy as well as provide health information on her infant following delivery. The participant will 
be asked to provide copies of clinic visits only if needed. The participants will receive no further 
study drug but may continue to receive antimicrobials if needed. The participants will continue 
safety monitoring and all follow up visits as noted in the study schedule.  
 
Principal investigator will notify study Independent Medical Monitor and PATH Medical Officer for 
participants that become pregnant during the study. Each pregnancy must be reported immediately 
(within 24 hours of identification) by e-mail or fax to the sponsor. 
 


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10.5 Study Halting Rules 
The study is planned to have each cohort of participants to be challenged at the same time. The 
greatest potential for risk is with the intentional cholera infection and with the dosing of the study 
drug (during the first 5 days post-challenge).  Should one of the study halting criteria be met, all 
participants of that cohort (and the following cohort, if applicable) will continue to be actively 
monitored and managed for their clinical illness (if during the inpatient period) and continued to be 
followed for safety information (if during the outpatient period).  
If any of the following events occurs during the study, administration of IP will be halted to all subjects 
in the study until a thorough joint review of the safety concern is undertaken and completed by the 
Safety Review Committee (SRC) and PI. The halting rules refer to suspected adverse reactions and will 
be triggered automatically if any of the events described below are met during the conduct of the 
study: 
x One or more participants experience a serious adverse event related to the study product.  
x Two or more participants experience the same severe (Grade 3) AE of the same organ class 
(systemic toxicity, or clinical laboratory tests or vital signs) deemed related to the study 
product within seven days following the study drug administration 
x One serious and unexpected, solicited or unsolicited, suspected or confirmed adverse 
reaction evaluated by the PI, Independent Medical Monitor, and PATH Medical Officer 
and determined to be an unacceptable risk to the health and safety of other 
investigational product recipients 
 
10.6 Safety Oversight 
Safety monitoring/oversight will be performed by a Safety Review Committee composed of three 
voting members (including two independent medical experts in the field): a chair, co-chair, and an 
independent medical monitor. The primary responsibility of the SRC is to monitor participant safety. 
The responsibilities of the SRC and safety oversight activities are described in the protocol and will 
be clearly outlined in the SRC Charter. The SRC considers study-specific data as well as relevant 
background information about the disease, test article, and target population under study. The SRC 
will review challenge data shortly after completion of the inpatient phase and will review the 
interim analysis after Cohort 1 has completed study activities before proceeding to the second 
cohort.  One of the SRC members, a physician, will be a designated Independent Medical Monitor.  
The SRC will also be empowered to suspend the study, recommend amendments to the protocol, 
recommend proceeding with the next Cohort, and/or to request further information for their 
Additionally, SRC will review the safety data only post enrollment of Cohort 2. 
The Independent Medical Monitor will be responsible for regularly reviewing the cumulative safety 
data, including a review of safety laboratory test results and adverse event reporting, should there 
be a halt in the study or at the request of the SRC.  The SRC is empowered with halting the study at 
any time. Should there be a halt in the study, no further challenges or study drug dosing will be 
performed; all enrolled participants will continue to be followed; for participants that are in the 
Clinical Research Unit, the management of diarrhea or other symptoms will continue until 
resolution. Antimicrobials will be administered immediately if a participant meets the criteria for 
severe cholera diarrhea. Further drug administration, in accordance with the protocol, may be 
resumed with the concurrence of the SRC, PATH Medical Officer and the PI. The sponsor will make 
the final determination as to whether the study will be stopped permanently. If the study is 
permanently stopped, participants will be followed until their next scheduled study visit for the 
safety assessments or until AE/SAE resolution, whichever is longer. 


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11 CLINICAL MONITORING 
Site monitoring is conducted to ensure that the rights and well-being of human participants are 
protected, that the reported trial data are accurate, complete, and verifiable, and that the conduct 
of the trial is in compliance with the currently approved protocol/amendment(s), with GCP, and with 
applicable regulatory requirement(s). Monitoring refers to the methods used by sponsors of 
investigational studies, or Contract Research Organizations (CROs) delegated site monitoring 
responsibilities, to oversee the conduct of, and reporting of data from, clinical investigations. Site 
monitoring includes ensuring appropriate clinical investigator supervision of study site staff and 
third party contractors.  
• 
Monitoring for this study will be performed by The Total Approach.  
• 
Monitoring visits will occur on-site throughout the study and will include 100% data 
verification of primary endpoint data (such as clinical signs and fluid output data) and 
excretion data. Targeted 10% data verification will be conducted for vital signs and 
other non-critical data.   
• 
PATH Medical Officer will be provided copies of monitoring reports within 14 days of 
visit.  
• 
Details of clinical site monitoring are documented in a Clinical Monitoring Plan. The 
Clinical Monitoring Plan describes in detail who will conduct the monitoring, at what 
frequency monitoring will be done, at what level of detail monitoring will be performed, 
and the distribution of monitoring reports.  
• 
Independent internal audits will also be conducted, as described in the Pharmaron 
Quality Management Plan. The results of the internal audit are reviewed with the PI, 
coordinator, and Clinical Research Manager; Corrective Action Plans are formulated in 
response to any issue.   
12 STATISTICAL CONSIDERATIONS 
 
12.1 Statistical and Analytical Plans 
The statistical analysis will be conducted following the principles as specified in the International 
Conference on Harmonization (ICH) Topic E9 (ICH, 1998). The final statistical analyses will be 
performed after database lock and final unblinding of the data. 
 
Full details of the statistical analysis and methods will be documented in a statistical analysis plan 
(SAP) which will be finalized prior the interim analysis.  The SAP will follow the general guidance of 
this protocol, and will further describe the selection of participants to be included in the analyses, 
detailed statistical and analytical plans for both the interim and final analyses, and procedures for 
accounting for missing, unused, and spurious data. 
 
12.2 Statistical Hypotheses 
The null (H0) and alternative (H1) hypotheses of the primary efficacy endpoint analysis are as follows: 
H0:  
The diarrheal stool output rate from participants treated with iOWH032 is not lower 
than that of participants treated with placebo. 
H1:  
The diarrheal stool output rate from participants treated with iOWH032 is lower than 
that of participants treated with placebo. 
 


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The diarrheal stool output rate is defined as the total volume of diarrheal stools (mL, Grade 3 and 
above) divided by the number of hours between initiation of study product dosing and initiation of 
antimicrobial therapy. The hypothesis is one-sided to permit the interim analysis and clear description 
of efficacy/futility boundaries. See Section 12.5.8 for a description of the one-sided alpha levels to be 
used for testing the primary endpoint. 
 
This hypothesis test will be supplemented with two-sided confidence intervals for the difference of 
medians, as described below. Secondary efficacy endpoints regarding AUC of diarrheal stool volume 
as well as quantitative culture of cholera organisms (defined by AUC and peak shedding) from stool 
samples will be analysed with similar methods. 
 
The null (H0) and alternative (H1) hypotheses for the secondary analysis of the proportion of 
participants with moderate or severe diarrhea are as follows: 
H0:  
iOWH032 and placebo do not differ in the proportion of participants that develop 
moderate or severe diarrhea 
H1:  
iOWH032 and placebo differ in the proportion of participants that develop with 
moderate or severe diarrhea 
 
12.3 Timing of Analyses 
When all Cohort 1 participants have completed the inpatient containment period and primary 
efficacy endpoint data are available, an interim analysis of the primary efficacy endpoint and 
accompanying safety data will be initiated. The study is designed with binding efficacy and futility 
thresholds, described in Section 12.5.8. Cohort 2 will be enrolled following this interim analysis if 
neither overwhelming efficacy, nor evidence of futility is indicated, with the final analysis resulting 
from the combined data from both cohorts. This process will include the review of the safety data 
by the SRC and determination of lack of major safety signal. 
Additionally, topline results will be conducted on the after the efficacy data data from Cohorts 1 and 
2 have been collected (i.e. including data prior to Visit 2).  These topline results will include analysis 
of the primary efficacy endpoint, as well as the analyses of the secondary endpoints of proportion of 
participants with MSD, attack rate of any diarrhea, AUC of stool volume, duration of episode (time 
to first formed stool), and total number of loose stools conducted on the mITT population. In 
addition, subject disposition and demographic and baseline characteristics tables will be provided. 
The topline results will be unblinded only to the treatment-level and not to the individual subject. To 
avoid identification of individual subjects, some summary statistics such as minimum and maximum 
will not be presented in the summary tables.  
The final statistical analyses will be performed after database lock and final unblinding of the data. 
12.4 Analysis Datasets 
Assignment of participants to analysis populations will be completed prior to unblinding, for each 
cohort.   
 
For purposes of analysis, the following populations are defined: 
 
Population 
Description 
Enrolled  
All participants who sign the ICF. 


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Population 
Description 
Intent to Treat (ITT) 
All participants who are randomized to study treatment 
Participants will be analysed according to their randomized 
treatment. 
Modified ITT (mITT) 
The subset of ITT who receive at least one dose of study drug.  Any 
participant displaying no indication of cholera infection (no 
diarrheal stool output of grade 3 or higher) within 48 hours of 
challenge will be removed from the mITT population, prior to 
unblinding of data. 
Per Protocol Population 
The subset of the mITT consisting of those participants who had no 
major protocol deviations and received all doses of assigned study 
drug.  
All protocol deviations will be assessed and documented on a 
case-by-case basis before the database lock, and deviations 
considered having a serious impact on the efficacy results will lead 
to the relevant participant being excluded from the per-protocol 
population. Before database lock, potential participant exclusions 
from per-protocol population will be reviewed by the Sponsor and 
documented. 
Safety 
The Safety Analysis Set includes all participants who received any 
study treatment. Safety analyses and demographic/baseline 
characteristic summaries will be based on the safety population, 
according to treatment received. 
 
12.5 Description of Statistical Methods 
12.5.1 General Approach 
This is a single-centre, double-blind, randomized, placebo-controlled trial comparing the efficacy and 
safety of iOWH032 to placebo after a cholera challenge in adult healthy volunteers.  
 
SAS version 9.4 or greater will be used for analysis. For descriptive statistics, continuous variables will 
be summarised by the number of non-missing observations, mean, median, standard deviation, and 
minimum and maximum.  Categorical variables will be summarised by presenting the frequency and 
percent.  Unless otherwise specified, inference such as confidence interval construction will be 
conducted with two-tailed Type I error level α = 0.05.  No adjustment for multiple comparisons across 
endpoints will be conducted.  All secondary endpoints will be considered as supportive evidence of 
efficacy and will be analysed without any procedures to account for multiple comparisons.   
 
In general, nonparametric analyses or exact methods (e.g., Fisher’s exact test) will be preferred for 
efficacy analyses, with confidence intervals for binary variables computed via the Clopper-Pearson 
exact method, and confidence intervals for continuous variables computed via the percentile 
bootstrap method, using n=10,000 replicates each. 
 
Primary efficacy analysis will be stratified by blood type status (O vs Non-O) as described below. All 
efficacy endpoints will be summarized, by group and by group and O vs Non-O status, using daily and 
overall summaries, where appropriate. Exploratory analyses will consider stratification by cohort to 
account for potential differences in challenge inoculum between cohorts. 


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Analysis of diarrheal stool output rate, diarrheal stool volume, cholera-containing diarrheal stool 
volume, and density of cholera organisms will be supplemented with reverse cumulative distribution 
curves. 
 
All data will be provided in listings, in addition to the summaries described below. 
12.5.2 Analysis of the Primary Efficacy Endpoint(s) 
The primary efficacy endpoint is “Diarrheal stool output rate”, defined as the total volume of diarrheal 
stools (mL, Grade 3 and above) divided by the number of hours between initiation of study product 
dosing and initiation of antimicrobial therapy. 
 
Statistical comparison of the primary endpoint between groups will be conducted with the stratified 
(by blood type group) version of the Wilcoxon rank-sum test, the Van Elteren test.  If a one-sided 
statistical test rejects H0 and the median is less for the iOWH032 group than for the placebo group, 
the study will have demonstrated that iOWH032 is superior. The hypothesis test will be supplemented 
by a two-sided confidence intervals for the difference in median diarrheal stool output rate, using the 
percentile bootstrap method, with n=10,000 replicates. See Section 12.5.8 for a description of the 
one-sided alpha levels to be used for testing the primary endpoint. 
 
The principal analysis will be performed using the mITT population. If greater than 20% of subjects are 
excluded from the mITT due to onset of symptoms after 48 hours, the primary endpoint will be 
calculated again including patients with symptom onset after 48 hours. For subjects with symptom 
onset after 48 hours, the time between onset of symptoms and administration of antimicrobials will 
define the denominator used to calculate the diarrheal stool output rate.   Supportive analysis will be 
performed using the per-protocol population. 
 
Analysis of the primary endpoint will be conducted at an interim analysis upon completion of 24 
participants, and again on the aggregate data after an additional 24 participants have completed the 
study. Methods defined in Section 12.5.8 will be used to control overall type I error rate at one-sided 
level alpha = 0.025. 
12.5.3 Analysis of the Secondary Efficacy Endpoints 
Secondary efficacy endpoints include a) the proportion of participants with moderate or severe 
diarrhea following cholera challenge defined as ˃3.0 L and ˃5.0 L of loose stools, as well as b) the 
attack rate of any diarrhea following cholera challenge defined as the number of participants with 2 
or more loose stools (grade 3-5) totalling >200 mL within 48 hours or 1 loose (grade 3-5) stool ˃ 300 
mL. 
 
Both endpoints will be computed as the proportion within each group satisfying the respective 
definitions, and will be accompanied by exact two-sided 95% confidence intervals. This will be 
supplemented with corresponding summaries within study arm and blood type group. Statistical 
inference for each endpoint will be conducted with the Cochran-Mantel-Haenszel test, stratified by 
blood type group. 
 
If a two-sided- statistical test with Type I error rate of 0.05 rejects H0 and the proportion of participants 
is less for the iOWH032 group than for the placebo group, the study will have demonstrated that 
iOWH032 is superior. 
 


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The AUC of diarrheal stool volume and cholera organisms will be computed via the trapezoidal rule. 
The secondary endpoints of AUC of cholera-containing diarrheal stool volume, AUC of cholera 
organisms, and peak shedding of cholera organisms (highest cfu counts for each participant) will be 
analysed in quantitative stool culture and will be summarized and analysed with methods similar to 
the primary efficacy endpoint, to be further defined in the SAP. 
 
Cholera illness during the interval immediately following challenge and prior to initiation of antibiotics 
will be summarized by group and by blood type group (O vs non O) within group. 
x 
Time (hours, post-challenge) to the last unformed stool (grade >=2, overall, as well as by-
grade), will be summarized with Kaplan-Meier methods and compared with the log-rank test 
x 
The total count of loose stools will be summarized as a continuous variable and compared 
separately for each blood type group with the Wilcoxon rank-sum test and jointly with the 
Van Elteren test, stratified by blood type group (O vs non O). 
x 
The presence of fever or vomiting will each be summarized as categorical variables, with 
between-group inference conducted separately for each blood type group with Fisher’s exact 
test, and jointly via the Cochran-Mantel-Haenszel test, stratified by blood type group (O vs 
non O). 
 
The principal analyses will be performed using the mITT population.  Supportive analysis will be 
performed using the per-protocol population.   
 
Detection of cholera in stool post-challenge will be summarized overall and by time point using 
categorical methods. Time to cessation of detectable cholera in stool (hours) will be analysed using 
Kaplan-Meier methods. Additional exploratory efficacy analyses may be identified, and should be 
described in SAP, prior to the interim analysis. 
12.5.4 Safety Analyses 
All safety analyses and demographic/baseline characteristic summaries will be based on the safety 
population, according to treatment received. 
 
Adverse Events: The summary of AEs will be based on treatment-emergent AEs (TEAEs) defined as AEs 
which start or worsen following the start of challenge and up until the follow-up visit. 
 
TEAE summaries will be based on system organ class and preferred term assigned to the event using 
the Medical Dictionary for Regulatory Activities (MedDRA). A summary table will be presented, by 
treatment group, for the numbers and percentages of participants with any TEAEs, related TEAEs, 
serious TEAEs, serious and related TEAEs, any severe TEAEs, and TEAEs leading to discontinuation 
from the study. TEAEs will also be summarized by preferred term/system organ class and severity by 
treatment group. This table will also be presented for TEAEs causally related to study treatment 
(defined as those described as possibly, probably or definitely related to study drug). Tables will also 
include an overall event count by treatment group. All subject-level percentages (solicited/unsolicited 
AEs, etc.) will be supplemented with two-sided 95% confidence intervals computed via the Clopper-
Pearson method. 
 
Solicited adverse events collected during the dosing phase will be summarized by causality and 
severity, using the maximum grade overall. 
 


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Clinical laboratory values will be graded per Appendix D, and abnormalities for each lab (and overall) 
will be summarized by maximum grade overall and per time point. A corresponding summary will be 
produced only for those considered to be clinically relevant. 
 
Physical exam abnormalities will be summarized. Clinically relevant abnormal vital signs will be 
summarized. Abnormal screening ECG results will be summarized. 
 
Continuous summaries of clinical lab values and vital signs, including change from baseline, will be 
computed by group and time point. 
12.5.5 Adherence and Retention Analyses 
Participants who prematurely discontinue from the study will not be included in analyses/summary 
statistics beyond the time of discontinuation.   
 
Rates of discontinuation and reason for discontinuation will be summarized in data listings or 
summary tables. 
12.5.6 Protocol Deviations 
Protocol deviations will be summarized by group and listed, with classification into major/minor of 
participant-specific deviations completed on blinded data prior to database lock. 
12.5.7 Baseline Descriptive Statistics 
Demographic data, including blood type, age, gender, height, weight, BMI, and race/ethnicity will be 
summarized using descriptive statistics by treatment group.  Continuous variables will be summarised 
by the number of non-missing observations, mean, median, standard deviation, and minimum and 
maximum. Categorical variables will be summarised by presenting the frequency and percent. 
 
Prior/ongoing and new concomitant medications will be summarized (separately) by anatomical-
therapeutic-chemical (ATC) class after coding with the WHO Drug dictionary. 
12.5.8 Planned Interim Analyses 
Once efficacy data are available from the completion of Cohort 1, an interim analysis of the primary 
efficacy endpoint will be conducted to guide the decision to move forward with Cohort 2. The 
interim analysis focusing on safety and efficacy of the iOWH032 will be partially blinded and will be 
shared with the SRC for a recommendation to the sponsor. Only the primary endpoint will be 
compared to the boundary values below for purposes of interim and final analysis. 
 
The following boundary values will be used at the interim analysis: 
If the one-sided Van Elteren test of the superiority of the diarrheal stool output rate 
(iOWH032 vs placebo) yields a p-value less than 0.0051, then the study product will be 
deemed superior to placebo, and the second cohort will not be enrolled 
If the one-sided Van Elteren test of the superiority of the diarrheal stool output rate 
(iOWH032 vs placebo) yields a p-value greater than 0.4585, this will be considered 
evidence of futility (lack of demonstrated efficacy), and the second cohort will not be 
enrolled 
In all other cases, the 2nd cohort will be enrolled 
 


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Due to the nonzero probability of concluding efficacy (under alternative hypotheses) at the interim 
analysis, the alpha level (type I error rate) for the final analysis needs to be adjusted to maintain 
overall one-sided level alpha = 0.025. The threshold to define success on the primary efficacy 
endpoint at the final analysis is one-sided alpha = 0.0238. 
12.5.9 Multiple Comparison/Multiplicity 
Adjustment for multiplicity for the group-sequential analysis of the primary efficacy endpoint is 
described in Section 12.5.8.  All secondary endpoints will be considered as supportive evidence of 
efficacy and will be analysed without any procedures to account for multiple comparisons. 
12.5.10 Exploratory Analyses 
Additional exploratory analyses may be identified, and should be described in SAP, prior to the interim 
analysis. 
12.5.11 Missing data 
All reasonable measures will be taken to ensure minimal missing data. Despite this, some data are 
likely to be missing at the end of the study. No imputation is planned for any endpoints in this study, 
and all data will be considered to be missing completely at random. Should observations indicate this 
is not likely to be true for a given endpoint, additional sensitivity analyses may be conducted, such as 
multiple imputation, to assess the impact of the missing data. 
 
12.6 Sample Size  
This study is a Phase 2a study to determine the presence and magnitude of treatment effect of 
iOWH032 on prevention of cholera sequela after a cholera challenge.  Data from placebo-vaccinated 
participants in a prior cholera challenge study conducted at the University of Maryland, including a 
mixture of type O and Non-O participants, demonstrated a mean total fluid output of 4448 ± 3178 mL 
over the post-challenge follow-up period(5). Type O participants had a minimum total diarrheal output 
volume of approximately 500 mL and a maximum of approximately 11,200 mL, with a roughly uniform 
distribution across this span. For non-Type O participants, the minimum was approximately 600 mL 
and the maximum was approximately 7,800 mL. The daily rate of diarrheal stool output between 
challenge and initiation of antibiotic therapy estimated from these same data yield a roughly uniform 
distribution between 100 mL/day (both groups) and 2,200 mL/day (non-type O) or 3,000 mL/day (Type 
O). For sample size computation, it was assumed that: 
 
x 
The placebo group in the present study would yield daily diarrheal stool rates uniformly 
distributed between 100 mL and 3,000 mL/day for Type O participants, and between 100 
mL/day and 2,200 mL for non-Type O participants 
x 
Treatment with iOWH032 would yield approximately a 50% reduction in daily diarrheal stool 
rate, with values uniformly distributed between 100 mL and ½ the upper limit for the placebo 
participants (1,500 mL/day and 1,100 mL/day for Type O and non-Type O participants, 
respectively) 
x 
Approximately 40% of participants will be blood Type O, and will be equally distributed across 
study arm and study stage 
 
This represents an assumption that all participants will respond to treatment, which is currently 
unknown. No prior data are available to inform the expected effect size, and the hypothesis selected 
here is expected to represent a feasible effect size for an impactful therapy. Randomization is 
stratified by blood type group (type O vs non-type O) to ensure balanced randomization, and 


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therefore a stratified analog of the Wilcoxon rank-sum test, the Van Elteren test, is employed for joint 
analysis across blood type group. 
 
Participants are to be excluded from analysis if there is lack of evidence of cholera infection following 
challenge, defined as no diarrheal stool output of grade 3 or higher within 48 hours of challenge. 
Additionally, participants displaying rapid onset of severe cholera may have diarrheal stool output 
volume truncated due to early initiation of antimicrobial therapy. The use of a rate endpoint, as well 
as the nonparametric test for analysis is expected to yield a result that is insensitive to the influence 
of these observations, which are expected to be rare. The distribution of diarrheal stool output 
volume and rate is not well-characterized, and cannot be assumed to be normally-distributed based 
on prior data, further implying the need for a nonparametric analysis. 
 
The primary efficacy endpoint is to be analysed at both an interim and a final analysis. Because of 
the multiplicity of testing and accompanying efficacy and futility thresholds at the interim analysis, a 
group-sequential framework is employed. Traditional methods for trial design are intractable in this 
case, due to the anticipated distribution of the endpoint and the use of stratified nonparametric 
testing for its evaluation. For this reason, a simulation-based framework was used to simulate data 
under both the null and alternative hypotheses. Power is computed as the proportion of simulations 
under the alternative hypothesis which yield a conclusion of superiority at either interim or, given 
that futility is not declared at interim, at the final analysis. Type I error is computed as the 
proportion of simulations under the null hypothesis which yield the same conclusion, erroneously. 
The simulation procedure was submitted to a constrained optimization routine that selected 
boundary values which ensure overall ≥90% power and type I error ≤0.025, simultaneously. Further 
simulations confirmed the adequacy of these boundaries to provide high power while maintaining 
nominal type I error rate. The boundary values that will be used at the interim analysis are: 
 
If the one-sided Van Elteren test of the superiority of the diarrheal stool output rate 
(iOWH032 vs placebo) yields a p-value less than 0.0051, then the study product will be 
deemed superior to placebo, and the second cohort will not be enrolled 
If the one-sided Van Elteren test of the superiority of the diarrheal stool output rate 
(iOWH032 vs placebo) yields a p-value greater than 0.4585, this will be considered 
evidence of futility (lack of demonstrated efficacy), and the second cohort will not be 
enrolled 
In all other cases, the 2nd cohort will be enrolled 
 
Due to the nonzero probability of concluding efficacy at the interim analysis, the alpha level (type I 
error rate) for the final analysis needs to be adjusted to maintain overall one-sided level alpha = 
0.025. The threshold to define success on the primary efficacy endpoint at the final analysis is one-
sided alpha = 0.0238. This hypothesis test will be supplemented by a two-sided 95% confidence 
interval for difference in median diarrheal stool output rate, using the percentile bootstrap method, 
with n=10,000 replicates. 
 
With the abovementioned boundary values and sample size, the power decreases to 70% for a 40% 
reduction in daily diarrheal stool rate and to 30% a reduction of 25% in daily diarrheal stool rate.  
However, and effect size of 50% reduction is expected to represent a feasible effect size for an 
impactful therapy. 
 
For the secondary endpoint of proportion of participants with moderate or severe diarrhea, with 24 
participants per group, and assuming 40% of participants are Type O, adequate power is available to 


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detect odds ratios of moderate or severe diarrhea in iOWH032-treated participants vs placebo which 
are significantly less than 1, using a Cochran-Mantel-Haenszel test stratified by blood type group, with 
two-sided level alpha = 0.05. For example, if 67% (47%) of the type O (non-type O) control participants 
experience moderate or severe diarrhea, and only 20% (10%) of type O (non-type O) iOWH032-
treated participants do (common odds ratio 0.123), then 80% power is available to detect a common 
odds ratio < 1. Only a single analysis will be conducted for efficacy endpoints other than the primary 
efficacy endpoint, upon collection of data from all participants included in the experiment. 
 
12.7  Measures to Minimize Bias 
12.7.1 Enrollment/ Randomization/ Masking Procedures 
This parallel group study is randomized and double-blinded to minimise bias, and includes a placebo 
control arm to enable those effects related to dosing of randomized study treatment to be more 
readily identified.  Participants will be randomized using a 1:1 ratio to iOWH032 or matching placebo.  
The randomization scheme will be generated by an independent statistician.  The study site will 
receive one set of randomization envelopes to be stored and used in the study clinic.  Clinic staff will 
assign these envelopes in sequential order, by envelope number, to eligible participants.   
 
Section 12.7.3 details the procedures for breaking the study blind.  
12.7.2 Evaluation of Success of Blinding 
This study is designed as a randomized, double blind, placebo controlled, parallel group study.  As 
such, participants will be allocated to either iOWH032 or matching placebo.  No specific evaluation of 
the success of study blinding is planned.  Any unblinding will be noted in case report forms.     
12.7.3 Breaking the Study Blind/Participant code 
In the event of a medical emergency, the site PI may require that the blind be broken for the subject 
experiencing the emergency when knowledge of the subject’s treatment assignment may influence 
the subject’s clinical care. Prior to unblinding, the site PI is encouraged (to the extent possible, without 
jeopardizing the subject’s health) to contact PATH Medical Officer (or designee) to discuss the decision 
to break the blind. In case of medical emergency, PATH must be notified within 24 hours after breaking 
the blind. The date and reason that the blind was broken must be recorded in the source 
documentation and case report form, as applicable. 
13 SOURCE DOCUMENTS AND ACCESS TO SOURCE 
DATA/DOCUMENTS 
Source data are all information, original records of clinical findings, observations, or other activities 
in a clinical trial necessary for validation of the clinical data.  Examples of these original documents 
and data records include, but are not limited to, hospital records, clinical and office charts, 
laboratory notes, memoranda, participants’ memory aid or evaluation checklists, pharmacy 
dispensing records, recorded data from automated instruments, copies or transcriptions certified 
after verification as being accurate and complete, microfiches, photographic negatives, microfilm or 
magnetic media, x-rays, and participant files and records kept at the pharmacy, at the laboratories, 
and medico-technical departments involved in the clinical trial.  All information on the Case Report 
Form (CRF) will be traceable to these source documents, which are generally maintained in the 
participant’s study file.  The source documents will include a copy of the signed Informed 
Consent/HIPAA authorization. The source document data collection forms for Screening, Inpatient 


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Pre-Challenge to Day of Challenge, Inpatient Post-Challenge, Flow Sheet of Stool and Emesis Record, 
Outpatient Visits and Adverse Events will also serve as Case Report Form (CRF) data collection 
instruments.  
14 QUALITY ASSURANCE AND QUALITY CONTROL 
All clinical trials conducted by Pharmaron are internally monitored for quality assurance and control.  
Quality Control (QC) and Quality Assurance (QA) procedures are designed to ensure that the site and 
investigator comply with applicable regulations, adhere to the IRB-approved protocol, generate 
quality data, protect data integrity, and safeguard the safety and well-being of study participants. 
The quality plan has the authority to enforce and correct deficiencies in clinical and laboratory 
conduct of a trial.  The QC process involves day-to-day logic and edits checks of source documents, 
case report forms, and laboratory documents.  The QA process involves periodic retrospective audits 
of study records and prospective reviews of clinical operations and assurance that all research 
required training has been completed as applicable.  These audits also involve the review of the 
regulatory file, consent forms/process, eligibility, study product accountability and storage, 
specimen collection, processing, storage, and shipping.  All observations are reviewed with the PI, 
coordinator, and Clinical Research Manager; Corrective Action Plans are formulated in response to 
any issue. 
15 ETHICAL CONSIDERATIONS (AND INFORMED 
CONSENT) 
15.1 Ethical Standard 
The investigator will ensure that this study is conducted in full conformity with the principles set 
forth in The Belmont Report: Ethical Principles and Guidelines for the Protection of Human Subjects 
of Research of the US National Commission for the Protection of Human Subjects of Biomedical and 
Behavioral Research (April 18, 1979) and codified in 45 CFR Part 46 and/or the ICH E6; 62 Federal 
Regulations 25691 (1997).   
15.2 Institutional Review Board 
The local institution must provide for the review and approval of this protocol and the associated 
informed consent documents by an appropriate ethics review committee or Institutional Review 
Board (IRB). Any amendments to the protocol or consent materials must also be approved before 
they are placed into use unless it is in the best interest of the participants’ safety to implement 
changes prior to approval. In both the United States and in other countries, only institutions holding 
a current U. S. Federal-Wide Assurance issued by OHRP may participate.  
Refer to: http://www.hhs.gov/ohrp/assurances/.   
Prior to enrollment of participants into this clinical study, the protocol and the informed consent 
form(s) will be reviewed and approved by the appropriate IRB. Any amendments to the protocol or 
consent materials will also be reviewed and approved by the appropriate IRB. The responsible 
official for the IRB will sign the IRB letter of approval of the protocol prior to the start of this clinical 
study. Should amendments to the protocol be required, the amendments will be submitted to the 
IRB; an IRB letter of approval of the amendment must be obtained prior to acting upon the 
amendment in the protocol. 


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15.3 Informed Consent Process 
15.3.1 Consent and Other Informational Documents Provided to 
Participants 
Consent forms describing in detail the study agent, study procedures, and risks are given to the 
participant and written documentation of informed consent is required prior to starting 
intervention/administering study product. The following consent materials are submitted with this 
protocol: 
x 
Recruitment script 
x 
Patient information sheet 
x 
Informed consent form 
x 
Study comprehension assessment test 
15.3.2 Consent Procedures and Documentation 
Informed consent is a process that is initiated prior to the individual’s agreeing to participate in the 
study and continues throughout the individual’s study participation.  Extensive discussion of risks 
and possible benefits of this therapy will be provided to the participant.  Consent forms describing in 
detail the study, study procedures and risks are given to the potential participant and written 
documentation of informed consent is required prior to starting any study procedure.  Consent 
forms will be IRB-approved and the participant will be asked to read and review the document. 
Upon reviewing the document, the investigator will explain the research study to the participant and 
answer any questions that may arise.  The participants will sign the informed consent document 
prior to any procedures being done specifically for the study.  As a part of the consenting procedures 
the participants should pass a written examination (comprehension assessment test) with a score 
≥70%, in order to demonstrate their comprehension of this study. If a participant scores at least 
50%, then they will be given one more opportunity to re-test after further re-education. The 
participant should have the opportunity to discuss the study with their surrogates or think about it 
prior to agreeing to participate.  The participant may withdraw consent at any time throughout the 
course of the trial.  A copy of the informed consent document will be given to the participant for 
their records.  The rights and welfare of the participant will be protected by emphasizing to them 
that the quality of their medical care will not be adversely affected if they decline to participate in 
this study. 
 
15.4 
Exclusion of Women, Minorities, and Children (Special 
Populations) 
This clinical study will include women, adults 18 years of age and older, and all minorities who meet 
the inclusion/exclusion criteria, regardless of religion, sex, or ethnic background.   
15.5 
Participant and Data Confidentiality 
Participant confidentiality is strictly held in trust by the investigators, their staff, and the Sponsor.  
This confidentiality is extended to cover testing of biological samples and other testing in addition to 
the clinical information relating to participant. 
The study protocol, documentation, data, and all other information generated will be held in strict 
confidence.  No information concerning the study or the data will be released to any unauthorized 
third party without prior written approval of the sponsor. 
The study monitor or other authorized representatives of the sponsor may inspect all documents 
and records required to be maintained by the investigator, including but not limited to, medical 


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records (office, clinic, or hospital) and pharmacy records for the participants in this study.  The 
clinical study site will permit access to such records. 
Participant identity data will be contained in paper study records which will be kept in a locked file 
cabinet and in a secure electronic database, accessible only to authorized users at each clinical site. 
The study database will be user-restricted and password-protected. The study database will identify 
participants by a coded study participant ID number assigned by clinical site personnel, thus 
participants will not be identified by name. 
15.5.1 Research Use of Stored Human Samples, Specimens, or Data 
 
x 
Intended Use: data collected under this protocol may be used to study cholera and other 
enteric health conditions.  
x 
Storage: Access to samples stored during the course of the study will be limited. Samples 
and data will be stored using codes assigned by the investigators. Data will be kept in 
password-protected computers. Only investigators will have access to the samples and data.  
x 
Tracking: Data will be tracked using a central inventory system. .  
Data collected for this study will be analyzed and stored at the Pharmaron study site. After the study 
is completed, the de-identified, archived data will be transmitted to and stored at PATH for use by 
other researchers including those outside of the study. Permission to transmit data to PATH will be 
included in the informed consent. When the study is completed, access to study data will be 
provided through PATH. 
 
15.6 Future Use of Stored Samples 
 
No stool, vomitus, or urine specimens will be stored after the end of the study for future use.  All 
blood specimens collected during the study will be stored, analyzed, and destroyed within 10 years. 
The informed consent form includes language to allow participants to agree for potential future 
research with their blood samples and data related to enteric diseases. The samples and data used 
for future research will be de-identified and labeled with study identification number and not any 
personally identifiable information. A Biorepository Governance Plan will be maintained by PATH to 
describe the disposition and access to samples being stored for future research. 
16 Data Handling and Recordkeeping 
16.1 Data Collection and Data Management Responsibilities 
The investigator is responsible for ensuring the accuracy, completeness, legibility, and timeliness of 
the data reported. Source documents are maintained for recording data for each participant 
enrolled in this clinical study. Study participants’ data collected on the CRF during the trial will only 
be identified by participant number.  If, as an exception, it is necessary for safety or regulatory 
reasons to identify the patient, both the Sponsor and the Investigator are bound to keep this 
information confidential. 
All the information required by the protocol should be provided; any omissions require explanation.  
Each source document and corresponding CRF should be completed and available for monitoring 
and/or collection within a timely manner so that the monitor may check the entries for 
completeness, accuracy and legibility, ensure the CRF is signed by the Investigator, and transmit the 
data to the Sponsor. 


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All source documents and CRF should be completed in a neat, legible manner to ensure accurate 
interpretation of data. Black or blue ink is required to ensure clarity of reproduced copies. When 
making a change or correction, cross out the original entry with a single line and initial and date the 
change. DO NOT ERASE, OVERWRITE OR USE CORRECTION FLUID OR TAPE ON THE ORIGINAL.  
PATH is responsible for data management activities and will develop a Data Management Plan 
(DMP) prior to participant enrollment. The DMP will describe roles of stakeholders and specific 
procedures to ensure appropriate handling of data at all steps of the data management process, to 
ensure a valid and high-quality database at the end of the study, ready for analysis. 
The Investigator or designee must enter the information required by the protocol onto the CRF 
provided.  The Sponsor’s clinical site monitor will review the CRF for completeness and accuracy and 
instruct site personnel to make any required corrections or additions. Source data will be entered 
into a Medidata RAVE® data management system, managed by Pharmaron. Medidata RAVE® is a 
secure cloud-based application for building an electronic database that provides a robust audit trail. 
The electronic data capture (EDC) system includes password protection and internal quality checks, 
such as automatic range checks, to identify data that appear inconsistent, incomplete, or inaccurate.  
Entered data will be verified by an independent monitoring CRO who will query the site for potential 
discrepancies following a review of the source data against the database for a percentage of the 
data. The site Investigator, or designee, will be responsible for the resolution of queries and 
appropriate documented changes in the database.  
Automated queries generated by the EDC system are used to flag data discrepancies or 
inconsistencies. The site investigator, or designee, is required to provide a satisfactory resolution to 
the queries within the EDC system. In addition, the medical reviewers will also review data for 
medical inconsistencies, which will be raised as manual queries within the EDC system. The Data 
Manager reviews all the discrepancies to ensure that these are adequately resolved and to identify 
corrective and preventive actions if required. The responsible data manager will perform all 
activities as per standard operating procedures (SOPs) and/or work instructions. All study specific 
processes will be described in the DMP. Quality control audits of all key safety, laboratory, and 
clinical data in the database will be made after data entry has been completed.  Coexistent medical 
conditions, adverse events and other medical events will be coded using MedDRA dictionary. 
Concomitant medications will be coded using WHO-DD dictionary. When the database has been 
declared to be complete and accurate, the database will be locked for final statistical analysis.  Any 
changes to the database after that time can only be made by joint written agreement of the study 
team. 
 
16.2 Study Records Retention 
 
Study documents should be retained for a minimum of 2 years after the last approval of a marketing 
application in an ICH region and until there are no pending or contemplated marketing applications 
in an ICH region or until at least 2 years have elapsed since the formal discontinuation of clinical 
development of the investigational product. These documents should be retained for a longer 
period, however, if required by local regulations. No records will be destroyed without the written 
consent of the sponsor, if applicable. It is the responsibility of the sponsor to inform the investigator 
when these documents no longer need to be retained. 
 
16.3 Protocol Deviations 
 


PATH iOWH032 – v4.0 
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CONFIDENTIAL 
Page 58 of 69 
 
A protocol deviation is any noncompliance with the clinical trial protocol, GCP, or MOP 
requirements. The noncompliance may be either on the part of the participant, the investigator, or 
the study site staff. As a result of deviations, corrective actions are to be developed by the site and 
implemented promptly. 
 
These practices are consistent with ICH E6:  
x 
4.5 Compliance with Protocol, sections 4.5.1, 4.5.2, and 4.5.3  
x 
5.1 Quality Assurance and Quality Control, section 5.1.1  
x 
5.20 Noncompliance, sections 5.20.1, and 5.20.2.  
 
It is the responsibility of the site to use continuous vigilance to identify and report deviations within 
48 hours of identification of the protocol deviation to PATH. All deviations must be addressed in 
study source documents, reported PATH. Protocol deviations must be sent to Advarra per their 
guidelines. The site PI/study staff is responsible for knowing and adhering to Advarra requirements. 
PATH Medical Officer will be responsible for PATH Research Ethics Committee correspondence, 
expedited and other reporting requirements. Further details about the handling of protocol 
deviations will be included in the MOP. 
 
16.4 Publications and Data Sharing Policy 
 
The study sponsor will register this trial on ClinicalTrials.gov in accordance with Section 801 of the 
FDA Amendments Act of 2007. 
 
It is understood by the investigators that the information generated in this study will be used by the 
sponsor in connection with the development of the product and therefore may be disclosed to 
government regulatory agencies in various countries.  The sponsor (and manufacturer) also 
recognizes the importance of communicating study findings and therefore encourages their 
publication in reputable scientific journals and presentation at seminars or conferences, while 
protecting the integrity of the ongoing trial.  Any publication, lecture, manuscripts of the findings of 
this study by any individual involved with the study will be governed by the procedure outlined in 
the Clinical Trial Agreement.   Within any presentation or publication, confidentiality of individual 
participants will be maintained, with identification by participant code number and initials, if 
applicable. 
17 Financing and Insurance 
The trial is supported by a grant from 
.  
 
PATH will provide locally admitted clinical trial insurance for the payment of any expenses related to 
the treatment of injuries directly attributable to participation in this study. 
18 Conflict of Interest Policy 
The independence of this study from any actual or perceived influence, such as by the 
pharmaceutical industry, is critical. Therefore, any actual conflict of interest of persons who have a 
role in the design, conduct, analysis, publication, or any aspect of this trial will be disclosed and 
managed. Furthermore, persons who have a perceived conflict of interest will be required to have 
such conflicts managed in a way that is appropriate to their participation in the trial. The study 
leadership has established policies and procedures for all study group members to disclose all 


PATH iOWH032 – v4.0 
13 March 2020 
 
 
CONFIDENTIAL 
Page 59 of 69 
 
conflicts of interest and will establish a mechanism for the management of all reported dualities of 
interest. 
 
 
 


PATH iOWH032 – v4.0 
13 March 2020 
 
 
CONFIDENTIAL 
Page 60 of 69 
 
19 References 
 
1. de Hostos EL, Choy RK, Nguyen T. Developing novel antisecretory drugs to treat infectious 
diarrhea. Future Med Chem. 2011. 3(10): 1317-25.  
2.  Muanprasat C, Sonawane ND, Salinas D, Taddei A, Galietta LJ, Verkman AS.  Discovery of 
glycine hydrazide pore-occluding CFTR inhibitors: mechanism, structure-activity analysis, 
and in vivo efficacy. J Gen Physiol. 2004. 124(2): 125-37.  
3. Weiglmeier PR, Rösch P, Berkner H. Cure and curse: E. coli heat-stable enterotoxin and its 
receptor guanylyl cyclase C. Toxins (Basel). 2010 2(9):2213-29.  
4. Schwertschlag U, Kumar A, Kochhar S,  Ings R, Ji Y, de Hostos E L, Choy R, Salam M, Khan W 
A, Lin P Pharmacokinetics and Tolerability of iOWH032, an Inhibitor of the Cystic Fibrosis 
Transmembrane Conductance Regulator (CFTR) Chloride Channel, in Normal Volunteers and 
Cholera Patients. Gastroenterology 2014. 146 (5): Supplement 1, S-633. 
5. Levin MM, Nalin DR, Craig JP, Hoover D, Bergquist EJ, Waterman D, Holley HP, Hornick RB, 
Pierce NP, Libonati JP. Immunity of cholera in man: relative role of antibacterial versus 
antitoxic immunity. Trans R Soc Trop Med Hyg. 1979;73(1):3-9. 
6. Levin MM, Black RE, Clements ML, Cisneros L, Nalin DR, Young CR. Duration of infection-
derived immunity to cholera. J Infect Dis. 1981 Jun;143(6):818-20. 
7. Clements ML, Levin MM, Young CR, Black RE, Lim YL, Robins-Browne RM, Craig JP. 
Magnitude, kinetics, and duration of vibriocidal antibody responses in North Americans 
after ingestion of Vibrio cholerae. J Infect Dis. 1982 Apr;145(4):465-73 
8. Black RE, Levin MM, Clements ML, Young CR, Svennerholm AM, Holmgren J. Protective 
efficacy in humans of killed whole-vibrio oral cholera vaccine with and without the B subunit 
of cholera toxin. Infect Immun. 1987 May;55(5):1116-20. 
9. Levin MM, Kaper JB, Herrington D, Losonsky G, Morris JG, Clements ML, Black RE, Tall B, Hall 
R. Volunteer studies of deletion mutants of Vibrio cholerae O1 prepared by recombinant 
techniques. Infect Immun. 1988 Jan;56(1):161-7. 
10. Levin MM, Kaper JB, Herrington D, Ketley J, Losonsky G, Tacket CO, Tall B, Cryz S. Safety, 
immunogenicity, and efficacy of recombinant live oral cholera vaccines, CVD 103 and CVD 
103-HgR. Lancet. 1988 Aug 27;2(8609):467-70. 
11. Tacket CO, Forrest B, Morona R, Attridge SR, LaBrooy J, Tall BD, Reymann M, Rowley D, 
Levin MM. Safety, immunogenicity, and efficacy against cholera challenge in humans of a 
typhoid-cholera hybrid vaccine derived from Salmonella typhi Ty21a. Infect Immun. 1990 
Jun;58(6):1620-7. 
12. Tacket CO, Losonsky G, Nataro JP, Cryz SJ, Edelman R, Kaper JB, Levin MM. Onset and 
duration of protective immunity in challenged volunteers after vaccination with live oral 
cholera vaccine CVD 103-HgR. J Infect Dis. 1992 Oct;166(4):837-41. 
13. Losonsky GA, Lim Y, Motamedi P, Comstock LE, Johnson JA, Morris JG Jr, Tacket CO, Kaper 
JB, Levin MM. Vibriocidal antibody responses in North American volunteers exposed to wild-
type or vaccine Vibrio cholerae O139 specificity and relevance to immunity. Clin Diagn Lab 
Immunol. 1997 May;4(3):264-9. 
14. Tacket CO, Kotloff KL, Losonsky G, Nataro JP, Michalski J, Kaper JB, Edeman R, Levin MM. 
Volunteer studies investigating the safety and efficacy of live oral El Tor Vibrio cholerae O1 
vaccine strain CVD 111. Am J Trop Med Hyg. 1997 May;56(5):533-7. 
15. Sack DA, Tacket CO, Cohen MB, Sack RB, Losonsky GA, Shimko J, Nataro JP, Edelman R, 
Levine MM, Giannella RA, Schiff G, Lang D. Validation of a volunteer model of cholera with 
frozen bacteria as the challenge. Infect Immun. 1998 May;66(5):1968-72. 


PATH iOWH032 – v4.0 
13 March 2020 
 
 
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Page 61 of 69 
 
16. Chen WH, Cohen MB, Kirkpatrick BD, et al. Single-dose Live Oral Cholera Vaccine CVD 103-
HgR Protects Against Human Experimental Infection With Vibrio cholerae O1 El Tor. Clin 
Infect Dis. 2016;62(11):1329-1335. 
17. Rennels MB, Levine MM, Daya V, et al. Selective vs. nonselective media and direct plating vs. 
enrichment technique in isolation of Vibrio cholerae: recommendations for clinical 
laboratories. J Infect Dis. 1980 Sep;142(3):328-31. 
 
 
 


PATH iOWH032 – v4.0 
13 March 2020 
 
 
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Page 62 of 69 
 
APPENDIX A: PROTOCOL REVISIONS 
 
Version  Date 
Significant Revisions
Ethics/Regulatory Approvals
1.0 
2.0 
 
 
3.0 
 
7/24/2019 
10/30/2019 
 
 
12/23/2019 
N/A 
Safety Monitoring, AE definition, 
cholera inoculum preparation 
procedures  
Exploratory endpoint, screening 
stool, Lab normal values, mITT 
description etc.  
 
 
Advarra IRB, FDA 
 
 
Advarra IRB, FDA 
 
4.0 
03/13/2020 Additional topline analyses
Advarra IRB, FDA
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


PATH iOWH032 – v4.0 
13 March 2020 
 
 
CONFIDENTIAL 
Page 63 of 69 
 
APPENDIX B: SCREENING TESTS  
 
Analyte 
Unit 
Acceptable Values for Safety Labs: 
White Blood Cell count (WBC)  
thou/mcL
3600–11000 (2800–11,000 for African-Americans)  
Absolute Neutrophil Count (ANC) 
thou/mcL
1500–8000  (1200-8000 for African-Americans) 
Hemoglobin (Females)  
g/dL 
F: 11.0–15.6  
Hemoglobin (Males)  
g/dL 
M: 13.0–17.2  
Platelet count  
per mm3 
125,000–450,000  
ALT 
IU/L 
<50 
AST 
IU/L 
<40 
Alkaline Phosphates 
IU/L 
44-147 
Sodium 
mEq/L 
135-145 
Potassium 
mEq/L 
3.5-5.5 
Glucose (nonfasting) 
mg/dL 
≤ 115 
Creatinine (females) 
mg/dL 
< 1.6  
Creatinine (males) 
mg/dL 
< 1.6  
Creatine phosphokinase (Males 
and females) 
U/L 
22-360 
Albumin 
g/dL 
<3.3 
Bilirubin, total  
mg/dL 
< 1.4 (unless known Gilbert’s syndrome, then <2.0) 
Glucose - urinalysis 
n/a 
trace or negative  
Protein - urinalysis 
n/a 
≤ 1+  
Occult blood - urinalysis 
n/a 
Small, trace or negative* 
RBC (Females) - urinalysis 
per hpf 
0–4*  
RBC (Males) - urinalysis 
per hpf 
0–4  
Serum β-HCG 
n/a 
Negative 
Hepatitis B surface antigen 
n/a 
Negative  
Hepatitis C virus ELISA 
n/a 
Negative 
HIV 
n/a 
Negative 
n/a, not applicable 
*presence is acceptable for menstruating women  
12-lead Electrocardiogram 
Must not have any of the following, in order to be acceptable:
x Pathologic Q wave abnormalities 
x Significant ST-T wave changes 
x Left ventricular hypertrophy 
x Right bundle branch block 
x Left bundle branch block 
x A-V heart block 


PATH iOWH032 – v4.0 
13 March 2020 
 
 
CONFIDENTIAL 
Page 64 of 69 
 
Analyte 
Unit 
Acceptable Values for Safety Labs: 
x Non-sinus rhythm, excluding isolated premature atrial 
contractions 


PATH iOWH032 – v4.0 
13 March 2020 
 
 
 
 
 
Page 65 of 69 
 
APPENDIX C: SCHEDULE OF STUDY VISITS AND EVALUATIONS  
Study visit Visit 1 
(screen) 
Inpatient Containment Period 
Visit 2 Visit 3 Visit 4 
Study Day D-85 to-7 
D-1 
D1 
D2 
D3 
D4 
D5 
D6 
D7 
D8 
D9 
D10 
D15±1 
D29±2 D180±14
Informed Consent 
√ 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Medical history, medications 
√ 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Comprehension Assessment Tool 
√ 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Physical examination 
√ 
 
√h 
√h 
√h 
√h 
√h 
√h 
√h 
√h 
√h 
√h 
√h 
√h 
 
Pregnancy testa 
√ 
√ 
 
 
 
 
 
 
 
 
 
 
 
 
 
Vital signs 
√ 
 
√g 
√ g 
√ g 
√ g 
√ g 
√ g 
√ g 
√ g 
√g 
√ g 
 
 
 
Screening laboratoryb 
18 mL 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
12-lead ECG 
√ 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Pre-challenge Stool Sample 
 
√ 
 
 
 
 
 
 
 
 
 
 
 
 
 
Safety laboratoryc 
 
10 mL 
 
 
 
 
 
 
 
 
 
 
10 mL
 
 
Eligibility confirmation 
√ 
√ 
√ 
 
 
 
 
 
 
 
 
 
 
 
 
Challenge 
 
 
√ 
 
 
 
 
 
 
 
 
 
 
 
 
Blinded Study Drug Administrationd 
 
 
(√) 
(√) 
(√) 
 
 
 
 
 
 
 
 
 
 
3-day course of antibioticse 
 
 
 
 
 
 
(√) 
(√) 
(√) 
 
 
 
 
 
 
Observe & manage illnessf 
 
 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
(√) 
(√) 
(√) 
 
 
 
Stool grading and culturesf 
 
 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
(√) 
(√) 
(√) 
 
 
 
pK analysisi 
 
 
√ 
 
√ 
 
 
 
 
 
 
 
 
 
 
Cumulative  blood volume 
18 mL 10 mL 2 mL 
 
2 mL 
 
 
 
 
 
 
 
10 mL
 
 
Solicited AEs 7 days post-challenge 
 
 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
 
 
 
 
 
Unsolicited AEs 
 
 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
 
SAEs 
 
 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
Concomitant medications 
 
 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
Interval medical history 
 
 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
Unanticipated problems (UPs) 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
a. For females of child bearing potential, a serum pregnancy test will be performed at screening and one day before challenge  
b. Screening laboratory to include: Complete Blood Count (CBC) for WBC, ANC, Hemoglobin, and Platelets; Chemistry panel for Na, K, ALT, AST, Creatinine, Albumin, ALP, CPK, 
Glucose and Total Bilirubin; Serology for HbSAg, anti-HCV, and HIV; Blood Typing; and Urinalysis 


PATH iOWH032 – v4.0 
13 March 2020 
 
 
 
 
 
Page 66 of 69 
 
c. Safety laboratory to include: Complete blood count (CBC) for WBC, ANC, Hemoglobin, and Platelets; and Chemistry panel for Na, K, ALT,AST,ALP, Creatinine, Albumin, and Total 
Bilirubin, CPK, Glucose 
d. Blinded therapeutic dosing will start at the onset of symptoms or by 48 hours after ingesting the cholera challenge inoculum.   
e. A 3-day course of antibiotics will be initiated upon satisfying the criteria for severe cholera diarrhea or approximately 4 day post-challenge, whichever is earlier 
f. 
The observation and management of cholera illness (includes measurement of vomiting) and stool grading and stool cultures are intended to be performed throughout the post-
challenge inpatient period, until discharge criteria are satisfied. For the duration of diarrhea, the participant will be requested to provide a urine specimen from every void that they 
experience; the specimen will be tested for urine specific gravity. In case of IV fluid replacement, serum electrolytes, BUN, and creatinine will be measured.   
g. Vital signs will be measured at least 3 times daily (approximately every 8 ±1 hours). Vital signs will also be measured every 4 hours when a participant has a fever ≥ 39qC 
(102.1qF), until the participant has 2 consecutive temperatures of ≤ 38qC (100.4qF). On Day 1, including the pre-challenge vitals assessment, there will be 4 vital signs 
assessments (1x pre-challenge and 3x post-challenge). 
h.  Focused physical examination at the discretion of the physician 
i. 
pK plasma samples for the levels of iOWH032 will be collected  7±1 hours after the first and last dosing in all participants 
 
 
 


PATH iOWH032 – v4.0 
13 March 2020 
 
 
 
 
 
Page 67 of 69 
 
APPENDIX D: CLINICAL SAFETY LABORATORY TOXICITY SCALES 
Test 
Mild 
Grade 1 
Moderate 
Grade 2 
Severe 
Grade 3 
Potentially Life 
Threatening 
Grade 4 
WBC, increase 
1.1 – 1.5 xULN 
1.6 – 2.0 xULN 
2.1 – 2.5 xULN 
≥ 2.6 xULN 
WBC, decrease* 
1.1 – 1.5 xLLN 
1.6 – 2.0 xLLN 
2.1 – 2.5 xLLN 
≥ 2.6 xLLN 
ANC decrease 
1.1 – 1.5 xLLN 
1.6 – 2.0 xLLN 
2.1 – 2.5 xLLN 
≥ 2.6 xLLN 
Hemoglobin decrease 
1.1 – 1.5 xLLN 
1.6 – 2.0 xLLN 
2.0 – 3.0 xLLN 
>3 xLLN 
Platelet count 
1.3 – 1.5 xLLN 
1.6 – 2.0 xLLN 
2.0 – 3.0 xLLN 
>3 xLLN 
Glucose  
Nonfasting, High 
116-160 
161-250 
251-500 
≥ 500 
Glucose, Low 
55-64 
40-54 
30-39 
< 30 
Sodium, low 
132 -134 
130 - 132 
125 - 129 
< 125 
Sodium, high 
146 – 148 
149 - 150 
151 - 152 
> 152 
Potassium, high 
5.6 – 5.8 
5.6 – 6.1 
6.2 – 6.5 
> 6.5 
Potassium, low 
3.3 – 3.4 
3.1 – 3.2 
2.9 – 3.0 
<2.9 
Creatinine* 
1.1 – 1.5 xULN 
1.6 – 2.0 xULN 
2.1 – 3.0 xULN 
> 3.0 xULN 
AST (SGOT) 
1.6 -.2.5 xULN 
2.6 – 4.0 xULN 
4.1 – 10.0 xULN 
>10 xULN 
ALT (SGPT) 
1.6 -.2.5 xULN 
2.6 – 4.0 xULN 
4.1 – 10.0 xULN 
>10 xULN 
Alkaline Phosphatase 
1.6 -.2.0 xULN 
2.1 – 3.0 xULN 
3.1 – 10.0 xULN 
>10 xULN 
Total Bilirubin 
1.2 -.1.5 xULN 
1.6 – 2.0 xULN 
2.0 - 4.0 xULN 
>4 xULN 
ULN = upper limit of normal range 
LLN = lower limit of normal range 
*if the baseline is outside of the reference range (e.g., African-American acceptable value), then relative changes 
to the baseline value will be evaluated for toxicity 
 
 
 


PATH iOWH032 – v4.0 
13 March 2020 
 
 
 
 
 
Page 68 of 69 
 
APPENDIX E: REACTOGENICITY AND SOLICITED ADVERSE EVENT TOXICITY SCALES 
Grading of Stool Consistency
normal stool 
loose or diarrheal stool
Grade 1 
Grade 2 
Grade 3
Grade 4
Grade 5
Well formed; 
does not take the 
shape of the 
container 
Soft; 
does not easily take 
the shape of the 
container 
Thick liquid stool; 
easily takes the 
shape of the 
container 
Opaque watery 
diarrheal stool 
Clear watery or “rice 
water” diarrheal 
stool 
 
Diarrhea Severity
Mild 
Moderate 
Severe 
2 or more loose stools of ≥ 200 mL 
within 48 hours 
or 
a single loose stool of ≥ 300 mL 
Cumulative loose stools of ≥ 3 liter 
Cumulative loose stools of ≥ 5 liters 
 
Objective 
Reactogenicity 
Mild 
Grade 1 
Moderate 
Grade 2 
Severe 
Grade 3 
Potentially Life 
Threatening 
Grade 4 
Diarrhea 
(refer to table above)
Requires 
hospitalization 
Vomiting (episodes/24 hour) 
1-2 
3-4
≥ 5
Fever, °C (°F) 
38.0 – 38.4
(100.4 – 101.1) 
38.5 – 38.9
(101.2 – 102) 
≥ 39
(≥ 102.1) 
Tachycardia, bpm 
101 - 115 
116 - 125
> 125 
Systolic Hypotension, mmHg 
85 - 89 
80 - 84
< 80
Subjective Reactogenicity, 
includes: abdominal 
pain/cramping, malaise, 
nausea, anorexia, arthralgia 
No interference 
with activity 
Some interference 
with activity 
Prevents daily 
activity 
Requires 
hospitalization 
 
 
 


PATH iOWH032 – v4.0 
13 March 2020 
 
 
 
 
 
Page 69 of 69 
 
APPENDIX F: EXAMPLES OF SUBSTRATES THAT ARE METABOLIZED BY CYP2C9 
 
Drug name 
 
Class 
x Irbesartan
x Angiotensin II blocker
x Losartan
x Angiotensin II blocker
x Phenytoin
x Antiepileptic
x Cyclophosphamide
x Alkylating agent
x Tamoxifen
x Anti-estrogen
x Fluvastatin
x Statin
x Celecoxib
x NSAID
x Diclofenac
x NSAID
x Ibuprofen
x NSAID
x Lornoxicam
x NSAID
x Meloxicam
x NSAID
x Naproxen
x NSAID
x Glibenclamide
x Sulfonylurea
x Glimepiride
x Sulfonylurea
x Glipizide
x Sulfonylurea
x Tolbutamide
x Sulfonylurea
x Warfarin
x Anticoagulant 
 
 
 
 

SAP_001.pdf: 
PATH
Statistical Analysis Plan Addendum
Updates of Secondary Efficacy Endpoints
Final 1.0, 25Nov2020
1
Statistical Analysis Plan
Addendum
A Phase 2a Randomized, Single-Center, Double-Blind, 
Placebo Controlled Study to Evaluate the Safety and 
Preliminary Efficacy of Oral iOWH032 Against 
Cholera Diarrhea in a Controlled Human Infection 
Model
Study Title 
A Phase 2a Randomized, Single-Center, Double-Blind, Placebo 
Controlled Study to Evaluate the Safety and Preliminary 
Efficacy of Oral iOWH032 Against Cholera Diarrhea in a 
Controlled Human Infection Model
Document Status͹
Final 1.0
Document Date͹
25Nov2020
Sponsor͹
PATH
CONFIDENTIAL
Information and data in this document contain trade secrets and privileged or confidential 
information, which is the property of Intra-Cellular Therapies, Inc. No person is authorized to 
make it public without the written permission of Intra-Cellular Therapies, Inc. These 
restrictions or disclosures will apply equally to all future information supplied to you that is 
indicated as privileged or confidential.
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	
National Clinical Trial (NCT) Identifier Number: NCT04150250


PATH
Statistical Analysis Plan Addendum
Updates of Secondary Efficacy Endpoints
Final 1.0, 25Nov2020
2
Statistical Analysis Plan Addendum Approval Form
Author:  
______
________________
Date (dd/mm/yyyy)
CR Medicon US, Inc.
Reviewer:
______
________________
Date (dd/mm/yyyy)
CR Medicon US, Inc.
Approver:
______
________________
Date (dd/mm/yyyy)
PATH, Center for Vaccine Innovation and Access
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	
	





PATH 
Statistical Analysis Plan Addendum 
Updates of Secondary Efficacy Endpoints 
Final 1.0, 25Nov2020 
 
3 
 
TABLE OF CONTENTS 
1 
INTRODUCTION .............................................................................................................................. 4 
2 
CONTENT OF UPDATES .................................................................................................................... 4 
3 
REPORTING OF ANALYSIS ................................................................................................................ 4 
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	


PATH 
Statistical Analysis Plan Addendum 
Updates of Secondary Efficacy Endpoints 
Final 1.0, 25Nov2020 
 
4 
 
  
1 
INTRODUCTION 
This SAP Addendum document is created to update secondary efficacy endpoints which 
were described in section 1.2.3 and section 4.4.2 of the final 1.1 statistical analysis plan 
(SAP), dated 23 March 2020. 
2 
CONTENT OF UPDATES  
2.1. Section 1.2.3: 
“Proportion of participants with moderate to severe diarrhea following cholera challenge, 
defined as >3 liters and >5 liters of loose stools, respectively, within 48 hours following 
challenge”  
will be updated to   
“Proportion of participants with moderate to severe diarrhea following cholera challenge, 
defined as >3 liters and >5 liters of loose stools, respectively, with onset within 48 hours 
following challenge.” 
 
2.2.  Section 4.2.2: 
 
“Secondary efficacy endpoints include a) the proportion of participants with moderate or 
severe diarrhea following cholera challenge defined as >3.0 L and >5.0 L of loose stools, 
as well as b) the attack rate of any diarrhea following cholera challenge defined as the 
number of participants with 2 or more loose stools (grade 3-5) totaling >200 mL within 
48 hours or 1 loose (grade 3-5) stool > 300 mL.” 
will be updated to   
 “Secondary efficacy endpoints include a) the proportion of participants with moderate or 
severe diarrhea following cholera challenge defined as ˃3.0 L and ˃5.0 L of loose stools, 
as well as b) the attack rate of any diarrhea following cholera challenge defined as the 
number of participants with 2 or more loose stools (grade 3-5) totalling >200 mL with 
onset within 48 hours or 1 loose (grade 3-5) stool ˃ 300 mL.” 
3 
REPORTING OF ANALYSIS 
The updates of secondary efficacy endpoints will be applied to all related analysis in 
Table, Figure and Listings (TFLs) and Clinical Study Report(CSR). 
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	


PATH  
Protocol DRG-032-PO-2-01-USA 
Statistical Analysis Plan  
Final v1.1, 23Mar2020 
 
 
 
 
Statistical Analysis Plan 
 
 
 
 
DRG-032-PO-2-01-USA 
 
A Phase 2a Randomized, Single-Center, Double-Blind, Placebo 
Controlled Study to Evaluate the Safety and Preliminary 
Efficacy of Oral iOWH032 Against Cholera Diarrhea in a 
Controlled Human Infection Model 
  
 
 
 
 
 
 
 
 
 
 
 
 
 Investigational Product: 
iOWH032 
 Reference Product˖ 
 Not Applicable 
Phase˖ 
2a 
Document Type˖ 
Statistical Analysis Plan 
Version˖ 
Final v1.1 
Date of Issue˖ 
23 March 2020 
 
 
 
 
Confidential  
Page 1 of 27 
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Protocol DRG-032-PO-2-01-USA
Statistical Analysis Plan
Final v1.1, 23Mar2020
Confidential
Page 2 of 27
 
 
 
Statistical Analysis Plan Approval Form
Author:  
___________
________________
   
Date (dd/mm/yyyy)
CR Medicon US, Inc. 
Reviewer:       
__________
________________
Date (dd/mm/yyyy)
CR Medicon US, Inc.
Approver:
_________
________________
Date (dd/mm/yyyy)
PATH, Center for Vaccine Innovation and Access
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PATH  
Protocol DRG-032-PO-2-01-USA 
Statistical Analysis Plan  
Final v1.1, 23Mar2020 
Confidential 
Page 3 of 27 
 
  
 
Document Version History 
 
Version Number 
Reason for Update 
Version Date 
Final v1.1 
 
x Add blood type group (O vs Non-O) in Time 
(hours, post-challenge) to the first formed 
stool, which will be summarized with Kaplan-
Meier methods and compared with the log-
rank test. 
x Adding timing of topline analyses. 
x Update the last unformed stool to the first 
formed stool. 
23 Mar 2020 
Final v1.0 
 
 
08 Jan 2020 
 
 
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Protocol DRG-032-PO-2-01-USA 
Statistical Analysis Plan  
Final v1.1, 23Mar2020 
Confidential 
Page 4 of 27 
 
  
 
 
TABLE OF CONTENTS 
DOCUMENT VERSION HISTORY -------------------------------------------------------------------- 3 
ABBREVIATIONS ------------------------------------------------------------------------------------------ 7 
1 
INTRODUCTION -------------------------------------------------------------------------------------- 9 
1.1 
Study Objectives ------------------------------------------------------------------------------------- 9 
1.1.1 Primary Efficacy Objective ----------------------------------------------------------------------- 9 
1.1.2 Primary Safety Objective ------------------------------------------------------------------------- 9 
1.1.3 Secondary Efficacy Objectives ------------------------------------------------------------------- 9 
1.1.4 Secondary Safety Objective ---------------------------------------------------------------------- 9 
1.1.5 Exploratory Objectives ---------------------------------------------------------------------------- 9 
1.2 
 Study Endpoints ------------------------------------------------------------------------------------- 9 
1.2.1 Primary Efficacy Endpoints ---------------------------------------------------------------------- 9 
1.2.2 Primary Safety Endpoint -------------------------------------------------------------------------- 9 
1.2.3 Secondary Efficacy Endpoints ------------------------------------------------------------------- 9 
1.2.4 Secondary Safety Endpoints --------------------------------------------------------------------- 10 
1.2.5 Exploratory Endpoints ---------------------------------------------------------------------------- 10 
1.3 
STUDY DESIGN ----------------------------------------------------------------------------------- 10 
1.3.1 General Study Design and Plan ----------------------------------------------------------------- 10 
1.3.2 Power Estimate ------------------------------------------------------------------------------------ 13 
1.3.3 Randomization and Blinding -------------------------------------------------------------------- 14 
2 
STATISTICAL CONSIDERATIONS ------------------------------------------------------------ 14 
2.1 
GENERAL CONSIDERATIONS --------------------------------------------------------------- 14 
2.2 
ANALYSIS SETS----------------------------------------------------------------------------------- 15 
3 
DATA HANDLING CONVENTIONS ----------------------------------------------------------- 15 
3.1 
DERIVED AND TRANSFORMED DATA --------------------------------------------------- 16 
3.1.1 Baseline and Change from Baseline Definition ----------------------------------------------- 16 
3.1.2 Study Day ------------------------------------------------------------------------------------------ 16 
3.1.3 Lab Parameter Results with Special Characters ---------------------------------------------- 16 
3.2 
ANALYSIS VISIT WINDOWS ----------------------------------------------------------------- 16 
3.3 
PREMATURE WITHDRAWAL AND MISSING DATA --------------------------------- 16 
3.4  
MULTIPLE COMPARISON/MULTIPLICITY -------------------------------------------- 16 
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PATH  
Protocol DRG-032-PO-2-01-USA 
Statistical Analysis Plan  
Final v1.1, 23Mar2020 
Confidential 
Page 5 of 27 
 
  
 
4 
STATISTICAL ANALYSES ----------------------------------------------------------------------- 17 
4.1 
SUBJECT INFORMATION --------------------------------------------------------------------- 17 
4.1.1 Disposition of Subjects --------------------------------------------------------------------------- 17 
4.1.2 Protocol Deviations ------------------------------------------------------------------------------- 17 
4.1.3 Demographics and Baseline Characteristics -------------------------------------------------- 17 
4.1.4 Serology -------------------------------------------------------------------------------------------- 17 
4.1.5 Pregnancy ------------------------------------------------------------------------------------------ 17 
4.1.6 Prior and Concomitant Medications ------------------------------------------------------------ 18 
4.1.7 Medical History ----------------------------------------------------------------------------------- 18 
4.1.8 Cholera Challenge --------------------------------------------------------------------------------- 18 
4.1.9 Study Drug Administration ---------------------------------------------------------------------- 18 
4.1.10 
Antibiotics treatment --------------------------------------------------------------------------- 18 
4.1.11 
Comprehension Assessment Tool and Venous Assessment------------------------------ 18 
4.2 
 EFFICACY ANALYSIS ------------------------------------------------------------------------- 19 
4.2.1 Analysis of the Primary Efficacy Endpoint --------------------------------------------------- 19 
4.2.2 Analysis of the Secondary Efficacy Endpoints ----------------------------------------------- 19 
4.3 
 SAFETY ANALYSIS ----------------------------------------------------------------------------- 21 
4.3.1 Adverse Events (AEs) ---------------------------------------------------------------------------- 21 
4.3.1.1 Overview of AEs ---------------------------------------------------------------------------- 21 
4.3.1.2 Incidence of TEAEs ------------------------------------------------------------------------- 21 
4.3.1.3 Incidence of TEAEs by Severity ---------------------------------------------------------- 22 
4.3.1.4 Incidence of Study Drug Related TEAEs ------------------------------------------------ 22 
4.3.1.5 Incidence of Study Related TEAEs ------------------------------------------------------- 22 
4.3.1.6 Serious Adverse Events (SAEs) ----------------------------------------------------------- 22 
4.3.1.7 TEAEs Leading to Discontinuation of Study Drug ------------------------------------- 22 
4.3.1.9 TEAEs Leading to Death ------------------------------------------------------------------- 22 
4.3.1.10 Solicited Adverse Events ------------------------------------------------------------------- 22 
4.3.1.11 Unanticipated Problems (UPs) ------------------------------------------------------------- 23 
4.3.2 Safety Laboratory Tests -------------------------------------------------------------------------- 23 
4.3.3 Urinalysis ------------------------------------------------------------------------------------------- 23 
4.3.4 Vital Signs ------------------------------------------------------------------------------------------ 23 
4.3.5 ECGs ------------------------------------------------------------------------------------------------ 23 
4.3.6 Physical Examination and Physical Measurement ------------------------------------------- 24 
4.4 
 Exploratory Analyses ----------------------------------------------------------------------------- 24 
4.4.1 Pharmacokinetic (PK) Properties of iOWH032 ---------------------------------------------- 24 
4.4.2 Evaluate Time of Cessation of Cholera Organism in Stool after Challenge -------------- 24 
4.4.3 Evaluate a decrease for oral rehydration solution and/or intravenous fluid replacement 
therapy need ------------------------------------------------------------------------------------------------ 24 
4.4.4 Exploratory analyses of diarrheal stool output ------------------------------------------------ 25 
5 
INTERIM ANALYSIS ------------------------------------------------------------------------------- 25 
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Protocol DRG-032-PO-2-01-USA 
Statistical Analysis Plan  
Final v1.1, 23Mar2020 
Confidential 
Page 6 of 27 
 
  
 
6 
SUMMARY OF MAJOR CHANGES IN THE PLANNED ANALYSES ---------------- 25 
7 
PROGRAMMING SPECIFICATIONS ---------------------------------------------------------- 26 
8 
TABLES, LISTINGS, AND FIGURES SHELLS ---------------------------------------------- 26 
APPENDIX A: CLINICAL SAFETY LABORATORY TOXICITY SCALES --------------- 27 
 
 
 
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PATH  
Protocol DRG-032-PO-2-01-USA 
Statistical Analysis Plan  
Final v1.1, 23Mar2020 
Confidential 
Page 7 of 27 
 
  
 
ABBREVIATIONS 
AE 
Adverse Event 
ALT 
Alanine aminotransferase 
ALP                 Alkaline phosphatase 
AMS                Accelerator Mass Spectrometry 
      ANC                Absolute neutrophil count  
AST 
      Aspartate aminotransferase 
ATC 
Anatomical Therapeutic Code 
AUC 
Area under the Concentration-time Curve 
BLQ                Below the Limit of Quantification 
BMI 
Body Mass Index 
BP                   Blood Pressure 
CBC                Complete Blood Count 
CRU 
Clinical Research Unit 
CPK                 Creatine phosphokinase 
CSR 
Clinical Study Report 
CV 
Coefficient of Variation 
eCRF 
Electronic Case Report Form 
ECG 
Electrocardiogram 
ET 
Early Termination 
HBsAg 
Hepatitis B Surface Antigen 
HCV Ab 
Hepatitis C Virus Antibody 
HIV 
Human Immunodeficiency Virus 
HPLC              High Performance Liquid Chromatography  
HR 
Heart Rate 
ITT                  Intent to Treat 
LLOQ              Lower Limit of Quantification 
MedDRA 
Medical Dictionary for Regulatory Activities 
mITT               Modified ITT 
MH                  Medical History 
ORS 
Oral Rehydration Solution 
PI 
Principal Investigator 
PK 
Pharmacokinetic 
PP                    Per Protocol 
PR 
Time between the Start of the P Wave and the Start of the QRS Complex 
PT 
Preferred Term 
QT 
Time between the Start of the Q Wave and the End of the T Wave 
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Protocol DRG-032-PO-2-01-USA 
Statistical Analysis Plan  
Final v1.1, 23Mar2020 
Confidential 
Page 8 of 27 
 
  
 
QTcF 
QT Interval Corrected by the Fridericia Formula 
RR                   R-R interval 
SAE 
Serious Adverse Events 
SAP 
Statistical Analysis Plan 
SD 
Standard Deviation 
SOC 
System Organ Class 
SRC                 Safety Review Committee 
TEAE 
Treatment Emergent Adverse Event 
ULN                Upper Limit of Normal   
WBC               White Blood Cell 
WHO 
World Health Organization 
 
 
 
 
 
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PATH  
Protocol DRG-032-PO-2-01-USA 
Statistical Analysis Plan  
Final v1.1, 23Mar2020 
Confidential 
Page 9 of 27 
 
  
 
 
1 
INTRODUCTION 
The purpose of this statistical analysis plan (SAP) is to provide details and specifications for 
statistical analyses of the study DRG-032-PO-2-01-USA “A Phase 2a Randomized, Single-
Center, Double-Blind, Placebo Controlled Study to Evaluate the Safety and Preliminary Efficacy 
of Oral iOWH032 Against Cholera Diarrhea in a Controlled Human Infection Model” as set 
forth in the clinical study protocol Version 4.0, dated 13 March 2020.  
1.1 
STUDY OBJECTIVES 
1.1.1 PRIMARY EFFICACY OBJECTIVE 
x To measure the rate and extent of diarrhea following cholera challenge, in participants 
treated with iOWH032 compared to placebo 
1.1.2 PRIMARY SAFETY OBJECTIVE 
x To evaluate safety of oral doses of iOWH032 compared to placebo 
1.1.3 SECONDARY EFFICACY OBJECTIVES 
x To evaluate additional measures of efficacy against cholera illness following cholera 
challenge, among those randomized to be treated with iOWH032 versus placebo 
1.1.4 SECONDARY SAFETY OBJECTIVE 
x To evaluate tolerability of oral doses of iOWH032 compared to placebo 
1.1.5 EXPLORATORY OBJECTIVES 
x To evaluate the pharmacokinetic (PK) properties of iOWH032 
x To evaluate time of cessation of cholera organism in stool after challenge 
x To evaluate a decrease for oral rehydration solution (ORS) and/or intravenous (IV) fluid 
replacement therapy need 
1.2 
 STUDY ENDPOINTS 
1.2.1 PRIMARY EFFICACY ENDPOINTS 
x Diarrheal stool output rate, defined as the total volume of diarrheal stools (mL, Grade 3 and 
above) divided by the number of hours between initiation of study product dosing and 
initiation of antimicrobial therapy 
1.2.2 PRIMARY SAFETY ENDPOINT 
x 
Frequency and incidence of serious adverse events (SAEs) throughout the study 
1.2.3 SECONDARY EFFICACY ENDPOINTS 
x Proportion of participants with moderate to severe diarrhea following cholera challenge, 
defined as >3 liters and >5 liters of loose stools, respectively, within 48 hours following 
challenge 
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Protocol DRG-032-PO-2-01-USA 
Statistical Analysis Plan  
Final v1.1, 23Mar2020 
Confidential 
Page 10 of 27 
 
  
 
x Attack rate of any diarrhea following cholera challenge, defined as the number of 
participants with either 2 or more loose stools (grades 3-5) totaling > 200 mL or 1 loose 
(grade 3-5) stool > 300 mL, respectively, within 48 hours following challenge 
x Area under the curve (AUC) of diarrheal stool volume between challenge dose and 
initiation of antibiotics 
x Density of cholera organisms in stool samples measured in stool samples via quantitative 
stool culture 
x To evaluate cholera illness in each participant after cholera challenge, using the following 
objective parameters: 
1. Duration of diarrheal episode as defined by time to first formed stool 
2. Total number of loose (Grades 3-5) stools 
3. Occurrence of fever 
4. Occurrence of vomiting 
1.2.4 SECONDARY SAFETY ENDPOINTS 
x To measure the occurrence of solicited adverse effects during the three days of oral dosing, 
through 8 hours following the last dose, which are attributed to iOWH032 and placebo, to 
include: 
1. Nausea 
2. Abdominal discomfort and pain 
3. Occurrence of abdominal cramps 
4. Occurrence of headache 
5. Occurrence of malaise 
6. Anorexia 
7. Pollakiuria 
8. Micturition urgency 
9. Sinus tachycardia 
10. 
Increased alertness 
x To measure the frequency and incidence of unsolicited adverse effects up to 28 days after 
the last dose. 
1.2.5 EXPLORATORY ENDPOINTS 
x To determine plasma levels of iOWH032 7±1 hours after the first and last dosing in all 
participants 
x Time (hours) to cessation of detectable cholera in stool, defined as the time of the first 
sample negative via quantitative stool culture, after which all following samples are also 
negative for cholera. 
x ORS and/or intravenous fluid replacement measured in liters 
1.3 STUDY DESIGN 
1.3.1 GENERAL STUDY DESIGN AND PLAN 
This clinical trial is designed to be a Phase 2a, which will document the preliminary efficacy 
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Protocol DRG-032-PO-2-01-USA 
Statistical Analysis Plan  
Final v1.1, 23Mar2020 
Confidential 
Page 11 of 27 
 
  
 
of oral doses of iOWH032 in diminishing the severity of illness of cholera, using a well-
established human challenge model. 
In Cohort 1, 24 eligible healthy male and female participants will be randomized 1:1 to be 
assigned to the iOWH032 (active treatment) or placebo. Following a favorable assessment of 
iOWH032 in an interim analysis, Cohort 2 will include an additional 24 healthy participants 
randomized 1:1 to iOWH032 or placebo. All participants will be challenged with 106 cfu of V. 
cholerae El Tor Inaba strain N16961. Blinded treatment will begin at the onset of (any) 
diarrhea symptoms or 48 hours after challenge, whichever is sooner, and consists of three oral 
doses per day, over three days. The dosage of iOWH032 is 500 mg (two 250 mg tablets) per 
dose. 
A 3-day course of antimicrobial therapy will be initiated at approximately 4 days post 
challenge or sooner if indicated. The Principal Investigator will be able to initiate antibiotics 
upon severe cholera definition being satisfied. The blinded study treatment will continue 
during antimicrobial therapy. For participants that remain asymptomatic, the last dose of 
iOWH032 and first dose of antibiotic will be separated by 1-2 hours. Discharge from the 
Clinical Research Unit will be contingent upon each participant meeting all the following 
criteria: 
x 3 consecutive stool cultures that are negative for growth of vibrios (with each culture 
separated by at least 12 hours) 
x The absence of moderate or higher grade objective reactogenicity (diarrhea, fever, and 
vomiting) for at least 12 hours prior to discharge 
x The completion of a 3-day course of antimicrobial (preferred agent, ciprofloxacin, 500 mg 
twice daily for 3 days) therapy 
The schedule of events is provided in Table 1. 
 
 
 
 
 
 
 
 
 
 
 
 
 
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Protocol DRG-032-PO-2-01-USA 
Statistical Analysis Plan  
Final v1.1, 23Mar2020 
Confidential 
Page 12 of 27 
 
  
 
 
Error! Reference source not found.:   Schedule of study visits and evaluations 
 
Study visit 
Visit 1 
(screen) 
Inpatient Containment Period 
Visit 2 Visit 3 Visit 4 
Study Day 
D-85 to-
D-1 D1 D2 D3 D4 
D5 D6 D7 D8 
D9 D10 D15±1 D29±2 D180±1
Informed Consent 
√   
  
  
 
 
 
 
 
 
 
 
 
 
 
Medical history, 
medications 
√   
  
  
 
 
 
 
 
 
 
 
 
 
 
Comprehension 
Assessment Tool 
√   
  
  
 
 
 
 
 
 
 
 
 
 
 
Physical examination 
√   
√h 
√h 
√h 
√h 
√h 
√h 
√h 
√h 
√h √h 
√h 
√h  
Pregnancy testa 
√    √ 
   
 
 
 
 
 
 
 
 
 
 
 
Vital signs 
√   
√g √ g √ g √ g √ g √ g √ g √ g 
√g √ g  
 
 
Screening laboratoryb 
18 mL   
  
  
 
 
 
 
 
 
 
 
 
 
 
12-lead ECG 
√   
  
  
 
 
 
 
 
 
 
 
 
 
 
Pre-challenge Stool 
 
√ 
 
 
Safety laboratoryc 
  
10 mL   
  
 
 
 
 
 
 
 
 
10 mL  
 
Eligibility confirmation 
√ 
√ √   
 
 
 
 
 
 
 
 
 
 
 
Challenge 
  
  
√   
 
 
 
 
 
 
 
 
 
 
 
Blinded Study Drug 
Administrationd  
  
  
(√) (√) (√)  
 
 
 
 
 
 
 
 
 
3-day course of 
antibioticse  
  
  
  
  
 
 
(√) (√) (√)  
 
 
 
 
 
Observe & manage 
illnessf  
  
  
√ 
√ 
√ 
√ 
√ 
√ 
√ (√) (√) (√)  
 
 
Stool grading and 
culturesf  
  
  
√ 
√ 
√ 
√ 
√ 
√ 
√ (√) (√) (√)  
 
 
pK analysisi  
  
  
√   
√  
 
 
 
 
 
 
 
 
 
Cumulative blood 
volume 
18 mL 10 mL 
2 
mL 
  
2 
mL 
 
 
 
 
 
 
 
10 mL  
 
Solicited AEs 7 days 
post-challenge 
  
  
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√  
 
 
 
 
Unsolicited AEs 
  
  
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√  
SAEs 
  
  
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
Concomitant 
medications 
  
  
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
Interval medical 
history 
  
  
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
Unanticipated 
problems (UPs) 
√ 
√ √ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
√ 
a. For females of child bearing potential, a serum pregnancy test will be performed at screening and one day before challenge 
b. Screening laboratory to include: Complete Blood Count (CBC) for WBC, ANC, Hemoglobin, and Platelets; Chemistry panel for Na, K, 
ALT, AST, Creatinine, Albumin, ALP, CPK, Glucose and Total Bilirubin; Serology for HbSAg, anti-HCV, and HIV; Blood Typing; and 
Urinalysis 
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Protocol DRG-032-PO-2-01-USA 
Statistical Analysis Plan  
Final v1.1, 23Mar2020 
Confidential 
Page 13 of 27 
 
  
 
c. Safety laboratory to include: Complete blood count (CBC) for WBC, ANC, Hemoglobin, and Platelets; and Chemistry panel for Na, K, 
ALT,AST,ALP, Creatinine, Albumin, and Total  
Bilirubin, CPK, Glucose 
d. Blinded therapeutic dosing will start at the onset of symptoms or by 48 hours after ingesting the cholera challenge inoculum. 
e. A 3-day course of antibiotics will be initiated upon satisfying the criteria for severe cholera diarrhea or approximately 4 day post-challenge, 
whichever is earlier 
f. The observation and management of cholera illness (includes measurement of vomiting) and stool grading and stool cultures are intended to 
be performed throughout the post-challenge inpatient period, until discharge criteria are satisfied. For the duration of diarrhea, the participant 
will be requested to provide a urine specimen from every void that they experience; the specimen will be tested for urine specific gravity. In 
case of IV fluid replacement, serum electrolytes, BUN, and creatinine will be measured. 
g. Vital signs will be measured at least 3 times daily (approximately every 8 ±1 hours). Vital signs will also be measured every 4 hours when a 
participant has a fever ≥ 39°C (102.1°F), until the participant has 2 consecutive temperatures of ≤ 38°C (100.4°F). On Day 1, including the 
pre-challenge vitals assessment, there will be 4 vital signs assessments (1x pre-challenge and 3x post-challenge). 
h. Focused physical examination at the discretion of the physician 
i. pK plasma samples for the levels of iOWH032 will be collected 7±1 hours after the first and last dosing in all participants 
 
1.3.2 POWER ESTIMATE  
Because of the multiplicity of testing and accompanying efficacy and futility thresholds at the 
interim analysis, a group-sequential framework is employed. Traditional methods for trial 
design are intractable in this case, due to the anticipated distribution of the endpoint and the 
use of stratified nonparametric testing for its evaluation. For this reason, a simulation-based 
framework was used to simulate data under both the null and alternative hypotheses. Power is 
computed as the proportion of simulations under the alternative hypothesis which yield a 
conclusion of superiority at either interim or, given that futility is not declared at interim, at the 
final analysis. Type I error is computed as the proportion of simulations under the null 
hypothesis which yield the same conclusion, erroneously. The simulation procedure was 
submitted to a constrained optimization routine that selected boundary values which ensure 
overall ≥90% power and type I error ≤0.025, simultaneously. Further simulations confirmed 
the adequacy of these boundaries to provide high power while maintaining nominal type I 
error rate. The boundary values that will be used at the interim analysis are: 
x 
If the one-sided Van Elteren test of the superiority of the diarrheal stool output 
rate (iOWH032 vs placebo) yields a p-value less than 0.0051, then the study product 
will be deemed superior to placebo, and the second cohort will not be enrolled 
x 
If the one-sided Van Elteren test of the superiority of the diarrheal stool output 
rate (iOWH032 vs placebo) yields a p-value greater than 0.4585, this will be 
considered evidence of futility (lack of demonstrated efficacy), and the second cohort 
will not be enrolled 
x 
In all other cases, the 2nd cohort will be enrolled 
Due to the nonzero probability of concluding efficacy at the interim analysis, the alpha level 
(type I error rate) for the final analysis needs to be adjusted to maintain overall one-sided level 
alpha = 0.025. The threshold to define success on the primary efficacy endpoint at the final 
analysis is one-sided alpha = 0.0238. This hypothesis test will be supplemented by a two-sided 
95% confidence interval for difference in median diarrheal stool output rate, using the 
percentile bootstrap method, with n=10,000 replicates. 
With the abovementioned boundary values and sample size (24 participants per group), the 
power decreases to 70% for a 40% reduction in daily diarrheal stool rate and to 30% for a 
reduction of 25% in daily diarrheal stool rate. However, an effect size of 50% reduction is 
expected to represent a feasible effect size for an impactful therapy. 
For the secondary endpoint of proportion of participants with moderate or severe diarrhea, 
with 24 participants per group, and assuming 40% of participants are Type O, adequate power 
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Protocol DRG-032-PO-2-01-USA 
Statistical Analysis Plan  
Final v1.1, 23Mar2020 
Confidential 
Page 14 of 27 
 
  
 
is available to detect odds ratios of moderate or severe diarrhea in iOWH032-treated 
participants vs placebo which are significantly less than 1, using a Cochran-Mantel-Haenszel 
test stratified by blood type group, with two-sided level alpha = 0.05. For example, if 67% 
(47%) of the type O (non-type O) control participants experience moderate or severe diarrhea, 
and only 20% (10%) of type O (non-type O) iOWH032-treated participants do (common odds 
ratio 0.123), then 80% power is available to detect a common odds ratio < 1. Only a single 
analysis will be conducted for efficacy endpoints other than the primary efficacy endpoint, 
upon collection of data from all participants included in the experiment. 
1.3.3 RANDOMIZATION AND BLINDING 
There will be 24 study participants challenged in the first cohort and an additional 24 study 
participants challenged in the second cohort, pending favorable results of an interim analysis. 
Subject treatment assignment will be based on a computer-generated randomization scheme 
using SAS V 9.4 or higher with a ratio of 1:1 stratified by blood type status (O vs. Non-O) to 
receive either iOWH032 500 mg every 8 hours for three days or matching placebo.  
The administration of iOWH032 and iOWH032 placebo will be double-blinded.   
Subjects, the Investigator and the Sponsor will be blinded to the treatment administered until 
enrollment is complete and the study database is locked. Only the unblinded Pharmaron CPC 
pharmacists will be provided with the randomized treatment sequences. 
2 
STATISTICAL CONSIDERATIONS 
2.1 
GENERAL CONSIDERATIONS 
SAS version 9.4 or greater (SAS Institute Inc., Cary, NC, SAS System) will be used for analysis. 
For descriptive statistics, continuous variables will be summarized by the number of non-missing 
observations, mean, median, standard deviation, and minimum and maximum. Categorical 
variables will be summarized by presenting the frequency and percent. Unless otherwise 
specified, inference such as confidence interval construction will be conducted with two-tailed 
Type I error level α = 0.05. All secondary endpoints will be considered as supportive evidence of 
efficacy and will be analyzed without any procedures to account for multiple comparisons. 
In general, nonparametric analyses or exact methods (e.g., Fisher’s exact test) will be preferred 
for efficacy analyses, with confidence intervals for binary variables computed via the Clopper-
Pearson exact method, and confidence intervals for continuous variables computed via the 
percentile bootstrap method, using n=10,000 replicates each. 
Primary efficacy analysis will be stratified by blood type status (O vs Non-O) as described 
below. All efficacy endpoints will be summarized, by group and by group and O vs Non-O 
status, using daily and overall summaries, where appropriate. Exploratory analyses will consider 
stratification by cohort to account for potential differences in challenge inoculum between 
cohorts. 
Analysis of diarrheal stool output rate, diarrheal stool volume, cholera-containing diarrheal stool 
volume, and density of cholera organisms will be supplemented with reverse cumulative 
distribution curves. 
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Protocol DRG-032-PO-2-01-USA 
Statistical Analysis Plan  
Final v1.1, 23Mar2020 
Confidential 
Page 15 of 27 
 
  
 
Unscheduled results will not be included in the summary tables except for determining baseline, 
but will be presented in data listings. 
All data listings will be sorted by group, subject and visit/timepoint where applicable. All dates 
will be displayed in DDMMMYYYY format. No algorithm for missing data imputation will be 
employed. 
2.2 
ANALYSIS SETS 
Assignment of participants to analysis populations will be completed prior to unblinding, for 
each cohort. 
For purposes of analysis, the following populations are defined: 
Population 
Description 
Enrolled 
All participants who sign the informed consent form (ICF). 
Intent to Treat (ITT) 
All participants who are randomized to study treatment 
Participants will be analyzed according to their randomized 
treatment. 
Modified ITT (mITT) 
The subset of ITT who receive at least one dose of study drug. 
Any participant displaying no indication of cholera infection (no 
diarrheal stool output of grade 3 or higher) within 48 hours of 
challenge will be removed from the mITT population, prior to 
unblinding of data. 
Per Protocol Population 
The subset of the mITT consisting of those participants who had 
no major protocol deviations and received all doses of assigned 
study drug. 
All protocol deviations will be assessed and documented on a 
case-by-case basis before the database lock, and deviations 
considered having a serious impact on the efficacy results will 
lead to the relevant participant being excluded from the per-
protocol population. Before database lock, potential participant 
exclusions from per-protocol population will be reviewed by the 
Sponsor and documented. 
Safety 
The Safety Analysis Set includes all participants who received 
any study treatment. Safety analyses and demographic/baseline 
characteristic summaries will be based on the safety population, 
according to treatment received. 
3 
DATA HANDLING CONVENTIONS 
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3.1 
DERIVED AND TRANSFORMED DATA 
3.1.1 BASELINE AND CHANGE FROM BASELINE DEFINITION 
The baseline value of a variable is defined as the last value obtained on or before the date and 
time of the dose of iOWH032 and iOWH032 placebo, including unscheduled assessments.  
Change from baseline will be derived for each subject as the post-baseline evaluation minus the 
baseline evaluation.  
3.1.2 STUDY DAY 
Study Day will be calculated from the reference start date (defined as the day of administration 
of study drug), and will be used to show start/stop day of assessments/events: 
x Study Day = (date of assessments/events - reference start date) if event/assessment is 
prior to the reference start; 
x Study Day = (date of assessments/events - reference start date + 1) if event/assessment is 
on or after the reference start. 
There is no study day 0. 
3.1.3 LAB PARAMETER RESULTS WITH SPECIAL CHARACTERS 
If any lab parameter results contain special characters such as ‘<’ or ‘>’, the numerical portion of 
the results will be used for descriptive summary. 
3.2 
ANALYSIS VISIT WINDOWS 
For analysis of data over time (wherever specified), the nominal study days/visits will be used as 
analysis days/visits. No windowing algorithm will be used.  
3.3 
PREMATURE WITHDRAWAL AND MISSING DATA 
The reason for participant discontinuation or withdrawal from the study will be recorded on the 
Case Report Form (CRF). Participants who sign the ICF and are randomized but do not receive 
the challenge may be replaced. Participants who sign the ICF, and are randomized and receive 
the challenge, and subsequently withdraw, or are withdrawn or discontinued from the study, will 
not be replaced. 
All reasonable measures will be taken to ensure minimal missing data. Despite this, some data 
are likely to be missing at the end of the study. No imputation is planned for any endpoints in 
this study, and all data will be considered to be missing completely at random. Should 
observations indicate this is not likely to be true for a given endpoint, additional sensitivity 
analyses may be conducted, such as multiple imputation, to assess the impact of the missing data. 
3.4  
MULTIPLE COMPARISON/MULTIPLICITY 
Adjustment for multiplicity for the group-sequential analysis of the primary efficacy endpoint is 
described in Section 5. All secondary endpoints will be considered as supportive evidence of 
efficacy and will be analyzed without any procedures to account for multiple comparisons. 
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Protocol DRG-032-PO-2-01-USA 
Statistical Analysis Plan  
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Confidential 
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Due to the nonzero probability of concluding efficacy (under alternative hypotheses) at the 
interim analysis, the alpha level (type I error rate) for the final analysis needs to be adjusted to 
maintain overall one-sided level alpha = 0.025. The threshold to define success on the primary 
efficacy endpoint at the final analysis is one-sided alpha = 0.0238. 
4 
STATISTICAL ANALYSES 
4.1 
SUBJECT INFORMATION 
4.1.1 DISPOSITION OF SUBJECTS 
Subject disposition will be summarized for all enrolled subjects by group. The number and 
percentage of subjects in all Populations, as well as number and percentage of subjects who 
completed the study and discontinued the study will be summarized along with the primary 
reason for discontinuation. The percentages will be based on the number of subjects in the Safety 
Population.  
A listing will present whether the subject is in the Safety, ITT, mITT and PP Population, whether 
the subject completed the study, date of completion/withdrawal, and the primary reason for 
discontinuation of study, if applicable. 
A subject eligibility listing will also be provided to describe if the subject meets all 
inclusion/exclusion criteria. 
4.1.2 PROTOCOL DEVIATIONS 
The study site will record all deviations that occurred during the conduct of the study. Protocol 
deviations will be classified as either minor or major by the Sponsor or designee prior to 
database lock.  
The protocol deviation data with the verbatim description, the reason for the deviation and 
whether the deviation is classified as minor or major will be summarized by group and listed. 
4.1.3 DEMOGRAPHICS AND BASELINE CHARACTERISTICS 
Demographics and baseline characteristics (blood type, age, gender, race, height (cm), weight 
(kg) and body mass index (BMI) (kg/m2)) will be summarized descriptively by treatment group 
for the Safety Population and mITT. Descriptive statistics will be presented for age, height, 
weight, and BMI. Frequency counts and percentage will be presented for blood type, sex, 
ethnicity origin, and race.  
The demographic and baseline characteristics data will also be presented as data listings by 
group and subject. 
4.1.4 SEROLOGY 
The screening serology for hepatitis B surface antigen (HBsAg), hepatitis C virus antibody (anti-
HCV) and human immunodeficiency virus antibodies (anti-HIV) will be listed by group and 
subject. 
4.1.5 PREGNANCY 
A serum pregnancy test (Serum β-HCG) at screening and one day before challenge will be listed 
by group and subject for all women. 
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Statistical Analysis Plan  
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4.1.6 PRIOR AND CONCOMITANT MEDICATIONS 
Concomitant medications will be coded using the World Health Organization (WHO) Drug 
Global September 1 2019. 
Prior medications will be those discontinued prior to the start of the study drug. Concomitant 
medications will be medications started prior to the start of the study drug and continued after 
administration of study drug or started during the study after administration of study drug.  
Prior and concomitant medications will be summarized (separately) by anatomical-therapeutic-
chemical (ATC) class and preferred term (PT) after coding with the WHO Drug dictionary. 
Prior and concomitant medication information will be listed by group and subject with verbatim 
text given by the investigator, WHO Drug Dictionary PT, indication, whether it is related to 
AE/MH and associated AE/MH number, start and stop date/time, dosage, route, and frequency. 
4.1.7 MEDICAL HISTORY  
A comprehensive medical history will be collected in screening by interview with the participant 
including participation in clinical trials, surgery, previous hospitalization, allergy to food/drugs, 
and history of any chronic or recurrent medical conditions. Interval medical history will inquiry 
regarding changes since the last medical history discussion including signs and symptoms. 
All medical history will be coded using Medical Dictionary for Regulatory Activities (MedDRA) 
22.. Medical history will be listed by group and subject. 
4.1.8 CHOLERA CHALLENGE 
The cholera challenge information will be listed by group and subject including whether 
participant was administered challenge, whether subject fasted for 90 minutes prior to challenge, 
date and time of challenge, and comments. 
4.1.9 STUDY DRUG ADMINISTRATION 
The study drug administration information will be listed by group and subject including date and 
time of dose, whether the drug was delayed and reason, AE number if AE, specify if other, and 
comments.  
4.1.10 ANTIBIOTICS TREATMENT 
Antibiotics treatment information will be listed by group and subject including day of treatment, 
date and time of dose, antibiotic name, prescribed dose, dosing frequency and comments.  
If the subject is terminated early from the study, antibiotic treatment information for early 
termination will be listed including day of treatment, date and time of dose, antibiotic name, 
prescribed dose, whether doses were provided for participant to take home, total dose provided 
for the participant to take home and comments.  
4.1.11 COMPREHENSION ASSESSMENT TOOL AND VENOUS ASSESSMENT 
Whether the subject passed the quiz of Comprehension Assessment Tool (≥70% correct answers) 
and assessment for venous will be listed by group and subject. 
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4.2 
 EFFICACY ANALYSIS  
4.2.1 ANALYSIS OF THE PRIMARY EFFICACY ENDPOINT  
The primary efficacy endpoint is “Diarrheal stool output rate”, defined as the total volume of 
diarrheal stools (mL, Grade 3 and above) divided by the number of hours between initiation of 
study product dosing and initiation of antimicrobial therapy. Diarrheal stool output rate will be 
summarized, by group and by group and O vs Non-O status, using daily and overall summaries. 
Statistical comparison of the primary endpoint between groups will be conducted with the 
stratified (by blood type group) version of the Wilcoxon rank-sum test, the Van Elteren test.  
A sample of SAS implementation code for the Van Elteren test is shown below: 
proc npar1way data=adeff; 
    strata stratum; /* O vs Non-O status */ 
    class trt01p; /* iOWH032 vs Placebo */ 
    var effrate;   /* Diarrheal stool output rate */ 
run; 
 
Due to the nonzero probability of concluding efficacy at the interim analysis, the alpha level 
(type I error rate) for the final analysis needs to be adjusted to maintain overall one-sided level 
alpha = 0.025. The threshold to define success on the primary efficacy endpoint at the final 
analysis is one-sided alpha = 0.0238. The hypothesis test will be supplemented by a two-sided 
confidence interval for the difference in median diarrheal stool output rate, using the percentile 
bootstrap method, with n=10,000 replicates. 
The principal analysis will be performed using the mITT population. If greater than 20% of 
subjects are excluded from the mITT due to onset of symptoms after 48 hours, the primary 
endpoint will be calculated again including patients with symptom onset after 48 hours. For 
subjects with symptom onset after 48 hours, the time between onset of symptoms and 
administration of antimicrobials will define the denominator used to calculate the diarrheal stool 
output rate. Supportive analysis will be performed using the per-protocol population. 
Analysis of diarrheal stool output rate will be supplemented with reverse cumulative distribution 
curves. The horizontal axis of the plot is the rate after logarithmic conversion, and the vertical 
axis is the percentage of individuals larger than this rate. 
Diarrhea Collection information will be listed by group and subject including date and time of 
collection, volume of Diarrhea collection, Grade and comments.  
4.2.2 ANALYSIS OF THE SECONDARY EFFICACY ENDPOINTS 
Secondary efficacy endpoints include a) the proportion of participants with moderate or severe 
diarrhea following cholera challenge defined as >3.0 L and >5.0 L of loose stools, as well as b) 
the attack rate of any diarrhea following cholera challenge defined as the number of participants 
with 2 or more loose stools (grade 3-5) totaling >200 mL within 48 hours or 1 loose (grade 3-5) 
stool > 300 mL. 
Both endpoints will be computed as the proportion within each group satisfying the respective 
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Statistical Analysis Plan  
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Confidential 
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definitions, and will be accompanied by exact two-sided 95% confidence intervals. This will be 
supplemented with corresponding summaries within study arm and blood type group. Statistical 
inference for each endpoint will be conducted with the Cochran-Mantel-Haenszel test, stratified 
by blood type group. 
If a two-sided statistical test with Type I error rate of 0.05 rejects H0 and the proportion of 
participants is less for the iOWH032 group than for the placebo group, the study will have 
demonstrated that iOWH032 is superior. 
The AUC of diarrheal stool volume and cholera organisms will be computed via the trapezoidal 
rule. The secondary endpoints of AUC of cholera-containing diarrheal stool volume, AUC of 
cholera organisms, and peak shedding of cholera organisms (highest cfu counts for each 
participant) will be analyzed in quantitative stool culture. Statistical comparison of the three 
secondary endpoints between groups will be conducted with the stratified (by blood type group) 
version of the Wilcoxon rank-sum test, the Van Elteren test. If a one-sided statistical test rejects 
H0 and the median is less for the iOWH032 group than for the placebo group, the study will 
have demonstrated that iOWH032 is superior. The hypothesis test will be supplemented by a 
two-sided 95% confidence interval for the difference in median AUC of cholera-containing 
diarrheal stool volume, AUC of cholera organisms, and peak shedding of cholera organisms , 
using the percentile bootstrap method, with n=10,000 replicates. 
Cholera illness during the interval immediately following challenge and prior to initiation of 
antibiotics will be summarized by group and by blood type group (O vs Non-O) within group. 
x Time (hours, post-challenge) to the first formed stool (grade <2, overall, as well as by blood 
type group(O vs Non-O) ), will be summarized with Kaplan-Meier methods and compared 
with the log-rank test by blood type group(O vs Non-O). 
x The total count of loose stools will be summarized as a continuous variable and compared 
separately for each blood type group with the Wilcoxon rank-sum test and jointly with the 
Van Elteren test, stratified by blood type group (O vs Non-O).The hypothesis test will be 
supplemented by a two-sided 95% confidence intervals for the difference in median total 
count of loose stools, using the percentile bootstrap method, with n=10,000 replicates. 
x The presence of fever or vomiting will each be summarized as categorical variables, with 
between-group inference conducted separately for each blood type group with Fisher’s 
exact test, and jointly via the Cochran-Mantel-Haenszel test, stratified by blood type group 
(O vs Non-O). 
The principal analyses will be performed using the mITT population. Supportive analysis will be 
performed using the per-protocol population. 
Analysis of diarrheal stool volume, cholera-containing diarrheal stool volume, and density of 
cholera organisms will be supplemented with reverse cumulative distribution curves. The 
horizontal axis of the plot is the volume and density after logarithmic conversion, and the vertical 
axis is the percentage of individuals larger than this rate. 
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Final v1.1, 23Mar2020 
Confidential 
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Stool collection information will be listed by group and subject including Not done, date and 
time of collection, type of Collection, weight of stool, Grade, result of quantitative culture and 
comments.  
Vomitus collection information will be listed by group and subject including none, date and time 
of collection, volume of vomitus, grade and comments.  
4.3 
 SAFETY ANALYSIS 
4.3.1 ADVERSE EVENTS (AES) 
AEs will be coded by system organ class (SOC) and preferred term PT according to MedDRA 
22..   
The summary of AEs will be based on Treatment-emergent AEs (TEAEs) are defined as AEs 
which start or worsen following the start of study medication and up until the follow-up visit. If 
the time of the AE is not available, then TEAEs will be those occurring on or after the first day of 
study medication.  
The summary of AEs will also include an overall event count by treatment group. The treatment 
percentages (solicited/unsolicited AEs, etc.) will be supplemented with two-sided 95% confidence 
intervals. 
4.3.1.1 Overview of AEs 
A summary table will be presented, by treatment group, for the numbers and percentages of 
participants with any AE, any TEAEs, study drug related TEAEs, study related TEAEs, serious 
TEAEs, serious and study drug related TEAEs, serious and study related TEAE, any severe 
TEAEs, TEAEs leading to discontinuation from the study and TEAE leading to death.  
4.3.1.2 Incidence of TEAEs 
A summary of the frequency (number and percentage of subjects) of TEAEs will be presented by 
treatment group and by SOC and PT. Multiple occurrences of the same AE (SOC or PT) will be 
counted only once when calculating the number and percentage of subjects. This summary will 
be sorted in decreasing order of overall frequency of SOC, and then in decreasing order of 
overall frequency of PT within the SOC. 
Number and percentage of subjects with TEAEs will also be presented by PT only. This 
summary will be sorted in decreasing order of overall frequency. 
All information pertaining to AEs will be listed by subject and all the details collected on the 
eCRF, including verbatim term given by the investigator, PT, SOC, onset date/time, end 
date/time, course, severity, solicited AE category if solicited AEs, seriousness, serious AE 
definition (congenital abnormality or birth defect, significant disability, death, hospitalization or 
prolongation of hospitalization, life threatening, other medically important event), autopsy 
performed and date of death if died, causality, study product administration, treatment required, 
and outcome. 
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Statistical Analysis Plan  
Final v1.1, 23Mar2020 
Confidential 
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4.3.1.3 Incidence of TEAEs by Severity 
The number and percentage of subjects reporting a TEAE will be tabulated by treatment group 
for SOC and PT and severity. 
A subject experiencing the same AE multiple times within same SOC/PT will be counted only 
once for that SOC/PT at the worst severity. 
4.3.1.4 Incidence of Study Drug Related TEAEs 
The number and percentage of subjects reporting a study drug related TEAE will be tabulated by 
treatment group for SOC and PT. 
4.3.1.5 Incidence of Study Related TEAEs 
The number and percentage of subjects reporting as study related (defined as those related to 
study drug or related to cholera) TEAE will be tabulated by treatment group for SOC and PT. 
4.3.1.6 Serious Adverse Events (SAEs) 
SAEs will be summarized and listed in the same manner described above for TEAEs (Section 
4.3.1.2). 
4.3.1.7 TEAEs Leading to Discontinuation of Study Drug 
TEAEs leading to discontinuation of study drug will be summarized and listed in the same 
manner described above for TEAEs (Section 4.3.1.2). 
4.3.1.9 TEAEs Leading to Death 
TEAEs leading to death will be listed in the same manner described above for TEAEs (Section 
4.3.1.2). 
4.3.1.10 
Solicited Adverse Events 
The occurrence of solicited adverse events during the three days of oral dosing, through 8 hours 
following the last dose, which are attributed to iOWH032 and placebo be summarized. 
Solicited adverse events collected during the dosing phase (three days of oral dosing through 8 
hours following the last dose) and considered to be related to study drug will be summarized by 
treatment group for causality and severity. For summary table by causality, subjects will be 
counted only once within each causality category, but can be counted in more than one causality 
category. For summary table by severity, subjects will only be counted once at the worst severity 
for an AE.  
All information pertaining to solicited AEs will be listed by subject and all the details collected 
on the eCRF, including solicited AE category, PT, SOC, onset date/time, end date/time, whether 
AE occurs during the dosing period, course, severity, seriousness, serious AE definition 
(congenital abnormality or birth defect, significant disability, death, hospitalization or 
prolongation of hospitalization, life threatening, other medically important event), autopsy 
performed and date of death if died, causality, study product administration, treatment required, 
and outcome. 
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Statistical Analysis Plan  
Final v1.1, 23Mar2020 
Confidential 
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4.3.1.11 
Unanticipated Problems (UPs) 
Unanticipated problems (UPs), following the definition the Office of Human Research 
Protections as described in Protocol Section 10.1.3, are collected for this study. A listing 
including event description, date/time of occurrence, corrective action, ongoing or not, and end 
date/time of occurrence will be provided. 
4.3.2 SAFETY LABORATORY TESTS 
Clinical laboratory values will be graded per Appendix A. 
The observed values and change from baseline of Complete blood count (CBC) for white blood 
cell (WBC), absolute neutrophil count (ANC), Hemoglobin, and Platelets; and Chemistry panel 
for Na, K, ALT, AST, ALP, Creatinine, Albumin, and Total Bilirubin, CPK, Glucose will be 
tabulated with descriptive statistics by visits if applicable.  
Abnormalities for each lab will be summarized by maximum grade overall and per time point. A 
corresponding summary will be produced only for those considered to be clinically relevant 
which corresponds to the abnormal clinically significant. 
The complete blood count (CBC) for WBC, ANC, Hemoglobin, and Platelets; and Chemistry 
panel for Na, K, ALT, AST, ALP, Creatinine, Albumin, and Total Bilirubin, CPK, Glucose will 
be listed by subject and visit, respectively. 
4.3.3 URINALYSIS 
The screening urinalysis for Glucose, Protein, Occult blood, RBC and specific gravity will be 
listed by group and subject. 
4.3.4 VITAL SIGNS 
Vital signs (blood pressure, pulse, and oral temperature) will be measured approximately every 8 
hours, unless more frequent monitoring is needed. Once a participant has passed a diarrheal stool 
(Grade 3 or higher), vital signs will be measured every 4 hours until the participant passes a 
Grade 1 or 2 stool or 24 hours have passed since the last Grade 3 – 5 stool, whichever comes 
first. 
Vital signs will also be measured every 4 hours when a participant has a fever ≥ 39°C (102.1°F), 
until the participant has 2 consecutive temperatures of ≤ 38°C (100.4°F). 
Any participant that complains of dizziness or lightheadedness upon standing will have 
orthostatic blood pressures assessed—BP after supine for ~5 minutes and BP after standing ~2-3 
minutes. Orthostatic hypotension is defined as a drop in systolic BP > 20 mmHg or in diastolic 
BP > 10 mmHg. 
Vital signs and their change from baseline will be tabulated using descriptive statistics by visits 
and time point. 
All vital signs including vital sign parameters and overall interpretations will be listed by subject. 
4.3.5 ECGS 
ECGs will be measured at screening. Subjects should rest in the supine position for at least 5 min 
before each 12-lead ECG recording. The results will include heart rate (HR), R-R interval (RR), 
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PR interval, QRS interval, QT interval, and QTcF interval. The corrected QT interval will be 
corrected for heart rate according to the following formula: 
Fridericia’s formula: QTcF = QT/RR0.33 
The listing will be provided for ECG including individual parameters, ECG interpretation and 
comments on findings if applicable. 
4.3.6 PHYSICAL EXAMINATION AND PHYSICAL MEASUREMENT 
Full physical examination will be done at Screening, Post-Challenge Observation Period (Day 1 
through discharge), visit2 and visit3. A symptom-directed physical examination may be 
performed at other times at the Investigator’s or Sub-Investigator's discretion. 
All subjects with any abnormal physical examination findings will be listed. 
Physical Measurement for weight will be done at Post-Challenge Observation Period (Day 1 
through discharge). All subjects with weight will be listed. 
4.4 
 EXPLORATORY ANALYSES 
4.4.1 PHARMACOKINETIC (PK) PROPERTIES OF IOWH032 
PK plasma samples for the levels of iOWH032 will be collected 7±1 hours after the first and last 
dosing in all participants. 
Concentration-time data will be summarized by treatment group using descriptive statistics (n, 
arithmetic mean, geometric mean, median, arithmetic standard deviation, minimum, maximum, 
and arithmetic coefficient of variation [%]) at each timepoint. At a particular timepoint, a 
concentration value reported as “<LLOQ” will be considered zero and a mean/median will not be 
calculated in the instance where >50% of the concentration values at a timepoint are reported as 
“<LLOQ”, then only the minimum and maximum will be reported. 
4.4.2 EVALUATE TIME OF CESSATION OF CHOLERA ORGANISM IN STOOL AFTER 
CHALLENGE 
Time (hours) to cessation of detectable cholera in stool is defined as the time of the first sample 
negative via quantitative stool culture, after which all following samples are also negative for 
cholera. 
Time to cessation of detectable cholera in stool (hours) will be analyzed using Kaplan-Meier 
methods. 
4.4.3 EVALUATE A DECREASE FOR ORAL REHYDRATION SOLUTION AND/OR INTRAVENOUS 
FLUID REPLACEMENT THERAPY NEED 
Statistical comparison of this exploratory endpoints will be conducted with the stratified (by 
blood type) version of the Wilcoxon rank-sum test, the Van Elteren test.  The hypothesis tests 
will be supplemented by a two-sided 95% confidence interval for the difference in median 
diarrheal stool output, using the percentile bootstrap method, with n=10,000 replicates. 
 
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Statistical Analysis Plan  
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Confidential 
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4.4.4 EXPLORATORY ANALYSES OF DIARRHEAL STOOL OUTPUT 
In addition to the primary and secondary analyses of diarrheal stool output rate, assessments of 
diarrheal stool output (volume) over 12- and 24-hour periods will aid interpretation of study 
results.  The following will be summarized by group and by blood type (O vs non-O) within 
group. 
x Diarrheal stool output in first 12 hours, after initiation of study product and prior to 
initiation of antibiotic 
x Diarrheal stool output in first 24 hours, after initiation of study product and prior to 
initiation of antibiotic 
x Diarrheal stool output during subject’s individual 24-hour window of peak output, after 
initiation of study product and prior to initiation of antibiotic 
x Diarrheal stool output during subject’s individual 24-hour window of peak output, after 
initiation of study product and including time after initiation of antibiotic 
x Diarrheal stool output in first 24 hours after initiation of antibiotic 
Statistical comparison of these exploratory endpoints will be conducted with the stratified (by 
blood type) version of the Wilcoxon rank-sum test, the Van Elteren test.  The hypothesis tests 
will be supplemented by a two-sided 95% confidence interval for the difference in median 
diarrheal stool output, using the percentile bootstrap method, with n=10,000 replicates. 
5 
INTERIM ANALYSIS 
Once efficacy data are available from the completion of Cohort 1, an interim analysis of the 
primary efficacy endpoint and accompanying safety data will be conducted to guide the decision 
to move forward with Cohort 2. The details of the interim analysis are described separately in an 
interim SAP.  
Additionally, topline results will be conducted after the efficacy data from Cohorts 1 and 2 have 
been collected (i.e. including data prior to Visit 2). These topline results will include analysis of 
the primary efficacy endpoint, as well as the analyses of the secondary endpoints of proportion of 
participants with MSD, attack rate of any diarrhea, AUC of stool volume, duration of episode 
(time to first formed stool), and total number of loose stools conducted on the mITT population. 
In addition, subject disposition and demographic and baseline characteristics tables will be 
provided. The topline results will be unblinded only to the treatment-level and not to the 
individual subject. To avoid identification of individual subjects, some summary statistics such 
as minimum and maximum will not be presented in the summary tables.  
 
The final statistical analyses will be performed after database lock and final unblinding of the 
data.  
6 
SUMMARY OF MAJOR CHANGES IN THE PLANNED ANALYSES  
The following updates from Protocol v4.0 were made in this SAP: 
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x TEAE definition has been revised to AEs which start or worsen following the start of 
study medication and up until the follow-up visit.  
x The Clinical Safety Laboratory Toxicity Scales defined in the Appendix D of the protocol 
have been revised as shown in Appendix A of this SAP. 
7 
PROGRAMMING SPECIFICATIONS 
Programming specifications will be provided in the SAP table, listing, and figures shells 
document. 
8 
TABLES, LISTINGS, AND FIGURES SHELLS 
The Tables, Listing, and Figures Shells will be provided as a separate document. 
 
 
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Statistical Analysis Plan  
Final v1.1, 23Mar2020 
Confidential 
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APPENDIX A: CLINICAL SAFETY LABORATORY TOXICITY SCALES 
 
Test 
Mild  
Grade 1 
Moderate  
Grade 2 
Severe  
Grade 3 
Potentially Life  
Threatening  
Grade 4 
WBC, increase 
1.1 – 1.5 xULN 
1.6 – 2.0 xULN 
2.1 – 2.5 xULN 
≥ 2.6 xULN 
WBC, decrease* 
LLN/1.5 – 1.1 
LLN/2.0 – 1.6 
LLN/2.5 – 2.1 
≤ LLN/2.6 
ANC decrease 
LLN/1.5 – 1.1 
LLN/2.0 – 1.6 
LLN/2.5 – 2.1 
≤ LLN/2.6 
Hemoglobin decrease 
  LLN/1.5 – 1.1 
LLN/2.0 – 1.6 
LLN/3.0 – 2.1 
< LLN/3 
Platelet count 
LLN/1.5 – 1.3 
LLN/2.0 –1.6 
LLN/3.0 – 2.1 
< LLN/3 
Glucose 
Nonfasting, High 
116-160 
161-250 
251-500 
> 500 
Glucose, Low 
55-64 
40-54 
30-39 
< 30 
Sodium, low 
132 -134 
130 - 132 
125 - 129 
< 125 
Sodium, high 
146 – 148 
149 - 150 
151 - 152 
> 152 
Potassium, high 
5.6 – 5.8 
5.9 – 6.1 
6.2 – 6.5 
> 6.5 
Potassium, low 
3.3 – 3.4 
3.1 – 3.2 
2.9 – 3.0 
<2.9 
Creatinine* 
1.1 – 1.5 xULN 
1.6 – 2.0 xULN 
2.1 – 3.0 xULN 
> 3.0 xULN 
AST (SGOT) 
1.6 -.2.5 xULN 
2.6 – 4.0 xULN 
4.1 – 10.0 xULN 
>10 xULN 
ALT (SGPT) 
1.6 -.2.5 xULN 
2.6 – 4.0 xULN 
4.1 – 10.0 xULN 
>10 xULN 
Alkaline Phosphatase 
1.6 -.2.0 xULN 
2.1 – 3.0 xULN 
3.1 – 10.0 xULN 
>10 xULN 
Total Bilirubin 
1.2 -.1.5 xULN 
1.6 – 2.0 xULN 
2.0 - 4.0 xULN 
>4 xULN 
 
ULN = upper limit of normal range  
LLN = lower limit of normal range 
*if the baseline is outside of the reference range (e.g., African-
American acceptable value), baseline value will replace LLN for 
WBC decrease, and replace ULN for Creatinine, to evaluate 
toxicity 
 
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