Conditions: Clostridium Difficile Infection (CDI)
Keywords: C Difficile Colitis Clostridium Difficile CDI C.Difficile Diarrhea Fecal Transplant Fecal Microbiota Transplant (FMT) Microbiota Restoration Therapy Diarrhea FMT Microbial Suspension
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 320
Healthy volunteers: no

Brief summary:
This is a prospective, multicenter, randomized, double-blinded, placebo-controlled Phase 3 study of a microbiota suspension of intestinal microbes. Patients who have had at least one recurrence after a primary episode and have completed at least one round of standard-of-care oral antibiotic therapy or have had at least two episodes of severe Clostridioides difficile infection (CDI) resulting in hospitalization within the last year may be eligible for the study. Subjects who are deemed failures following the blinded treatment per the pre-specified treatment failure definition may elect to receive an unblinded dose of RBX2660.

Detailed description:
This is a prospective, multicenter, randomized, double-blinded, placebo-controlled Phase 3 study of a microbiota suspension of intestinal microbes. The primary assessments for this study are (i) efficacy of RBX2660 as compared to a Placebo in preventing recurrent episodes of CDI and (ii) safety via assessment of adverse events. The primary efficacy analysis of the study will be a Bayesian hierarchical model, which formally incorporates data from a previous randomized Phase 2b study (Protocol 2014-01, NCT02299570) of RBX2660.

Follow-up office visits occur at weeks 1-, 4- and 8 after completing the blinded study treatment. Telephone assessments for adverse events occur during weeks 2, 3 and 6 after the study treatment and at months 3 and 6. Patients who have had at least one recurrence after a primary episode and have completed at least one round of standard-of-care oral antibiotic therapy or have had at least two episodes of severe CDI resulting in hospitalization within the last year may be eligible for the study. Study Subjects who are deemed failures following the blinded treatment per the pre-specified treatment failure definition may elect to receive an unblinded dose of RBX2660.

Arms:
arm 1: Placebo is an suspension of normal saline. Packaging and labeling are identical to the packaging and labeling for RBX2660 to support the study blinding arm 2: RBX2660 is a rectally administered microbiota suspension in a 0.9% sodium chloride irrigation United States Pharmacopeia (USP) solution and cryoprotectant

Interventions: intervention 1: RBX2660 intervention 2: Placebo intervention 3: Open label RBX2660 (only for confirmed CDI recurrence)
Intervention descriptions: intervention 1: RBX2660 is a rectally administered microbiota suspension intervention 2: Placebo is normal saline solution administered rectally intervention 3: RBX2660 administered as a second treatment after confirmed CDI recurrence following the initial dose of placebo or RBX2660

Protocol document:
Prot_000.pdf: 
Rebiotix Inc.  
Phase 3 Study Protocol 2017-01 
RBX2660 Microbiota Suspension 
Version: 7.0 
06 August 2019 
CONFIDENTIAL 
Page 1 of 77 
A Phase 3 Prospective, Randomized, Double-blinded, Placebo-controlled Clinical Study to 
Evaluate the Efficacy and Safety of Rebiotix RBX2660 (microbiota suspension) for the 
Prevention of Recurrent Clostridium difficile Infection 
NCT03244644
Sponsor: 
Rebiotix Inc. 
2660 Patton Road 
Roseville, MN 55113 USA 
P:  1-651-705-8770 
Study Product: 
RBX2660 (microbiota suspension) 
Protocol #:  
2017-01 
Version #: 
7.0;  06 August 2019 
This document contains confidential information belonging to Rebiotix Inc. the study sponsor.  
Except as otherwise agreed to in writing by the sponsor, by accepting or reviewing this 
document, you agree to hold this information in confidence and not copy or disclose it to others 
or use it for unauthorized purposes.  In the event of any actual or suspected breach of this 
obligation, Rebiotix must be promptly notified. 


Rebiotix Inc.  
 
Phase 3 Study Protocol 2017-01 
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Version: 7.0 
 
 
 
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CONFIDENTIAL  
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Table of Contents 
Revision History ............................................................................................................................. 2 
Investigator’s Acknowledgement of the Clinical Study Protocol................................................... 3 
List of Abbreviations and Acronyms .............................................................................................. 8 
Study Synopsis ................................................................................................................................ 9 
1. 
Introduction ........................................................................................................................... 19 
1.1. 
Background .................................................................................................................... 19 
1.2. 
Investigational Agent ..................................................................................................... 20 
1.3. 
Placebo ........................................................................................................................... 20 
1.4. 
Preclinical Data .............................................................................................................. 20 
1.5. 
Clinical Data to Date ...................................................................................................... 21 
1.5.1. Phase 2 Study Protocol 2013-001 Final Data ............................................................. 21 
1.5.2. Phase 2B Study Protocol 2014-01 Interim Study Data ............................................... 24 
1.5.3. Phase 2B Study Protocol 2015-01 Interim Study Data ............................................... 26 
1.6. 
Treatment Rationale and Risk/Benefit ........................................................................... 28 
2. 
Study Objectives and Endpoints ........................................................................................... 29 
2.1. 
Primary Objective .......................................................................................................... 29 
2.2. 
Secondary Objective ...................................................................................................... 29 
2.3. 
Other Objectives ............................................................................................................ 29 
2.4. 
Primary Efficacy Endpoint ............................................................................................ 29 
2.5. 
Secondary Efficacy Endpoint ........................................................................................ 29 
2.6. 
Safety Endpoints ............................................................................................................ 30 
2.7. 
Other Efficacy Endpoints ............................................................................................... 30 
3. 
Study Design ......................................................................................................................... 30 
3.1. 
Study Purpose ................................................................................................................ 30 
3.2. 
General Study Design .................................................................................................... 31 
3.3. 
Definition of Recurrent CDI for Study Entry ................................................................ 33 
3.4. 
Definition of CDI Diarrhea for Use in Enrolled Subjects Throughout the Study ......... 34 
3.5. 
Definition of Treatment Success .................................................................................... 34 
3.6. 
Definition of Sustained Clinical Response .................................................................... 34 
3.7. 
Definition of Treatment Failure ..................................................................................... 34 
3.8. 
Management of Treatment Failure................................................................................. 35 
3.9. 
Determination of Treatment Success/Failure................................................................. 36 
3.10. Endpoint Adjudication Committee ................................................................................ 36 
3.11. Data and Safety Monitoring Board ................................................................................ 36 


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3.12. Medical Monitor ............................................................................................................ 36 
3.13. Blinding.......................................................................................................................... 37 
4. 
Subject Selection and Withdrawal ........................................................................................ 38 
4.1. 
Inclusion Criteria ........................................................................................................... 38 
4.2. 
Exclusion Criteria .......................................................................................................... 38 
4.3. 
Subject Recruitment and Screening ............................................................................... 40 
5. 
Investigational Product ......................................................................................................... 40 
5.1. 
RBX2660 Description .................................................................................................... 40 
5.2. 
Placebo Description ....................................................................................................... 40 
5.3. 
Rebiotix Kit Description ................................................................................................ 41 
5.4. 
Treatment Regimen ........................................................................................................ 41 
5.5. 
Subject Follow-up Requirements ................................................................................... 42 
5.6. 
Concomitant Therapy..................................................................................................... 42 
5.7. 
Preparation of Investigational Product ........................................................................... 42 
5.8. 
Packaging ....................................................................................................................... 42 
5.9. 
Ordering, Receipt and Storage, Dispensing, Returning, and Tracking Investigational 
Product ...................................................................................................................................... 42 
5.9.1. Ordering Investigational Product ................................................................................ 42 
5.9.2. Receipt and Storage of Investigational Product .......................................................... 42 
5.9.3. Dispensing and Administering Investigational Product ............................................. 43 
5.9.4. Returning IP ................................................................................................................ 43 
5.9.5. Product Accountability and Tracking ......................................................................... 43 
6. 
Study Procedures .................................................................................................................. 44 
6.1. 
General Information ....................................................................................................... 44 
6.2. 
Informed Consent........................................................................................................... 46 
6.3. 
Subject Diary ................................................................................................................. 46 
6.4. 
Optional Stool Samples for Additional Testing by Rebiotix ......................................... 46 
6.5. 
Screening Visit ............................................................................................................... 47 
6.6. 
Randomization ............................................................................................................... 48 
6.7. 
Baseline Visit and Enema Administration ..................................................................... 49 
6.8. 
In Office Visit (1-Week) ................................................................................................ 51 
6.9. 
In Office Visit (4-Week) ................................................................................................ 51 
6.10. In Office Visit (8-Week) ................................................................................................ 51 
6.11. Telephone Assessments (Weeks 2, 3 and 6) .................................................................. 52 
6.12. Telephone Assessments (3- and 6- Months) .................................................................. 52 


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6.13. Unscheduled Possible Recurrence Visit ........................................................................ 53 
6.14. Unblinded RBX2660 ..................................................................................................... 55 
6.15. Study Exit....................................................................................................................... 56 
6.16. Subject Withdrawal or Termination ............................................................................... 57 
7. 
Assessment of Safety ............................................................................................................ 57 
7.1. 
Adverse Event Management .......................................................................................... 57 
7.2. 
Definitions...................................................................................................................... 58 
7.3. 
Anticipated Adverse Events ........................................................................................... 59 
7.4. 
Solicited Events ............................................................................................................. 60 
7.5. 
Preexisting Condition..................................................................................................... 60 
7.6. 
Serious Adverse Event Reporting – Procedures for Investigators ................................. 61 
7.6.1. Initial Reports.............................................................................................................. 61 
7.6.2. Follow-Up Reports...................................................................................................... 61 
7.7. 
Grading Event Severity .................................................................................................. 62 
7.8. 
Causality ........................................................................................................................ 65 
7.9. 
Pregnancy Reporting ...................................................................................................... 66 
7.10. Medical Monitor ............................................................................................................ 66 
7.11. Data and Safety Monitoring Board (DSMB) ................................................................. 67 
7.12. Study Stopping Rules for Safety .................................................................................... 67 
7.13. Sponsor Reporting of Adverse Events ........................................................................... 68 
7.13.1. 
Timeline for Reporting Requirements................................................................. 68 
7.13.2. 
Additional Reporting Requirements.................................................................... 69 
7.13.3. 
Reporting AEs to FDA and Health Canada ........................................................ 69 
8. 
Sample Size and Statistical Considerations .......................................................................... 69 
8.1. 
Assessment of Efficacy .................................................................................................. 69 
9. 
Study Administration ............................................................................................................ 71 
9.1. 
Institutional Review Board / Research Ethics Board Approval..................................... 71 
9.2. 
Form 1572 and Financial Disclosure ............................................................................. 71 
9.2.1. Canadian Sites:  Qualified Investigator Undertaking Form ........................................ 72 
9.3. 
Subject Confidentiality .................................................................................................. 72 
9.4. 
Study Oversight ............................................................................................................. 72 
9.5. 
Study Site Qualification ................................................................................................. 73 
9.6. 
Investigator / Site Training ............................................................................................ 73 
9.7. 
Data Management .......................................................................................................... 73 
9.8. 
Monitoring ..................................................................................................................... 74 


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9.9. 
Direct Access to Source Data/Documents ..................................................................... 74 
9.10. Investigator Responsibilities .......................................................................................... 74 
9.11. Investigator Records ...................................................................................................... 75 
9.12. Investigator Reports ....................................................................................................... 75 
9.13. Study Site Termination .................................................................................................. 76 
9.14. Protocol Amendments .................................................................................................... 76 
9.15. Protocol Deviations ........................................................................................................ 76 
9.16. Inspection/Auditing........................................................................................................ 76 
10. 
Publication Plan ................................................................................................................. 77 
11. 
References .......................................................................................................................... 77 
 
 
 


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List of Abbreviations and Acronyms 
AE – adverse event 
AIDS – Acquired Immunodeficiency Syndrome 
ALT – alanine aminotransferase 
AST – aspartate transaminase 
BUN – blood urea nitrogen 
CBC – complete blood count 
CDAD – Clostridium difficile-associated diarrhea 
CDI – Clostridium difficile infection 
CFR – Code of Federal Regulations 
CMP – comprehensive metabolic panel 
CRP – C-reactive protein 
DSMB – Data and Safety Monitoring Board 
EAC – Endpoint Adjudication Committee 
eCRF – electronic case report form 
EIA – enzyme immunoassay 
EVA – ethylene vinyl acetate 
FDA – Food and Drug Administration 
FT – fecal transplant 
GCPs – Good Clinical Practices 
GDH – glutamate dehydrogenase  
HIV – human immunodeficiency virus 
IBD – inflammatory bowel disease 
IBS – irritable bowel syndrome 
ICF – informed consent form 
ICH – International Council on Harmonisation 
ICU – intensive care unit 
IgG – Immunoglobulin  
IP – Investigational product 
IRB – Investigational Review Board 
ISF – Investigator Site File 
ITT – Intent-to-Treat 
IV – intravenous  
IVIG – intravenous immunoglobulin 
MDRO – multi-drug resistant organism  
mL – milliliter 
NA – not applicable 
PAL – Product Accountability Log 
PCR – Polymerase chain reaction 
PP – Per Protocol 
REB – Research Ethics Board 
SAE – serious adverse event 
SAP – Statistical Analysis Plan 
SAR – suspected adverse reaction 
VRE – vancomycin-resistant enterococci 


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Phase 3 Study Protocol 2017-01 
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Version: 7.0 
 
 
 
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 Study Synopsis 
 
Title 
A Phase 3 Prospective, Randomized, Double-blinded, Placebo-controlled 
Clinical Study to Evaluate the Efficacy and Safety of Rebiotix RBX2660 
(microbiota suspension) for the Prevention of Recurrent Clostridium 
difficile Infection.  
Investigational 
Product and 
Indication for Use 
RBX2660 (microbiota suspension).  RBX2660 is being studied for the 
prevention of recurrent Clostridium difficile infection (CDI) in 
individuals with prior recurrent Clostridium difficile infection resolved 
following antibiotic treatment. 
Control Product 
Placebo (enema) of normal saline.   
Study Purpose 
The purpose of this study is to confirm the efficacy and safety of 
RBX2660 for the prevention of recurrent CDI in subjects who have had 
prior recurrent Clostridium difficile infection that was resolved with 
antibiotic treatment. 
Definition of 
Recurrent CDI for 
Study Entry 
The definition of recurrent CDI for study entry is a documented diagnosis 
of: 
1. CDI diarrhea; the passage of three or more unformed/loose stools 
in 24 or fewer consecutive hours for at least two consecutive 
days, that began within 8 weeks after completion of previous 
CDI treatment; 
2. and at least one positive stool test for the presence of toxigenic 
C. difficile, which must be within 30 days prior to or on the date 
of enrollment (informed consent).   
 
To be considered for enrollment, a subject must have medical record 
documentation of recurrent CDI per the above study definition that 
includes either:  
a) at least one recurrence after a primary episode and has completed 
at least one round of standard-of-care oral antibiotic therapy or;  
b) had at least two episodes of severe CDI resulting in 
hospitalization within the last year. 
Definition of CDI 
Diarrhea 
The definition of CDI diarrhea for use in enrolled subjects throughout the 
study includes: 
1. The passage of three or more unformed/loose stools (i.e. Bristol 
Stool Scale type 6-7) in 24 or fewer consecutive hours for at least 
two consecutive days; 
2. and a positive stool test for the presence of C. difficile toxin; 
documented at the time of the diarrhea. 


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Version: 7.0 
 
 
 
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CONFIDENTIAL  
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Objectives 
Primary Objective: To confirm the efficacy of RBX2660 as compared to 
a Placebo in preventing recurrent episodes of CDI through 8 weeks. 
Secondary Objective: To evaluate the sustained clinical response rate of 
RBX2660 as compared to Placebo after blinded treatment. 
Endpoints 
Primary Efficacy Endpoint:  Recurrence of CDI within 8 weeks of 
blinded treatment. 
Secondary Efficacy Endpoint:   Loss of sustained clinical response 
through 6 months after blinded treatment. 
Safety Endpoints: 
1. Number of adverse events per subject 
2. Timing of attributable adverse event post-treatment exposure 
(treatment emergent adverse event TEAE) 
3. Duration of TEAE 
4. Relatedness of TEAE 
5. Severity of TEAE 
6. Causality of TEAE to IP, enema, C difficile or prior condition  
7. Number of each of the following through 8 weeks post blinded 
treatment: death, septic shock, toxic megacolon, colonic 
perforation, emergency colectomy, and ICU admission.  
8. Onset of new chronic conditions relative to blinded treatment 
administration  
Study Design 
This is a prospective, multicenter, randomized, double-blinded, placebo-
controlled Phase 3 study to confirm the efficacy and safety of RBX2660 
for the prevention of recurrent CDI.   
Up to 270 subjects may be randomized and treated in this study. 
Randomization will be at a 2:1 ratio (RBX2660:Placebo); approximately 
180 subjects will be randomized to RBX2660 treatment and 90 subjects 
randomized to Placebo.  Consented subjects who meet all eligibility 
criteria (inclusion and exclusion) will be randomized.  Consented 
subjects who do not meet the eligibility criteria will be considered screen 
failures and will not count towards the total randomized.  Subjects who 
are randomized but exit prior to the administration of the first blinded 
enema may be replaced with additional subjects which could cause 
overall number of subjects randomized to be greater than the 270 
anticipated, however no more than 270 subjects will be treated with a 
blinded enema.  Any replacement subjects will be randomized to ensure 
maintenance of proper blinding.  
Potential subjects are expected to already be taking or have just been 
prescribed antibiotics to control recurrent CDI symptoms at the time of 
enrollment per the investigator’s standard of care.  This means patients 
who have already completed their prescribed course of antibiotics to treat 


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recurrent CDI, are not eligible for enrollment unless they have another 
recurrence that is treated with antibiotics.   
Once enrolled (informed consent obtained), in order to be randomized 
and treated:  
1. Subject must meet all the inclusion and none of the exclusion 
criteria; 
2. Antibiotics will have been administered for a minimum of 10 
consecutive days prior to the washout period; 
3. CDI symptoms must be under control leading into the antibiotic 
washout period.  
Control of CDI symptoms is defined as no longer meeting the 
symptomatic criteria for CDI diarrhea which is the passage of three or 
more unformed/loose (i.e. Bristol Stool Scale type 6-7) stools in 24 or 
fewer consecutive hours for at least two consecutive days while taking 
antibiotics.  Thus, at minimum the Subject Diary (completed from time 
of enrollment until day of treatment) must show that symptoms are under 
control the two days prior to the washout period in order for the subject 
to be treated.   
Once screening is completed and all inclusion and exclusion verified, the 
subject can be randomized.  The assigned study treatment will be 
scheduled to consist of a single, blinded study enema according to the 
randomization assignment.  Study treatment is to be completed as soon as 
possible, but no more than 14 calendar days following randomization.   
Ultimately, the study treatment needs to be administered within 21 days 
of the Screening visit. A minimum of 24hr to maximum of 72hr 
antibiotic washout period is required prior to administration of the 
assigned study treatment.    
In-office study follow-up visits occur at weeks 1, 4 and 8 after 
completing the blinded study treatment.  Telephone assessments for 
adverse events occur during weeks 2, 3 and 6 after the study enema and 
at months 3 and 6.  Subjects are required to keep a detailed diary to 
assess for solicited events from the date of enrollment (informed consent) 
to the 1-week follow-up visit.  The diary will be collected and reviewed 
at the baseline visit prior to blinded enema administration. The subject 
will continue to complete the diary following enema administration and it 
will be collected and reviewed at the 1-week follow-up visit.  
Subjects who are deemed failures following the blinded treatment per the 
pre-specified treatment failure definition may elect to receive an 
unblinded RBX2660 enema.  This unblinded enema is to be administered 
within 21 calendar days of failure determination.  If a subject receives an 


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unblinded RBX2660 enema, the follow-up requirements will restart from 
the day of the unblinded RBX2660 enema received according to the same 
schedule as required for the blinded portion of the study.   
A primary efficacy and safety analysis will be conducted when all the 
randomized subjects have completed their 8-week follow-up assessment 
after receiving the blinded treatment. One or more interim analyses will 
be conducted to evaluate the primary efficacy endpoint for possible early 
study stopping for success or futility.  The first interim analysis will 
occur after a minimum of 160 subjects have been treated and evaluated 
for efficacy.  A second interim analysis could occur when 220 subjects 
have been treated and evaluated for efficacy if success is not achieved at 
the first interim analysis.  The details of the  planned interim analyses are 
included in the statistical analysis plan. 
A final analysis and report will be completed once the last subject has 
completed the 6-month follow-up visit for their study treatment. This 
includes 6-month follow-up after an unblinded RBX2660 study 
treatment. At that time the study will be considered complete.  
Randomized Study 
Dosing 
Placebo Arm – Group A 
• Placebo dosing consists of one enema of Placebo given in a 
blinded manner.   
RBX2660 Arm – Group B 
• Active treatment consists of one enema of RBX2660 given in a 
blinded manner.   
Definition of 
Treatment Success 
Treatment success is defined as:  
• The absence of CDI diarrhea for 8 weeks after completing a study 
treatment.   
Definition of 
Sustained Clinical 
Response 
Sustained Clinical Response is defined as: 
• Treatment success of the presenting CDI recurrence and no new 
CDI episodes for greater than 8 weeks through 6 months after 
completing a study treatment.        
Definition of 
Treatment Failure 
(CDI recurrence) 
Treatment failure (CDI recurrence) is defined as:  
• The presence of CDI diarrhea within 8 weeks of administration of 
a study enema, which includes a positive stool test for C. difficile 
toxin at the time of the diarrhea using the study required C. 
difficile testing algorithm.  
If a recurrence is suspected, an in-office visit is required and the subject 
must provide a fresh stool sample.  The stool sample is to be sent to the 
central laboratory for analysis. In the case of suspected treatment failure 
where there is an immediate concern for subject safety, including the 


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potential for serious adverse events, an additional C. DIFF QUIK CHEK 
COMPLETE® Test will be permitted to be conducted at the site or other 
laboratory known to the site in addition to sending a sample to the 
central laboratory for testing.   
Management of 
Treatment Failures 
Following blinded treatment, subjects meeting the protocol definition of 
treatment failure may be scheduled for administration of an unblinded 
RBX2660 enema within 21 calendar days of failure determination.  In 
order to receive the unblinded RBX2660 enema, the site must provide 
documentation of meeting all treatment failure criteria as defined by the 
protocol.   
If antibiotics are given to control symptoms, a 24-72hr washout period 
prior to administration of an unblinded RBX2660 enema is required. If 
elected, the unblinded enema is to be administered within 21 calendar 
days of failure determination.  The use of antibiotics prior to an 
unblinded RBX2660 enema is at the discretion of the investigator.  
If a subject receives an unblinded RBX2660 enema, the follow-up 
requirements will restart from the day of administration of the unblinded 
RBX2660 enema according to the same schedule as required for the 
blinded portion of the study.  This results in completion of a new Subject 
Diary for up to 7 days after treatment; in-office visits at 1-, 4- and 8-
weeks as well as telephone calls at 2, 3 and 6 weeks and at 3-and 6 
months after the unblinded RBX2660 treatment.   
Treatment failures who do not receive an unblinded RBX2660 enema 
will continue to follow their original schedule of assessments for the 
duration of the study based on the blinded study enema.  
Concomitant 
Therapy 
The subject must agree not to take any oral vancomycin, metronidazole, 
fidaxomicin, rifaximin, nitazoxanide, and IVIG through the 8-week 
follow-up assessment unless newly prescribed by a treating investigator 
during the course of the study as a result of recurrent CDI diagnosis.   
Determination of 
Success/Failure: 
(Investigator and 
EAC) 
The site investigator makes the initial determination of success or failure 
based on the pre-defined study definitions.  The site investigator’s 
assessment will then be provided to the Endpoint Adjudication 
Committee (EAC) for independent, blinded adjudication of treatment 
success or failure that will be utilized for study analysis and reporting 
purposes.   


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Medical Monitor  
The Medical Monitor provides blinded review of serious adverse events 
to assess accuracy of the reporting as related to seriousness and causality, 
and periodic review of adverse events for trends.  The Medical Monitor’s 
review will be provided to the Data and Safety Monitoring Board 
(DSMB) for analysis purposes and as applicable for the proper 
adjudication of potential study stopping rules.  
Data and Safety 
Monitoring Board 
(DSMB) 
The DSMB will review safety data for trends and may be unblinded to 
adjudicate certain adverse events and for review of both the primary 
efficacy and safety analysis and the final analysis. Details regarding 
DSMB responsibilities related to analysis oversight and data reporting 
will be provided in the DSMB Charter. 
Blinding 
Subjects, site personnel, the DSMB, EAC and the Medical Monitor are 
blinded to the randomization assignment.  Blinding will be maintained 
through the data cutoff for the primary efficacy analysis after which the 
blind may be lifted for the DSMB.  The DSMB may be unblinded to 
individual assignments earlier as needed to adjudicate certain adverse 
events.  Individual randomization assignments will not be revealed to the 
subjects, site personnel, EAC or the Medical Monitor until the study is 
completed.   
Subjects will be randomized to receive either RBX2660 or placebo 
through an Interactive Response Technology (IRT) System prior to the 
blinded study treatment.  Randomization assignment is provided to the 
Rebiotix Operations Department personnel for product preparation and 
shipment.  The Rebiotix Kit is shipped to the site in a sealed carton that is 
only opened by the site IP administrator at the time of administration, out 
of view of the subject and other site personnel.  While the site IP 
administrator will not have access to the actual randomization 
assignment, it may be possible to guess through visualization at the time 
of enema preparation and administration.  Additionally, subjects will be 
instructed to disregard any product characteristics that may become 
evident during or after administration such as the presence or absence of 
color or odor.  To limit study bias, the IP administrator is not allowed to 
perform any of the subject study assessments from completion of the 
enema administration visit through the subject’s final study visit. 
Sample Size  
To demonstrate a 69% success with one dose of the RBX2660 treatment 
group vs. 47% success in the Placebo control group, 240 subjects are 
required (power 90%).  Up to an additional 30 subjects will be enrolled 
to allow for a 10% lost-to-follow up rate, for a total of up to 270 subjects 
randomized and treated with a blinded enema.  Considering the 2:1 


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randomization, this results in approximately 180 subjects in the 
RBX2660 arm and 90 subjects in the Placebo arm.   
Potential subjects who have signed consent but do not meet all inclusion 
and/or exclusion criteria will be considered screen failures.  Randomized 
subjects who withdraw for any reason prior to administration of the 
blinded enema will be replaced without counting toward the sample size 
cap which may result in more than 270 subjects being randomized, 
however no more than 270 subjects will be treated with a blinded enema.  
Replacement subjects will be randomized in the same manner as other 
subjects.   
Statistical 
Considerations 
 
The primary efficacy analysis will be performed on all pre-defined 
analysis populations comparing the RBX2660 arm to the Placebo arm, 
Intent-to-Treat (ITT), modified Intent-to-Treat (mITT) and Per-Protocol 
(PP).  However, the determination of meeting the pre-defined primary 
efficacy endpoint will utilize the mITT population results.  The primary 
endpoint analysis will be based on Pearson’s chi-square test with a 0.05 
alpha level for this comparison.  Secondary efficacy endpoints will also 
be completed on all analysis populations.  Finally, all safety analyses will 
be performed on the Safety population (SP) which includes all subjects in 
whom a blinded enema was at least opened and attempted.   
A primary efficacy and safety analysis will be conducted when all 
subjects have completed their 8-week follow-up assessment after 
receiving the blinded treatment.  Safety analysis will be completed for 
safety endpoints defined at or prior to 8 weeks following blinded 
treatment along with any longer term safety data available at the time of 
analysis.  One or more interim analyses will be conducted to evaluate the 
primary efficacy endpoint for possible early study stopping for success or 
futility.  The first interim analysis will occur after a minimum of 160 
subjects have been treated and evaluated for efficacy.  A second interim 
analysis could occur when 220 subjects have been treated and evaluated 
for efficacy if success is not achieved at the first interim analysis.  The 
details of the planned interim analyses are included in the statistical 
analysis plan. 
Final long-term safety results as well as unblinded RBX2660 efficacy 
data will be analyzed and reported in a final report after the last subject 
has completed the 6-month follow-up visit for their study treatment.  This 
includes 6-month follow-up after an unblinded RBX2660 study 
treatment.  At that time the study will be considered complete.   


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Study Stopping 
Rules 
The DSMB will determine whether enrollment will be paused, the study 
terminated, or other action taken based on their assessment that:  
1. There is probable cause that the IP or enema procedure (e.g., due to 
transfer from an RBX2660 donor) contributed to a new pathogenic 
intestinal infection in the stool of any subject, or 
2. Any series of events of major significance such as death or other 
serious outcome for which a causal connection with the IP is 
plausible represents an excess of the important adverse event(s) in 
one of the study arms.  
In the event that one of the preceding rules appears to be met based on 
site reported data and/or medical monitor review, the Rebiotix 
Operations Department will review the donor history and batch 
processing/release records for the product unit(s) administered in these 
cases.  A report of the review will be forwarded to the DSMB Chair for 
blinded review.  If probable cause is suspected, the DSMB Chair will 
convene the entire DSMB for event review.   
Upon the DSMB’s determination of action, the Chair will notify the 
Rebiotix Clinical Department, who will notify the study sites.  If 
enrollment is stopped, Rebiotix and the study sites will assess for the 
occurrence of similar events, and evaluate IP and study records for 
possible root cause(s).  Enrollment may be re-started upon further review 
and approval from the DSMB.  As appropriate, the DSMB may 
recommend study termination or measures short of termination with the 
objective of reducing the risk of adverse events.  Details regarding 
DSMB responsibilities will be provided in the DSMB Charter. 
Key Inclusion 
Criteria 
1. ≥ 18 years old.  
2. Medical record documentation of recurrent CDI per the study 
definition, that includes either: a) at least one recurrence after a 
primary episode and has completed at least one round of standard-of-
care oral antibiotic therapy or b) has had at least two episodes of 
severe CDI resulting in hospitalization within the last year.  
3. A positive stool test for the presence of toxigenic C. difficile within 
30 days prior to or on the date of enrollment. 
4. Is currently taking or was just prescribed antibiotics to control CDI 
related diarrhea at the time of enrollment.   
Note: Subject’s CDI diarrhea must be controlled (<3 unformed/loose 
stools/day, i.e. Bristol Stool Scale type 6-7, for two consecutive days) 
while taking antibiotics during screening  
5. Willing and able to have an enema(s). 
6. Willing and able to complete the stool and serum testing required for 
the study. 
7. Agrees not to take non-dietary probiotics through 8 weeks after 
receiving the last study enema (including OTC and prescription). 


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8. Agrees not to take any oral vancomycin, metronidazole, fidaxomicin, 
rifaximin, nitazoxanide and IVIG through the 8-week follow-up 
assessment unless newly prescribed by a treating investigator during 
the course of the study as a result of recurrent CDI diagnosis.   
9. Agrees to practice a form of effective contraception during study 
participation, does not apply to persons with documented non-child 
bearing potential. 
10. Has a negative urine pregnancy test at the time of enrollment and on 
the day of each enema prior to administration (persons of child-
bearing potential only). 
11. Willing and able to provide informed consent and local privacy 
authorization as applicable. 
12. Willing and able to complete the required Subject Diary. 
13. Willing and able to meet all study requirements, including attending 
all assessment visits and telephone calls. 
Key Exclusion 
Criteria 
1. A known history of refractory CDI. 
2. Currently has continued CDI diarrhea despite being on antibiotics 
prescribed for CDI treatment. 
3. Requires antibiotic therapy for a condition other than CDI. 
4. Previous fecal transplant, RBX2660 treatment, receipt of CDI 
vaccine or treatment with CDI monoclonal antibodies prior to study 
enrollment. 
5. History of inflammatory bowel disease (IBD), e.g., ulcerative colitis, 
Crohn’s disease, or microscopic colitis. 
6. Diagnosis of irritable bowel syndrome (IBS) as determined by Rome 
III criteria. 
7. History of chronic diarrhea. 
8. History of celiac disease. 
9. Disease symptoms (diarrhea) caused by a confirmed intestinal 
pathogen other than C. difficile. 
10. Have a current colostomy. 
11. Intraabdominal surgery within the last 60 days. 
12. Evidence of active, severe colitis. 
13. History of short gut syndrome or motility disorders. 
14. Requires the regular use of medications to manage bowel 
hypermotility. 
15. Planned therapy in the next 3 months that may cause diarrhea (e.g., 
chemotherapy). 
16. Planned surgery requiring perioperative antibiotics within 6 months 
of study enrollment. 
17. Life expectancy of < 6 months.  
18. Compromised immune system (e.g., HIV infection with CD4 count 
<200/mm3; inherited/primary immune disorders; immunodeficient or 
immunosuppressed due to a medical condition or medication). 
Note: Eligible HIV patients who have a CD4 count >200/mm3 who 
are on stable, highly active anti-retroviral therapy may be considered 
for enrollment.  


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19. Taking systemic steroids > 20 mg a day or prednisone-equivalent, or 
is expected to be on steroids (> 20 mg a day or equivalent) after 
enrollment through 8 weeks after completing the assigned study 
treatment. 
Note: Eligible patients taking a steroid dose equivalent to prednisone 
20 mg/day for >2 weeks, antimetabolites (e.g., azathioprine, 6-
mercaptopurine, or low-dose methotrexate for autoimmune disease), 
calcineurin inhibitors (e.g., tacrolimus and cyclosporine), or 
mycophenolate mofetil may be enrolled only after consultation with 
the Medical Monitor, and only if the doses have been stable (except 
for drug therapeutic monitoring adjustments for calcineurin 
inhibitors) for 90 days and have not been associated with diarrhea 
prior to the current episode of CDI. 
20. An absolute neutrophil count of <1000 cells/µL during screening. 
21. Known or suspected current (< 90 days) illicit drug use.  Note: 
marijuana use is allowed. 
22. Participant is unable to discontinue opioids (unless on a stable dose 
with no increase in dose planned for the duration of the study). 
Note: Opioids are permitted as needed as long as participants are on 
a stable dose at the time of randomization and expect to maintain the 
same dose until the 8 week follow-up visit OR if the participant has 
been on short-term (i.e., ≤ 14 days) opioid treatment and there is 
anticipation of a dose decrease or cessation of use during the course 
of the study.  Participants who only receive a few doses at the time of 
presentation of CDI may be considered for participation.  
Investigator should consult on any clarification of the opioid 
doses/treatment with the Medical Monitor. 
23. Pregnant, breastfeeding, or intends to become pregnant during study 
participation. 
24. Participating in a clinical trial of another investigational product 
(drug, device or other) and has not completed the required follow-up 
period. 
25. Subject, in the opinion of the investigator, for whatever reason, 
should be excluded from the study. 
Study Sites 
Approximately 80 US and Canadian sites will participate in this study. 
Study Sponsor 
Rebiotix Inc. 
2660 Patton Road 
Roseville MN  55113 USA 
www.rebiotix.com 
651-705-8770 
Contract Research 
Organization 
Medpace, Inc. 
 
 


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1. Introduction 
1.1. Background 
RBX2660 (microbiota suspension) is an intestinal microbial suspension prepared from 
human stool obtained from carefully and thoroughly screened healthy human donors.  It 
is being studied for the treatment of recurrent Clostridium difficile infection (CDI).  
RBX2660 is prepared from a standardized amount of stool mixed with saline and a 
cryoprotectant.  It is stored at the manufacturer in a frozen state and is shipped frozen to 
the clinical site for administration via enema. 
 
The donor selection process is multifaceted in its original screening of candidate donors 
as well as the ongoing assessment of donated samples prior to release for use in a 
clinical study.  Some of these measures include the following:  
1. Donor qualification screening includes questions that specifically address risk factors 
for colonization with multi-drug resistant organisms (MDROs), and individuals at 
higher risk of colonization with MDROs are excluded from donation. Examples of 
persons at higher risk for colonization with MDROs include: 
a. Health care workers 
b. Persons who have recently been hospitalized or discharged from long term care 
facilities 
c. Persons who regularly attend outpatient medical or surgical clinics 
d. Persons who have recently engaged in medical tourism 
2. Donor stool testing includes MDRO testing to exclude use of stool that tests positive 
for MDRO. The MDRO tests include at minimum extended spectrum beta-lactamase 
(ESBL)-producing Enterobacteriaceae, vancomycin-resistant enterococci (VRE), 
carbapenem-resistant 
Enterobacteriaceae 
(CRE), 
and 
methicillin-resistant 
Staphylococcus aureus (MRSA).  
3. All drug product currently in storage has undergone screening and stool testing for 
MDROs as described above. 
4. The informed consent process for subjects being treated with RBX2660 product as 
part of this study describes the risks of MDRO transmission and invasive infection as 
well as the measures implemented for donor screening and stool testing. 
 
Rebiotix has concluded the first clinical study of RBX2660 (n=34) in an open-label, 
non-controlled Phase 2 study demonstrating the safety of the product.  Additionally, 
Rebiotix has completed enrollment and interim reports of two Phase 2 clinical studies. 
Protocol 2014-01 is a prospective, multicenter, randomized, double-blinded, placebo-
controlled, 3-arm Phase 2B study (n=127 treated) designed to demonstrate the efficacy 
and safety of RBX2660 for the treatment of recurrent CDI.  Primarily, the Phase 2B 
Central Laboratory 
 
Medpace Central Labs LLC 
 


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study compared the efficacy of RBX2660 to placebo, and secondarily compared the 
efficacy of two planned enemas of RBX2660 to one planned enema of RBX2660 with 
one enema of placebo.  The study has completed enrollment and primary efficacy 
analysis and continues in long term follow-up.  Protocol 2015-01 is an open label study 
comparing the efficacy and safety of two planned RBX2660 enemas (n=136 treated) to a 
historical control (n=110) is in long-term follow-up.  
 
Finally, to complete the RBX2660 clinical development program, Rebiotix is initiating a 
Phase 3 study.  The purpose of this Phase 3 study is to confirm the efficacy and safety of 
RBX2660 for the prevention of recurrent CDI in subjects who have had prior recurrent 
Clostridium difficile infection that was resolved with antibiotic treatment. 
 
The study will be conducted in compliance with this protocol as approved by the federal 
Food and Drug Administration and each site’s governing Institutional Review Board or 
Research Ethics Board (Canada); in accordance to relevant regulations in 21 CFR Part 
11, 50, 54, 56 and 312; and the ICH E6 Good Clinical Practice: Consolidated Guidance. 
 
1.2. Investigational Agent 
RBX2660 (microbiota suspension) 50g/150mL in an enema bag within a brown opaque 
sleeve, which is to remain in place over the bag and tubing during administration and 
disposal.  Each bag contains one dose.  Each dose contains minimum 107 microbes/mL 
of suspension in a saline/polyethylene glycol 3350 vehicle.   
 
Note:  For this protocol, the term “Investigational Product (IP)” refers to both 
RBX2660 and the Placebo. 
 
1.3. Placebo 
The Placebo consists of 150 mL of normal saline, but without the polyethylene glycol 
3350 or drug substance, human stool.  It is supplied in an enema bag within a brown 
opaque sleeve, which is to remain in place over the bag and tubing during administration 
and disposal.  Each bag contains one dose. 
 
1.4. Preclinical Data 
Fecal transplantation is being used today in many highly respected hospitals throughout 
the world with no significant reports of adverse events or other safety-related 
complications.  Rebiotix has conducted an extensive literature review from the first 
published cases in 1958 (Eiseman et al., 1958) to the present.  The literature presents 
significant evidence of the safe and effective use of FT for over 500 cases of recurrent 
Clostridium difficile infection (CDI) with no product-related adverse events reported to 


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date and only a few procedure-related events, none of which have occurred in subjects 
receiving FT via enema (Bakken, 2015). Based on the significant human clinical 
experience documented in the literature, it has been determined that preclinical studies 
are not warranted. 
 
1.5. Clinical Data to Date 
1.5.1. Phase 2 Study Protocol 2013-001 Final Data 
On July 12, 2013, Rebiotix received permission from FDA to proceed with the Phase 2 
clinical study entitled “A Phase 2 Open-label Clinical Trial Demonstrating the Safety of 
RBX2660 Microbiota Suspension for the Treatment of Recurrent Clostridium difficile-
associated Diarrhea.”  This study ended with the last subject’s 6-month assessment in 
July 2014.   
It was a prospective, multi-center, open-label, non-controlled Phase 2 study designed to 
demonstrate the safety of RBX2660 (50 g/150 mL) for the treatment of recurrent CDI.  
The target population was adults (≥ 18 years old) with recurrent CDI who had either a) 
at least two recurrences after a primary episode (primary episode + ≥ two recurrences, 
i.e., at least three episodes) and had completed at least two rounds of standard-of-care 
oral antibiotic therapy or b) had at least two episodes of severe CDI resulting in 
hospitalization.  The Rebiotix Phase 2 study was the first study assessing the safety of 
RBX2660, although similar fecal transplant products have been used in humans for 
many years, as discussed in the literature.  The study’s primary objective was to assess 
the safety of RBX2660 but additional information was gathered on efficacy and the 
therapy’s impact on quality of life.  Eligible subjects who proceeded to treatment 
received one treatment with RBX2660.  If their CDAD returned before Day 56 after 
receiving one treatment, they were eligible to receive a second treatment if the re-
treatment occurred within ten days of recurrence.   
Follow-up visits occurred at Days 7, 30 and 60, and subjects remained in the study for 
telephone assessments for adverse events at 3 and 6 months.  If a subject received a 
second treatment, the follow-up schedule was re-set, so that they came in for visits at 7, 
30 and 60 days after the second treatment and receive 3- and 6-month telephone 
assessments.  Subjects also kept a written diary for the first 60 days after each treatment 
with RBX2660, including the collection of detailed information on solicited adverse 
events for the first eight days after receiving an RBX2660 enema. 
Rebiotix performed a final analysis of the Phase 2 data for the generation of the Final 
Clinical Study Report.  As of the report’s data lock on July 28, 2014, 40 subjects were 
enrolled.  Of these, six did not proceed to receive a treatment with RBX2660 (screen 
failures).  Of the 34 treated subjects, two subjects terminated their participation in the 
study around their respective 7-day visit.  One subject, 02-RBX01-007, died 35 days 


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after receiving her second treatment with RBX2660 from respiratory failure, which was 
not related to RBX2660 or the administration procedure.  As of July 28, 2014, 31 
subjects remained in active follow-up and all completed the study (6-month telephone 
assessment after their last treatment with RBX2660).  Here are the clinical study data 
and conclusions as presented in the Final Report. 
Efficacy Results 
The primary efficacy parameter of this study was treatment success, defined as the 
absence of CDAD (passage of three or more unformed stools in 24 or fewer consecutive 
hours for at least two consecutive days) at 56 days after the last treatment with 
RBX2660. Of the 34 subjects who received at least one treatment with RBX2660, 31 of 
them were presented in this efficacy analysis (31 had efficacy data available).  Twenty-
seven (27) out of 31 subjects, or 87.1%, were considered a treatment success.  Sixteen 
(16) subjects or 50.0% (N=16/32) were considered a treatment success after their first 
treatment with RBX2660.  Of the 16 subjects who failed their first treatment, 15 subjects 
proceeded to receive a second enema of RBX2660. Of those, 11 subjects or 78.6% 
(N=11/14) were considered a treatment success (one subject was not counted because 
she did not reach the 56-day endpoint).  The reasons for the increased rate of success 
after the second enema were not fully understood.  It may be due to a number of factors, 
including changes in the subject’s microbiome after one treatment, complete elimination 
of vancomycin from the subject’s stool over time, or other reasons not yet determined. 
Safety Results 
The subjects enrolled in the Rebiotix Phase 2 study had significant pre-existing 
comorbidities.  Even with their recurrent CDI symptoms temporarily controlled by 
standard-of-care antibiotic therapy, they remained very sick people.  The incidence, 
severity, and seriousness of the adverse events observed in this study represented the 
first attempt to systematically and rigorously collect, and consistently analyze and 
characterize them in a subject group suffering from recurrent CDI who were treated with 
an intestinal microbiota fecal transplant product.  There were 188 adverse events 
reported by 28 subjects, which were gathered through the very rigorous solicitation of 
events.  The adverse events reported were mostly mild-to-moderate in severity and were 
primarily related to gastrointestinal disorders as was expected in this population.  Most 
resolved within the 7-day interval after RBX2660 administration. This was true whether 
the subject received one or two treatments with RBX2660.   
 
Of the reported solicited events, which were collected during the 8-day period inclusive 
of the day of dosing and the subsequent 7-day follow-up period, the incidence and 
severity of these events declined over the 8-day period.  The proportion of subjects 
reporting solicited AEs was lower on Day 7 in subjects who received two treatments 
(41.7%; N=5/12) with RBX2660 than in subjects who received one treatment (51.5%; 
N=17/33), yet the efficacy rate after two treatments rose to 78.6% from 50% after one 


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treatment.  There were seven subjects who experienced a total of 20 SAEs, only one of 
which had a possible relationship to RBX2660; most were probably or definitely related 
to a pre-existing condition, including CDI. Overall, adverse events declined over time; 
66.5% of AEs occurred from baseline through the 7-day visit.  This was the first known 
study to prospectively, systematically, consistently and aggressively solicit, record, and 
classify adverse events in subjects who received an intestinal microbiota fecal transplant 
product.  It was therefore expected that the incidence rate of reported AEs might be 
higher than that reported in the literature, but it represented the true rate and types of 
events experienced by this population after treatment with an intestinal microbiota fecal 
transplant product. 
Figure 1-1 shows the decline in the proportion of subjects reporting solicited adverse 
events over the 8 days from the day of dosing (Day 0) to the seventh day after dosing 
(Day 7). 
 
 
 
Figure 1-1:  Proportion of Subjects Reporting Solicited AEs from Day 0-Day 7 
 
Conclusions 
The RBX2660 efficacy rate of 87.1% represented a significant improvement over 
reported success rates for oral antibiotics and was within the range of results reported in 
the literature for similar fecal microbiota products. This was the first known study to 
prospectively, systematically, consistently and aggressively solicit, record, and classify 
adverse events in subjects who received an intestinal microbiota fecal transplant product.  
It was therefore expected that the incidence rate of reported AEs (188 events in 28 
subjects) might be higher than that reported in the literature but it represented the true 


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rate and types of events experienced by this population receiving this therapy. The 
incidence rate and type of adverse events were not unexpected, given the high number of 
comorbidities that existed in this very sick, primarily elderly population.  None of the 
SAEs were determined to be probably or definitely related to RBX2660 and most were 
related to a pre-existing condition, primarily C. difficile infection.  Efficacy rose after 
two treatments with RBX2660 yet the proportion of subjects reporting solicited AEs was 
lower on Day 7 in subjects who received two treatments than in subjects who received 
one treatment.  The efficacy benefit of RBX2660 outweighed the incidence and type of 
adverse events, especially the risk of recurrent CDI, thus providing a satisfactory 
benefit: risk ratio. 
 
1.5.2. Phase 2B Study Protocol 2014-01 Interim Study Data 
EFFICACY RESULTS:  The efficacy objectives of the study were analyzed using the 
n=127 treated subjects (n=41 Group A, n=44 Group B, n=42 Group C) in the ITT 
population.  In particular the assessment of RBX2660 as compared to placebo for 
subjects with a history of recurrent CDI and the assessment of further treatment on 
subjects who are confirmed treatment failures.  
 
While the anticipated primary efficacy endpoint of two (2) enemas of RBX2660 
administered 7 ±2 days apart (61.0%) as compared to placebo (45.5%) in the blinded 
phase was not met, the key secondary efficacy endpoint of one (1) enema of RBX2660 
(66.7%) as compared to the placebo group (45.5%) was met with a p-value of 0.048.  All 
other secondary objectives for the study were also met. The active RBX2660 treatment 
groups A and C in the were compared to each other with a p-value of 0.589 indicating 
no difference between one RBX2660 enema and two (2) administered 7 ±2 days apart.  
As such, an analysis using the combined RBX2660 treatment groups A and C with a 
success rate of 63.9% (53/83) was compared to the placebo (45.5%) also with a 
significant p-value of 0.046 by Pearson’s chi-square.   
 
Additionally, analysis of the ITT population was done to establish the overall efficacy 
rate for RBX2660 which took into consideration both blinded and open label treatments.  
The resulting overall efficacy for all subjects treated with RBX2660 (n=107) was 88.8% 
which compared to the placebo rate of 45.5% was also significant with a p-value of 
<0.0001. 
 
Finally, an analysis of the ITT placebo group subjects using them as their own control 
by comparing blinded treatment success (placebo treatment) to open label success (after 
RBX2660 treatment) showed a significant increase in success rate from 45.5% (20/44) 
in the blinded phase to 87.5% (21/24) after treatment with RBX2660 with a resulting p-
value of <0.0001. 
 


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Confirmation is provided by the Per Protocol (PP) population analysis in which the 
combined RBX2660 treatment groups A and C with a success rate of 75.0% (45/60) 
were compared to the placebo 52.9% (18/34) with a significant p-value of 0.029 by 
Pearson’s chi-square during the blinded treatment phase. Further, overall RBX2660 
efficacy rate in PP subjects who received at least 1 RBX2660 enema either blinded or in 
open label (n=76) was 93.4% (71/76).   When compared to the PP Control Group B rate 
of 52.9%, a resulting p-value of <0.0001 further substantiates that RBX2660 is an 
effective treatment for the prevention of recurrent CDI. 
 
Overall the efficacy data confirms the following primary conclusions: 
• Treatment with RBX2660 following a standard course of antibiotics 
significantly increases the success rate for prevention of recurrent 
Clostridium difficile infection in subjects with a history of multi-recurrent 
disease.    
• Appropriate treatment with RBX2660 includes a single enema administered 
following completion of a standard course of antibiotics with another 
RBX2660 enema administered only if CDI symptoms return.  
 
Not only do these results prevent a recurrence of CDI within the 56 day (8 week) 
timeline as established by the CDC, the results are maintained longer term.  The long 
term CDI free rate at the time of this analysis was 95.8% (91/95) for subjects 
successfully treated with RBX2660 at an average follow-up of 8.2 months (range: 1.6 – 
14.9). 
 
Lastly, the donor screening and manufacturing process appears to properly ensure the 
quality of the RBX2660 product as consistent results in treatment success are shown 
regardless of donor. 
 
These efficacy results also validate previous results from the Phase 2 study which 
reported an 87.1% treatment success with RBX2660.  
 
SAFETY RESULTS:  The Safety Population (SP) consists of any subject who had a 
study enema attempted (n=128) whether placebo or RBX2660.  Adverse events were 
independently reviewed by a Medical Monitor with additional oversight by a DSMB for 
evaluation of safety trends and stopping rules. The DSMB completed an interim review 
of safety data as reported during the double-blind period.  Following review, the DSMB 
recommends continuation of the study as no pre-defined study stopping rules have been 
met or other safety concerns identified.   
 
Adverse events (n=580 in 94 subjects) were collected by the investigational sites and 
solicited via subject diary following administration of a study enema.  The adverse 


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events reported were mostly mild-to-moderate in severity (90.8%) and were primarily 
related to gastrointestinal disorders (41.9%) as would be expected in this population.  
 
Death has been reported in seven (7) subjects at the time of the analysis, none (0) of 
which were reported as related to the study enemas or procedure.  Six (6) of the seven 
(7) were reported as related to a pre-existing condition.  Further, one (1) of the seven (7) 
subjects died prior to receiving the assigned study treatment.   
There were 45 (7.8%) serious adverse events (SAEs) in 26 subjects reported.  Of the 
SAEs only three (3) were reported as possibly related to the study enema.  Additionally, 
of the 345 enemas administered in the study, zero (0) had a report of SAE related to the 
enema procedure.  While 31.1 % of SAEs were reported as related to CDI and 77.8% 
reported as related to a pre-existing condition.  No differences in the rate of adverse 
events between treatment groups was identified in the number of subjects experiencing 
an adverse event.  Additionally, only five (5) subjects were hospitalized for CDI 
recurrence during the blinded period, none (0) resulting in ICU admission.  
 
A detailed review of subjects with adverse events relative to the number of exposures to 
RBX2660 (0-4 exposures) was completed which showed there was no significant 
increase in the number or rate of adverse events with increased exposure to RBX2660.  
There were 108 subjects (84.4%) in the safety population who were exposed to 
RBX2660 with a total of 223 exposures for an average of two (2) exposures per subject.  
 
Analysis of the donor screening process indicated no adverse events subsequent to 
treatment with RBX2660 were linked to transfer of conditions to a subject from an 
RBX2660 enema or its constituents.  
 
Overall the safety data indicates that treatment with an RBX2660 enema followed by an 
additional treatment with RBX2660 if needed due to recurrence of CDI symptoms can 
be safely administered as it does not increase the risk for adverse events or result in a 
transfer of donor conditions.  
 
OVERALL RESULTS:  These results conclude that a single enema of RBX2660 
followed by an additional RBX2660 enema if needed due to a recurrence of CDI 
symptoms is a safe and effective treatment for the prevention of recurrent CDI following 
a standard course of antibiotics. 
 
1.5.3. Phase 2B Study Protocol 2015-01 Interim Study Data 
EFFICACY RESULTS:  The efficacy objectives of the study were analyzed using the 
n=246 treated subjects (n=136 treated with at least one enema of RBX2660, and 110 
historical controls) in the Full Analysis Set defined as all subjects who received any 
study treatment.  In particular the assessment of RBX2660 as compared to historical 


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controls for subjects with a history of recurrent CDI and the assessment of further 
treatment on subjects who are confirmed treatment failures. Historical controls were 
subjects who received antibiotics for recurrent CDI, not RBX2660 or other enema 
treatment, and were identified through review of patient records.  
 
The primary efficacy endpoint analysis of treatment success for the RBX2660 treatment 
administered 7 ±2 days apart (79.4%) as compared to historical controls (51.8%) 
reached statistical significance (p < 0.0001).   
 
Further sensitivity analysis was completed in which the RBX2660 improvement in 
treatment success remained statistically significant versus the control group after 
adjusting for age (p<0.0001), race (p-value 0.0003), sex (p<0.0001), number of previous 
episodes of CDI at baseline (p-value 0.0002). 
 
Overall the efficacy data confirms the following primary conclusions: 
• Treatment with RBX2660 following a standard course of antibiotics 
significantly increases the success rate for prevention of recurrent 
Clostridium difficile infection in subjects with a history of multi-recurrent 
disease.    
 
SAFETY RESULTS:  The Safety Population (SP) consists of any subject who had a 
study enema attempted (n=136) of RBX2660.  Adverse events were independently 
reviewed by a Medical Monitor. Following review, the Medical Monitor did not see an 
emergence of new adverse events or change in the occurrence rate.   
A total of 482 adverse events were reported in 99 subjects (72.8% of subjects) in the 
RBX2660 treatment group.  This was similar to the number and percentages of adverse 
events documented in the historical control group (n=691 events in 69 subjects; 62.7% 
of subjects). 
There were 385 treatment emergent adverse events (TEAEs) in 94 subjects (69.1% of 
subjects) in the RBX2660 treated group.  The TEAEs reported were mostly mild-to-
moderate in severity (89.1%) and primarily related to gastrointestinal disorders (37.4%) 
as would be expected in this population 
Three deaths were reported in RBX2660 treated subjects at the time of the interim 
analysis data cutoff date, 12 January 2017, one of which was reported as possibly related 
to the RBX2660 or procedure but definitely related to C. difficile disease and pre-
existing condition(s).  The other two recorded deaths were reported as definitely related 
to a pre-existing condition.  Death was reported in eight of the 110 subjects in the 
historical control group (7.3%). 
In the RBX2660 treated group, there were 82 Serious Adverse Events (SAEs) reported 
in 25 subjects.  Two of these SAEs occurred prior to administration of the first enema, 
leaving 80 treatment emergent SAEs (20.8% of all TEAEs) in 23 subjects (16.9%).   Of 
the treatment emergent SAEs, 11 (13.8%) were reported as possibly related to the 


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investigational product, and 5 (6.3%) were reported as possibly related to the enema 
procedure.  Additionally, 23 (28.8%) of the treatment emergent SAEs were reported as 
related to CDI disease and 62 (77.5%) were reported as related to a pre-existing 
condition. No adverse events subsequent to treatment with RBX2660 were linked to 
transmission of disease to a subject from an RBX2660 enema or its constituents. 
In the historical control group, there were 80 reported SAEs in 31 subjects (28.2% of 
subjects).  
As of the interim analysis, none of the pre-identified study stopping rules were met as 
determined by the medical monitor and the study continues as planned. 
 
OVERALL RESULTS:  These results indicate that treatment with RBX2660 is a safe 
and effective treatment for the prevention of recurrent CDI following a standard of care 
course of antibiotics as compared to a historical control. 
 
1.6. Treatment Rationale and Risk/Benefit 
RBX2660 microbiota suspension was developed after careful, controlled component and 
process testing performed by Rebiotix using applicable scientific methods.  RBX2660 
(microbiota suspension) 50 g/150 mL is supplied in an ethylene vinyl acetate enema bag.  
Each bag of RBX2660 contains one enema; each enema of RBX2660 consists of a 
suspension of minimum 107 live organisms/mL in polyethylene glycol 3350/0.9% 
Sodium Chloride Irrigation, USP, solution to ensure a consistent product is delivered.  
This study is confirming the optimal dosing strategy of RBX2660 enemas (one enema 
followed by a second if needed due to a recurrence of CDI symptoms) as compared to a 
placebo enema.  In an assessment of risk of recurrent CDI, which often results in severe 
and serious consequences, the benefit of preventing recurrent episodes by administering 
multiple enemas of RBX2660 outweigh the risk of the predominately mild to moderate 
AEs experienced by subjects in the Phase 2 and Phase 2B studies.  The enema route was 
chosen over other potential routes of administration because the risk of procedural 
complications for the subject is low and the method is easy to perform and requires no 
special equipment.  In addition, the enema route for fecal transplants is routinely used 
and has high efficacy rates reported in the literature with no reports of safety issues.   
 
Based on the results of the Rebiotix Phase 2 and Phase 2B studies, and the known, 
documented, safe and highly effective use of fecal transplantation for recurrent CDI with 
other products administered either via enema, colonoscopy or nasogastric/duodenal tube, 
the rigor of Rebiotix’s donor screening process, and implementation of quality assurance 
testing at all stages of manufacturing, the anticipated risks to subjects are acceptable in 
relation to the potential benefits of RBX2660. 
 
 
 


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2. Study Objectives and Endpoints 
2.1. Primary Objective 
To confirm the efficacy of RBX2660 as compared to a Placebo in preventing recurrent 
episodes of CDI through 8 weeks. 
 
2.2. Secondary Objective 
To evaluate the sustained clinical response rate of RBX2660 as compared to Placebo 
after blinded treatment. 
 
2.3. Other Objectives 
1. To confirm the safety and tolerability of RBX2660. 
2. To identify baseline characteristics predictive of efficacy outcomes. 
3. To characterize the changes from baseline fecal microbial composition in 
subjects treated with RBX2660 as compared to Placebo. 
4. To characterize the changes from baseline comorbidities in subjects treated 
with RBX2660 as compared to Placebo. 
5. To evaluate health-related quality of life for CDI as measured by the Cdiff32 
questionnaire. 
6. To characterize the baseline severity of CDI in subjects with documented CDI 
recurrence. 
7. Evaluate treatment success of RBX2660 in Placebo subjects who are 
documented study treatment failures then went on to receive RBX2660. 
8. Assess the ability of more than one dose of RBX2660 to prevent CDI 
recurrence. 
9. Assess the combined treatment success of all subjects receiving a single dose 
of RBX2660 during the study both to prevent recurrent CDI as well as prevent 
new CDI episodes.  
10. Assess the clearance rate of vancomycin resistant enterococcus in subjects 
who are carriers at baseline. 
11. Assess the clearance of C. difficile following enema treatment at 4 and 8-
weeks and 3 and 6 months after blinded study treatment in subjects receiving 
RBX2660 and those receiving Placebo. 
 
2.4. Primary Efficacy Endpoint 
Recurrence of CDI within 8 weeks of blinded treatment. 
 
2.5. Secondary Efficacy Endpoint 
Loss of sustained clinical response through 6 months after blinded treatment.  
  


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2.6. Safety Endpoints 
1. Number of adverse events per subject 
2. Timing of attributable adverse event post-treatment exposure (treatment 
emergent adverse event TEAE) 
3. Duration of TEAE 
4. Relatedness of TEAE 
5. Severity of TEAE 
6. Causality of TEAE to IP, enema, C. difficile or prior condition  
7. Number of each of the following through 8 weeks post blinded treatment: death, 
septic shock, toxic megacolon, colonic perforation, emergency colectomy, and 
ICU admission  
8. Onset of new chronic conditions relative to blinded treatment administration 
 
2.7. Other Efficacy Endpoints 
1. Baseline characteristics  
2. Subject fecal microbial composition at Screening, and 4-weeks, 8-weeks, 3 
months and 6 months after blinded study treatment. 
3. Charlson Comorbidity Index at Screening, 8-week, 3-, and 6- month phone 
assessments.  
4. Cdiff32 questionnaire at Screening, Week 1, 4, 8, month 3 and 6 
5. ATLAS score for CDI severity of qualifying CDI event. 
6. Recurrence of CDI within 8 weeks of unblinded (open-label) RBX2660 
treatment in Placebo subjects who were documented blinded study treatment 
failures. 
7. Recurrence of CDI within 8 weeks of unblinded (open-label) RBX2660 
treatment in subjects who received a blinded dose of RBX2660. 
8. Occurrence of CDI through 6 months in all subjects receiving a single dose of 
RBX2660. 
9. Concentration of vancomycin resistant enterococcus in stool samples for 
subjects who were carriers at baseline. 
10. Presence of C. difficile in stool samples at Screening, and 4-weeks, 8-weeks, 3 
months and 6 months after study treatment.  
 
 
3. Study Design 
3.1. Study Purpose 
The purpose of this study is to confirm the efficacy and safety of RBX2660 for the 
prevention of recurrent CDI in subjects who have had prior recurrent Clostridium 
difficile infection that was resolved with antibiotic treatment. 
 


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3.2. General Study Design 
This is a prospective, multicenter, randomized, double-blinded, placebo-controlled 
Phase 3 study to confirm the efficacy and safety of RBX2660 for the prevention of 
recurrent CDI.  
Up to 270 subjects may be randomized and treated in this study. Randomization will be 
at a 2:1 ratio (RBX2660:Placebo) which means approximately 180 subjects randomized 
to treatment with RBX2660 and 90 subjects randomized to Placebo.  Consented subjects 
who meet all inclusion and none of the exclusion criteria will be randomized.  Potential 
subjects who are consented but do not meet the inclusion and/or exclusion criteria will 
be considered screen failures and will not count towards total randomized.  Subjects who 
are randomized but exit prior to the administration of the first blinded enema may be 
replaced with additional subjects which could cause overall number of subjects 
randomized to be greater than the 270 anticipated, however no more than 270 subjects 
will be treated with a blinded enema.  Replacement subjects will be randomized to 
ensure maintenance of proper blinding. 
Potential subjects are expected to already be taking or just have been prescribed 
antibiotics to control recurrent CDI symptoms at the time of enrollment per the 
investigator’s standard of care.  This means patients who have already completed their 
prescribed course of antibiotics to treat recurrent CDI, cannot be considered for 
enrollment unless they have another recurrence. 
Once enrolled (informed consent obtained), in order to be randomized and treated; 
1. Subject must meet all the inclusion and none of the exclusion criteria; 
2. Antibiotics will have been administered for a minimum of 10 consecutive 
days prior to the washout period; 
3. CDI symptoms must be under control leading into the antibiotic washout 
period.  
 
Control of CDI symptoms is defined as no longer meeting the symptomatic criteria for 
CDI diarrhea which is the passage of three or more unformed/loose (i.e. Bristol Stool 
Scale type 6-7) stools in 24 or fewer consecutive hours for at least two consecutive days 
while taking antibiotics.  Thus, at minimum the diary must show that symptoms are 
under control the two days prior to the washout period in order for the subject to be 
treated.   
Once screening is completed and all inclusion and exclusion verified, the subject can be 
randomized.  The assigned study treatment will be scheduled to consist of a single 
blinded study enema according to the randomization assignment.  Study treatment is to 
be completed as soon as possible, but no more than 14 calendar days following 
randomization.  A minimum of 24hr to maximum of 72hr antibiotic washout period is 
required prior to administration of the assigned study treatment. Ultimately, the study 
treatment needs to be administered within 21 days of the Screening visit.   


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In-office study follow-up visits occur at weeks 1-, 4- and 8 after completing the blinded 
study treatment.  Telephone assessments for adverse events occur during weeks 2, 3 and 
6 after the study enema and at months 3 and 6.  Subjects are required to keep a detailed 
diary to assess for solicited events from the date of enrollment (informed consent) to the 
1-week follow-up visit.  The diary will be collected and reviewed at the baseline visit 
prior to blinded enema administration. The subject will continue to complete the diary 
following enema administration and it will be collected and reviewed at the 1-week 
follow-up visit. 
Subjects who are deemed failures following the blinded treatment per the pre-specified 
treatment failure definition may elect to receive an unblinded RBX2660 enema.  This 
unblinded enema is to be administered within 21 calendar days of failure determination.  
If a subject receives an unblinded RBX2660 enema, the follow-up requirements will 
restart from the day of the unblinded RBX2660 enema received according to the same 
schedule as required for the blinded portion of the study.   
A primary efficacy and safety analysis will be conducted when 100% of the randomized 
subjects in each group have completed their 8-week follow-up assessment after 
receiving the blinded treatment. One or more interim analyses will be conducted to 
evaluate the primary efficacy endpoint for possible early study stopping for success or 
futility.  The first interim analysis will occur after a minimum of 160 subjects have been 
treated and evaluated for efficacy.  A second interim analysis could occur when 220 
subjects have been treated and evaluated for efficacy if success is not achieved at the 
first interim analysis.  The details of the planned interim analyses are included in the 
statistical analysis plan.  
A final analysis and report will be completed once the last subject has completed the 6-
month follow-up visit for their study treatment. This includes 6-month follow-up after 
an unblinded RBX2660 study treatment. At that time the study will be considered 
complete.   
See Figure 3-1 for an illustration of the Study Design. 
 


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Figure 3-1: Phase 3 Study Design. 2:1 randomization refers to two subjects randomized to receive RBX2660 
(Group B) for every one subject randomized to Placebo (Group A) 
  
3.3. Definition of Recurrent CDI for Study Entry 
The definition of recurrent CDI for study entry is a documented diagnosis of: 
1. CDI diarrhea; the passage of three or more unformed/loose stools in 24 or 
fewer consecutive hours for at least two consecutive days, that began within 8 
weeks after completion of previous CDI treatment; 
2. and at least one positive stool test for the presence of toxigenic C. difficile; 
which must be documented within 30 days prior to or on the date of 
enrollment (informed consent).   
To be considered for enrollment, a subject must have medical record documentation of 
recurrent CDI per the above study definition that includes either:  
a. at least one recurrence after a primary episode and has completed at least 
one round of standard-of-care oral antibiotic therapy or;  
b. had at least two episodes of severe CDI resulting in hospitalization within 
the last year. 


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3.4. Definition of CDI Diarrhea for Use in Enrolled Subjects Throughout the Study 
1. The passage of three or more unformed/loose (i.e. Bristol Stool Scale type 6-
7) stools in 24 or fewer consecutive hours for at least two consecutive days; 
2. and a positive stool test for the presence of C. difficile toxin; documented at 
the time of the diarrhea. 
 
3.5. Definition of Treatment Success 
Treatment success is defined as: 
• The absence of CDI diarrhea for 8 weeks after completing a study 
treatment.  
 
3.6. Definition of Sustained Clinical Response  
Sustained clinical response is defined as:  
• Treatment success of the presenting CDI recurrence and no new CDI 
episodes through 6 months after completing a blinded treatment.       
    
3.7. Definition of Treatment Failure 
Treatment failure (CDI recurrence) is defined as:  
• The presence of CDI diarrhea within 8 weeks of administration of a 
study enema, which includes a positive stool test for C. difficile toxin 
at the time of the diarrhea.  
 
If a recurrence is suspected, an in-office visit is required, and the subject must provide a 
fresh stool sample.  Site staff will send this stool sample to the central laboratory for 
analysis. To ensure consistency across clinical sites for the determination of study failures, 
the central laboratory will perform the required C. DIFF QUIK CHEK COMPLETE® Test 
rapid enzyme immunoassay for glutamate dehydrogenase (GDH) and toxins A and B.  If 
the test is inconclusive, the central laboratory will conduct a second confirmatory test by 
PCR.  
Specifically, regarding interpretation of results, if the GDH and toxin are both positive per 
the rapid enzyme immunoassay test during the presence of diarrhea then the subject is 
considered a treatment failure.   
However, if the subject experiences diarrhea and the GDH and toxin are both negative per 
the test, then the subject is not considered a treatment failure as the presence of C. difficile 
was not established.  The study investigator will need to determine the appropriate 
treatment for the subject as to avoid the possibility of serious adverse events.   


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Alternatively, if the GDH and toxin results per the rapid enzyme immunoassay test are 
inconclusive (one is positive and the other is negative) the central lab will perform a PCR 
test.  If the PCR test is negative, the subject will not be considered a treatment failure as 
the presence of C. difficile was not established.  If the PCR test is positive, then the subject 
will be considered a treatment failure.   
Finally, if the rapid enzyme immunoassay test provides an invalid result such that the 
control line is not visible, the test will be repeated.  
For suspected treatment failures, a sample must be sent to the central laboratory.  In the 
case of suspected treatment failure where there is an immediate concern for subject safety, 
including the potential for serious adverse events, a C. DIFF QUIK CHEK COMPLETE® 
Test will be permitted to be conducted at the site or other laboratory known to the site in 
addition to sending a sample to the central laboratory for testing.  If the C. DIFF QUIK 
CHEK COMPLETE® Test is not available, a testing algorithm that directly assesses the 
presence of C. difficile toxin for laboratory confirmation of recurrent CDI should be used. 
In the event, the test results from the central laboratory differ from that of a local 
laboratory, the results from the central laboratory will be used in determination of 
treatment failure. For subjects who are suspected of treatment failure, but for whom there 
is not a test result from the central laboratory for the suspected episode, a deviation will 
be added to the database, and due documentation of the subject’s symptoms and all 
available data will be sent to the Endpoint Adjudication Committee (EAC) for 
independent adjudication of treatment failure.   
 
3.8. Management of Treatment Failure 
Following blinded treatment, subjects meeting the protocol definition of treatment 
failure may be scheduled for administration of an unblinded RBX2660 enema within 21 
calendar days of failure determination.  In order to receive the unblinded RBX2660 
enema, the site must provide documentation of meeting all treatment failure criteria as 
defined by the protocol.  If the subject has a subsequent treatment failure after the 
unblinded RBX2660 enema, the subject can be given another therapy as deemed 
appropriate by the treating investigator.   
Without conclusive failure documentation, an unblinded RBX2660 will not be 
provided for administration. 
Treatment failures are required to be entered in the study database shortly after 
determination of failure to allow for sponsor awareness and assurance that an unblinded 
RBX2660 can be provided if requested.  
If antibiotics are given to control symptoms, a 24-72hr washout period prior to 
administration of an unblinded RBX2660 enema is required. If the subject will have the 
unblinded enema, it needs to be administered within 21 calendar days of failure 


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determination.  The use of antibiotics prior to an unblinded RBX2660 enema is at the 
discretion of the investigator. 
If a subject receives an unblinded RBX2660 enema, the follow-up requirements will 
restart from the day of the unblinded RBX2660 enema according to the same schedule 
as required for the blinded portion of the study.  This results in completion of a new 
subject diary for up to 7 days after treatment; in-office visits at 1-, 4- and 8-weeks as 
well as telephone calls at 2-, 3- and 6- weeks and at 3 and 6 months after the last 
RBX2660 treatment. 
Treatment failures who do not receive an unblinded RBX2660 enema will continue to 
follow their original schedule of assessments for the duration of the study based on the 
blinded study enema. 
3.9. Determination of Treatment Success/Failure 
The site investigator makes the initial determination of treatment success or failure 
based on the pre-defined study definitions.  The site investigator’s assessment will then 
be provided to the EAC for independent, blinded adjudication of treatment success or 
failure that will be utilized for study analysis and reporting purposes. This may be done 
in an on-going basis or a single meeting as deemed appropriate. Details of the EAC 
logistics, activity and responsibilities will be included in a EAC charter.   
3.10. Endpoint Adjudication Committee 
The Endpoint Adjudication Committee (EAC) provides independent, blinded 
adjudication of treatment success or failure that will be used for study analysis and 
reporting purposes. The EAC is comprised of three physicians specializing in infectious 
diseases or gastroenterology who have experience managing subjects with recurrent CDI 
and are not investigators in the study.  Details of the EAC logistics, activity and 
responsibilities will be included in a EAC charter.   
3.11. Data and Safety Monitoring Board 
The DSMB will review safety data for trends and may be unblinded as needed to 
adjudicate certain adverse events and for review of both the primary efficacy and safety 
analysis and the final analysis. Details regarding DSMB responsibilities related to 
analysis oversight and data reporting will be provided in the DSMB Charter.  
3.12. Medical Monitor 
The Medical Monitor provides blinded review of serious adverse events or events 
reported by the site as related to the investigational product or enema procedure to assess 
accuracy of reporting as related to seriousness and causality, and periodic review of 
adverse events for trends.  Details of this review are documented in a Safety 
Management Plan.  The Medical Monitor will remain blinded throughout the study.  The 


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Medical Monitor’s review will be provided to the DSMB for analysis purposes and as 
applicable for the proper adjudication of potential study stopping rules. 
3.13. Blinding 
Subjects, site personnel, the DSMB, EAC and the Medical Monitor are blinded to the 
randomization assignment.  Blinding will be maintained through the data cutoff for the 
primary efficacy and safety analysis after which the blind may be lifted for the DSMB.  
The DSMB may be unblinded to individual assignments earlier as needed to adjudicate 
certain adverse events.  Individual randomization assignments will not be revealed to the 
subjects, site personnel, EAC or the Medical Monitor until the study is completed. Sites 
will be provided an emergency unblinding code if it becomes medically necessary to 
know the treatment group assigned for a particular subject.     
Subjects will be randomized to receive either RBX2660 or placebo through an 
Interactive Response Technology (IRT) System 4-14 calendar days prior to the blinded 
study treatment.  Randomization assignment is revealed to Rebiotix Operations 
Department personnel for product preparation and shipment.  The Rebiotix Kit is 
shipped to the site in a sealed carton that is only opened by the IP administrator at the 
time of administration, out of view of the subject and other site personnel.  While the IP 
administrator will not have access to the actual randomization assignment, it may be 
possible to guess through visualization at the time of enema preparation and 
administration.  To limit study bias, the IP administrator should not perform any of the 
subject study assessments from completion of the enema administration visit through the 
subject’s final study visit.   
The Rebiotix Kit remains sealed at the site until opened at the time of administration by 
the IP Administrator.  The IP bag is blinded by an opaque brown sleeve secured over it 
at Rebiotix at the time of shipment, and the tube set is covered by another brown opaque 
sleeve, which is placed by the IP Administrator at the time of preparation at the site; 
preparation occurs out of sight from the subject and other site personnel.  The IP is 
covered by the opaque sleeves throughout the administration process to avoid 
inadvertent visualization by the subject or other personnel who may accidentally view it.  
Subjects will be instructed to disregard the presence or absence of odor or color, as these 
characteristics may or may not indicate which product is being administered.  Product is 
placed in an opaque biohazard bag that is sealed to avoid accidental visualization.   
 
 
 


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4. Subject Selection and Withdrawal 
4.1. Inclusion Criteria 
All responses must be “yes” to include a subject in the study: 
1. 
≥ 18 years old.  
2. 
Medical record documentation of recurrent CDI per the study definition, that 
includes either: a) at least one recurrence after a primary episode and has 
completed at least one round of standard-of-care oral antibiotic therapy or b) 
has had at least two episodes of severe CDI resulting in hospitalization within 
the last year.  
3. 
A positive stool test for the presence of toxigenic C. difficile within 30 days 
prior to enrollment. 
4. 
Is currently taking or was just prescribed antibiotics to control CDI related 
diarrhea at the time of enrollment.   
Note: Subject’s CDI diarrhea must be controlled (<3 unformed/loose, i.e. 
Bristol Stool Scale type 6-7, stools/day for 2 consecutive days) while taking 
antibiotics during screening  
5. 
Willing and able to have an enema(s). 
6. 
Willing and able to complete the stool and serum testing required for the 
study. 
7. 
Agrees not to take non-dietary probiotics through 8 weeks after receiving the 
last study enema (including OTC and prescription). 
8. 
Agrees not to take any oral vancomycin, metronidazole, fidaxomicin, 
rifaximin, nitazoxanide and IVIG through the 8-week follow-up assessment 
unless newly prescribed by a treating investigator during the course of the 
study as a result of recurrent CDI diagnosis. 
9. 
Agrees to practice a form of effective contraception during study 
participation, does not apply to persons with documented non-child bearing 
potential. 
10. Has a negative urine pregnancy test at the time of enrollment and on the day 
of each enema prior to administration (persons of child-bearing potential 
only). 
11. Willing and able to provide informed consent and local privacy authorization 
as applicable. 
12. Willing and able to complete the required Subject Diary. 
13. Willing and able to meet all study requirements, including attending all 
assessment visits and telephone calls. 
 
 
4.2. Exclusion Criteria 
All responses must be “no” to include a subject in the study: 
1. 
A known history of refractory CDI. 
2. 
Currently has continued CDI diarrhea despite being on a course of antibiotics 
prescribed for CDI treatment. 
3. 
Requires antibiotic therapy for a condition other than CDI. 
4. 
Previous fecal transplant, RBX2660 treatment, receipt of CDI vaccine or 
treatment with CDI monoclonal antibodies prior to study enrollment.  


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5. 
History of inflammatory bowel disease (IBD), e.g., ulcerative colitis, Crohn’s 
disease, or microscopic colitis. 
6. 
Diagnosis of irritable bowel syndrome (IBS) as determined by Rome III 
criteria. 
7. 
History of chronic diarrhea. 
8. 
History of celiac disease. 
9. 
Disease symptoms (diarrhea) caused by a confirmed intestinal pathogen other 
than C. difficile. 
10. Currently has a colostomy. 
11. Intraabdominal surgery within the last 60 days. 
12. Evidence of active, severe colitis. 
13. History of short gut syndrome or motility disorders. 
14. Requires the regular use of medications to manage bowel hypermotility. 
15. Planned therapy in the next 3 months that may cause diarrhea (e.g., 
chemotherapy). 
16. Planned surgery requiring perioperative antibiotics within 6 months of study 
enrollment. 
17. Life expectancy of < 6 months.  
18. Compromised immune system (e.g., HIV infection with CD4 count 
<200/mm3; inherited/primary immune disorders; immunodeficient or 
immunosuppressed due to a medical condition or medication).  
Note: Eligible HIV patients who have a CD4 count >200/mm3 who are on 
stable, highly active anti-retroviral therapy may be considered for 
enrollment. 
19. Taking systemic steroids > 20 mg prednisone a day or prednisone-equivalent, 
or is expected to be on steroids (> 20 mg prednisone a day or equivalent) 
after enrollment through 8 weeks after completing the assigned study 
treatment.  
Note: Eligible patients taking a steroid dose equivalent to prednisone 20 
mg/day for >2 weeks, antimetabolites (e.g., azathioprine, 6-mercaptopurine, 
or low-dose methotrexate for autoimmune disease), calcineurin inhibitors 
(e.g., tacrolimus and cyclosporine), or mycophenolate mofetil may be 
enrolled only after consultation with the Medical Monitor, and only if the 
doses have been stable (except for drug therapeutic monitoring adjustments 
for calcineurin inhibitors) for 90 days and have not been associated with 
diarrhea prior to the current episode of CDI. 
20. An absolute neutrophil count of <1000 cells/µL during screening. 
21. Known or suspected current (< 90 days) illicit drug use.  Note: marijuana use 
is allowed. 
22. Participant is unable to discontinue opioids (unless on a stable dose with no 
increase in dose planned for the duration of the study).  
Note: Opioids are permitted as needed as long as participants are on a 
stable dose at the time of randomization and expect to maintain the same 
dose until the 8 week follow-up visit OR if the participant has been on short-
term (i.e., ≤ 14 days) opioid treatment and there is anticipation of a dose 
decrease or cessation of use during the course of the study.  Participants who 
only receive a few doses at the time of presentation of CDI may be 


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considered for participation.  Investigator should consult on any clarification 
of the opioid doses/treatment with the Medical Monitor.   
23. Pregnant, breastfeeding, or intends to become pregnant during study 
participation. 
24. Participating in a clinical trial of another investigational product (drug, 
device or other) and has not completed the required follow-up period. 
25. Subject, in the opinion of the investigator, for whatever reason, should be 
excluded from the study. 
 
4.3. Subject Recruitment and Screening 
Subjects are recruited by qualified and trained site personnel when they are identified as 
recently diagnosed with or experiencing recurrent CDI within 8 weeks of completing the 
treatment for a previous CDI episode.  Potential subjects are to be fully informed as to 
this study’s purpose, requirements, randomization schedule, blinding, anticipated risks, 
etc., and are to be given the chance to review the informed consent form and receive 
satisfactory answers to all questions.  Subjects sign the study-specific, IRB/REB-
approved Informed Consent and local privacy authorization, which is the point at which 
the potential subject is considered enrolled in the study.   
Subjects are eligible to receive study treatment upon confirmation that they have met all 
inclusion/exclusion criteria listed in Sections 4.1 and 4.2.  Antibiotics being taken for 
their recurrent CDI symptoms must be stopped for a 24-72 hour washout period prior to 
receiving the blinded study enema. 
 
 
5. Investigational Product 
5.1. RBX2660 Description  
RBX2660 microbiota suspension 50 g/150 mL in an enema bag.  Each bag of RBX2660 
provides one enema.  Each enema of RBX2660 consists of a suspension of minimum 
107 live organisms/mL in polyethylene glycol 3350/0.9% Sodium Chloride Irrigation, 
USP, solution. 
 
5.2. Placebo Description  
The Placebo is an enema of normal saline.  Placebo packaging and labeling are identical 
to the packaging and labeling for RBX2660 to support the study blinding. 
 


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5.3. Rebiotix Kit Description  
Investigational product is supplied in a single-enema bag fitted with a spike port and a 
rectal tube assembly that is used to administer IP as an enema; these components are 
packaged together in the Rebiotix Kit.  Rebiotix Kit components include: 
 1 bag of RBX2660 or Placebo with a spike port contained in a brown opaque 
sleeve 
 1 rectal tube assembly 
 1 brown opaque sleeve for the tube assembly 
 1 adhesive strip to secure opaque sleeves 
 1 biohazard disposal bag 
 1 Instructions for Use 
5.4. Treatment Regimen 
Randomization is to occur about 4-14 calendar days prior to the blinded study treatment 
to allow for notification to Rebiotix and shipment of product. Ultimately, the study 
treatment needs to be administered within 21 days of the Screening visit. Subjects are 
randomized to one of the following groups:  
• Group A:  Placebo  
• Group B:  RBX2660  
 
Randomization will occur in a 2:1 fashion with two subjects randomized to receive 
RBX2660 for every one subject randomized to Placebo.  
Once completed, assigned study treatment consists of a single blinded enema of either 
RBX2660 or Placebo as per the randomization assignment.  Product is to only be 
administered by the authorized IP Administrator who must be a qualified and trained 
health care professional following the Instructions for Use and standard site procedures.  
Subjects are to remain at the site under supervision for at least one hour post-enema 
administration for vital sign assessment (temperature, heart rate, blood pressure, 
respiratory rate) and observation.   
Subjects with a documented treatment failure following blinded treatment may be 
scheduled for administration of an unblinded RBX2660 enema.  In order to receive an 
unblinded RBX2660 enema, a subject must have documentation of meeting all treatment 
failure criteria as defined by the protocol (Section 3.7).  
If, in the opinion of the investigator, antibiotics are warranted for the control of CDI 
symptoms prior to receiving the unblinded RBX2660 enema, they can be administered 
but need to be stopped 24-72 hours before enema administration.  The total time from a 
treatment failure (CDI recurrence) determination to administration of an unblinded 
enema is within 21 calendar days.   
See Section 6 for the Schedule of Study Procedures and detailed description of the 
requirements. 


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5.5. Subject Follow-up Requirements 
Subjects are assessed at office visits at 1, 4 and 8 weeks and via telephone calls from the 
study coordinator at 2, 3 and 6 weeks and 3 and 6 months from their last study enema.  
See Section 6 for follow-up assessments and phone calls, and Section 6.16 for subject 
withdrawal/termination. 
5.6. Concomitant Therapy 
The subject must agree not to take any oral vancomycin, metronidazole, fidaxomicin, 
rifaximin, nitazoxanide, and IVIG through the 8 week follow-up assessment unless 
newly prescribed by a treating investigator during the course of the study as a result of 
recurrent CDI diagnosis.  Medication information will be collected at the screening visit 
and changes in medications will be recorded throughout the study.  Subjects should be 
encouraged to notify their general practitioners of study participation and the 
requirements for concomitant therapy as able.  
5.7. Preparation of Investigational Product 
Instructions for Use with detailed directions for IP preparation and administration are in 
each Rebiotix Kit. 
5.8. Packaging 
Investigational product is supplied in an enema bag fitted with a spike port and a rectal 
tube assembly that is used to administer the product via enema; these components are 
packaged together in the Rebiotix Kit.  
5.9. Ordering, Receipt and Storage, Dispensing, Returning, and Tracking Investigational 
Product 
5.9.1. Ordering Investigational Product  
Authorized site personnel will randomize subjects using the Interactive Response 
Technology (IRT) System which automatically triggers ordering of IP.  If a subject is a 
confirmed treatment failure and will proceed to unblinded RBX2660 treatment, the site 
will document the failure in the IRT which automatically triggers ordering of RBX2660 
IP.  
5.9.2. Receipt and Storage of Investigational Product 
Note:  Each unit of IP is designated to be administered to a specific subject on a 
specific date; administration of IP designated for one subject to another subject is 
prohibited.  The Rebiotix Kit must only be opened by the IP Administrator and 
only at the time of product preparation immediately before administration away 
from subjects and other study personnel. 
 


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Personnel from the Rebiotix Operations Department confirm the randomization 
assignment and ship a unit of IP to the authorized person/ department as specified on the 
Delegation of Authority log at the site in a temperature-controlled shipping package.  
The authorized person opens the shipping package, visually confirms that the unit of IP 
arrived intact, confirms that the temperature indicator has not been tripped and records 
receipt and inspection on the Product Accountability Log.  IP is to be thawed and stored 
in the refrigerator until administration.   
IP must be kept in a secure location under the control of the Investigator or authorized 
designee at all times; see Section 5.9.5 for details.  IP must be administered to the 
subject by the expiration date as specified on the label.  If it is not administered by the 
expiration date, it must be destroyed by site staff per the institution’s processes and be 
recorded on the Product Accountability Log. 
 
5.9.3. Dispensing and Administering Investigational Product 
Dispensing procedures vary from site to site, but each site’s study investigator is 
responsible for delegating the administration of IP to a qualified and trained health care 
professional; this person(s) (“IP Administrator”) must be identified on the Delegation of 
Authority Log and receive product-specific training on IP administration.   
IP is to be administered by the IP Administrator after the site Investigator confirms all 
inclusion/exclusion criteria have been met, the subject had a negative pregnancy test (if 
applicable) and the 24-72 hour washout period has been completed.  Administration is 
via enema following the Instructions for Use in each product package and the site’s 
standard procedures.   
 
5.9.4. Returning IP 
Do not return IP to Rebiotix. For IP received but not administered to a subject, it must 
be destroyed by site staff per the institution’s processes.  Destruction must be recorded 
on the Product Accountability Log.  
 
5.9.5. Product Accountability and Tracking 
Each unit of IP is sent to the site for administration to an assigned subject and is labeled 
with a batch number to be recorded in the eCRF and on the Product Accountability Log.  
Each unit must be tracked on the Product Accountability Log by the person(s) 
authorized on the Delegation of Authority Log to perform that step.  IP must be kept in a 
secure location under the control of the site investigator or authorized designee as 
recorded on the Delegation of Authority Log.  The Product Accountability Log and 
Delegation of Authority Log are to be maintained by the site and confirmed during 


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monitoring visits.  The steps to be tracked include dates of receipt and dispensing, batch 
number, subject number, storage in refrigerator, and destruction if not administered to a 
subject.  The IP Administrator must ensure the correct batch number is administered to 
the correct subject. 
 
 
6. Study Procedures 
6.1. General Information 
Study information is collected on study-specific electronic case report forms (eCRFs) by 
the site.  Study monitoring occurs at regular intervals to ensure the protection of subject 
rights and safety, data integrity and accuracy, and proper study conduct in compliance to 
the protocol and applicable regulations including 21 CFR 312 and ICH E6 GCPs.  Study 
visits and telephone assessments must occur with the window specified in Table 6-1; 
visits and calls out of window or missed are to be reported as protocol deviations. The 
study-required procedures are to be conducted as shown in Table 6-1. 


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Table 6-1 Schedule of Events 
Activity 
Screening 
(Enrollment) 
Baseline / 
Enema 
Administration (≤ 21 
days from screening) 
Follow-up Visits 
1-, 4- and 8-
Week (± 3 days) 
Assessments1  
Weekly Phone 
Assessment 
(Weeks 2, 3 and 
6  (± 3 days) 
Unscheduled 
Possible 
Recurrence 
Visit 
Unblinded Enema 
Administration1 (≤ 
21 calendar days 
post Tx Failure) 
Phone 
Assessment at 3 
and 6 months  
(± 14 days) 
Informed consent obtained 
X 
 
 
 
 
 
 
Demographics, medical history 
X 
 
 
 
 
 
 
Prescribe/continue antibiotics for 
CDI symptom control 
X 
 
 
 
 
 
 
Modified physical exam conducted 
 
X 
 
 
 
 
 
Stool sent to Rebiotix by subjects 
for testing and archiving (optional) 
X 
 
X 
 
X 
 
X 
Central Lab CBC w/differential 
testing3 
X3 
X 
 
 
 
 
 
Central Lab CMP & CRP testing 
X 
 
 
 
 
 
 
C. difficile testing4 
X4 
 
 
 
X4 
 
 
Central Lab stool and blood testing  
 
X 
 
 
 
 
 
Urine pregnancy testing performed 
at site (if applicable) 
X 
X 
 
 
 
X 
 
Cdiff32 Questionnaire 
X 
 
X 
 
X 
 
X 
Inclusion/exclusion criteria 
confirmed 
X 
X 
 
 
 
 
 
Randomization assignment 
X5 
 
 
 
 
 
 
24-72hr washout period confirmed 
 
X 
 
 
 
X 
 
Enema administered 
 
X 
 
 
 
X 
 
Product complaint (if applicable) 
 
X 
 
 
 
X 
 
Recurrence or New CDI symptoms 
assessed 
 
X 
X 
X 
X 
X 
X 
Vital signs assessed 
X 
X 
 
 
X 
X 
 
Subject Diary discussed/reviewed 
X 
X 
X6 
 
 
X 
 
Employment status assessed 
X 
 
X7 
 
 
 
X 
Concomitant medications 
X 
X 
X 
X 
X 
X 
X 
Medical History Assessment 
X 
 
X7 
 
X 
 
X 
Adverse events assessed 
 
X 
X 
X 
X 
X 
X 
Solicited events assessed2 
 
X 
X2 
 
 
X2 
 
Protocol deviations (if applicable) 
X 
X 
X 
X 
X 
X 
X 
1 Documented treatment failures may receive an unblinded RBX2660 enema. If an unblinded enema is administered, the follow-up visit requirement re-start based on the date of last 
enema administration.  
2 Solicited events are collected in the Subject Diary from the day of administration of any study enema (blinded or unblinded) until the day prior to the 1-week visit. The Subject Diary 
is collected and reviewed at the 1-week visit.  Solicited events that increase in severity from screening should be assessed for a possible adverse event. 
3 Exclusion criteria for absolute neutrophil count should be assessed based on the CBC collected at the screening visit. 
4 Perform within 30 days prior to or at enrollment and if CDI recurrence is suspected. 
5 Randomization should occur after screening criteria have been assessed and eligibility confirmed (4-14 calendar days from randomization to blinded study treatment). 
6 Subject Diary is reviewed only at the 1-week follow-up visit. 
7 Collected at the 8-week follow-up visit only. 


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6.2. Informed Consent 
All subjects must sign the study-specific IRB/REB-approved Informed Consent/HIPAA 
Form; see Section 4.3.  The original signed Form is to be placed in the subject’s study 
file.  A copy of the signed Form is also to be given to the subject for his/her records. 
 
6.3. Subject Diary  
The Subject Diary serves as a tool for the subject to record pre- and post-treatment 
health and information that may indicate adverse events or the recurrence of CDI.  The 
subject also documents changes in medications.  
At the time of enrollment (informed consent) subjects are provided a Subject Diary that 
they are to complete until the day prior to the blinded study enema.  This may mean up 
to 21 days depending on how long it takes to determine subject eligibility for the study.  
The diary will be collected and reviewed at the baseline visit prior to enema 
administration.  
A diary is also given to the subject on the day of the blinded study enema administration 
and is collected at the 1-week office visit.  The Subject Diary is reviewed for solicited 
event occurrence and severity, changes to medications, occurrence of adverse events, 
and possible recurrence of CDI. The following information is entered into the study 
database:  
• solicited event severity 
• changes to medication 
• occurrence of adverse events   
 
If a subject is a documented treatment failure and s/he elects to receive unblinded 
RBX2660 treatment, the follow-up visit requirements start over with the receipt of an 
unblinded enema. The subject will be given additional diary pages to complete 
beginning on the day of the unblinded enema until the 1-week visit. This additional 
information is also entered into the study database. 
 
6.4. Optional Stool Samples for Additional Testing by Rebiotix 
Subjects are requested to provide stool samples directly to Rebiotix at:  
• Screening (between the time of enrollment and prior to treatment) 
• at the time of the 1, 4 and 8 week office visits;  
• at the time of the 3 and 6 month phone calls;  
• at the time of possible treatment failure symptoms. 


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Subjects collect the samples at home in pre-labeled containers given to them by the 
study team.  Rebiotix provides the study sites with kits containing all the materials for 
collecting and shipping the samples, including subject directions, container labels, and 
prepaid, preaddressed mailing labels.  Samples are identified by the label on the 
container that contains only the subject’s study identification number and date/time of 
the sample; samples will not contain any personal information that would identify the 
subject.  Samples may be stored for approximately five years. The stool samples will be 
used for research about the microorganisms in the human gut. Some of the research will 
include testing for microorganisms (Clostridium difficile and vancomycin-resistant 
enterococcus) both before and after receiving the study enema.  Failure to send in a 
sample does not require a protocol deviation, however subjects should be reminded of 
this request.   
 
6.5. Screening Visit 
The following study activities/data will be collected at the screening visit: 
• Subjects are told about the study and informed consent is obtained 
(enrollment) 
• Standard demographics (gender, ethnicity, date of birth, race.) 
• Detailed medical history, including Charlson Comorbidity Index variables 
• Assess inclusion/exclusion criteria, including C. difficile stool testing 
o At the time of enrollment or study entry, a positive stool test for 
toxigenic C. difficile must be or have been performed and 
documented no more than 30 days before entry and must be on file 
at the site.  C. difficile laboratory testing according to the site’s 
standard of care is acceptable for enrollment, however, the use the 
C. DIFF QUIK CHEK COMPLETE® Test rapid enzyme 
immunoassay test for enrollment is preferred.  If the rapid enzyme 
immunoassay test is utilized, the same interpretation of results as 
defined for treatment failures is required.  
• Core laboratory blood sample is collected for CBC w/differential and 
comprehensive metabolic panel (CMP: Sodium, potassium, chloride, 
BUN, creatinine, albumin, AST, ALT, alkaline phosphatase, bilirubin 
(direct, indirect and total), glucose) and C-reactive protein (CRP) testing  
• Onsite urine pregnancy test for subjects of child bearing potential   
• For subjects who consented to the Optional Stool Sample collection, 
remind them to obtain and ship a stool sample to Rebiotix anytime 
between their enrollment date and their blinded enema administration visit 
• Assess vital signs: height, weight, temperature, blood pressure, pulse, 
respiration. 
• Complete the Cdiff32 health-related quality of life questionnaire 


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• Document concomitant medications 
• Completion of the Subject Diary is explained and how to complete it is 
demonstrated; see Section 6.3.   
• Verify employment status 
• Assess for protocol deviations  
 
6.6. Randomization 
Once enrolled (informed consent obtained), in order to be randomized and treated:  
1. Subject must meet all of the inclusion and none of the exclusion criteria; 
2. Antibiotics will have been administered for a minimum of 10 consecutive 
days prior to the washout period; 
3. CDI symptoms must be under control leading into the antibiotic washout 
period. 
 
After enrollment and confirmation of inclusion/exclusion criteria compliance, including 
criteria confirmed by central laboratory testing, the study coordinator or other trained 
and authorized site staff randomize the subject in the Interactive Response Technology 
(IRT) System and ensures study treatment is completed within 4-14 calendar days of 
randomization. Ultimately, the study treatment needs to be administered within 21 days 
of the Screening visit. 
The randomization schedule will be created using randomized blocks within four strata 
based on antibiotics used at screening (Vancomycin alone, Vancomycin in combination, 
Fidaxomicin or Other), and randomized subjects will be assigned one of two treatments 
(Group A or Group B) within the appropriate stratum. The randomization will not be 
stratified by site, so each site will draw from the same set of blocks.  A subject will be 
assigned the next available treatment allocation from the randomization list.  The 
randomization code and the date and time of randomization will be captured in the IRT. 
Enrolled (consented) subjects who are randomized but exited prior to attempting the 
blinded treatment may be re-enrolled if another CDI recurrence occurs and thus 
randomized under a different subject identification numbers (re-randomized).  If this 
occurs, they will be included in the analysis using their second randomization 
assignment and subsequent assessments and will only be counted once towards the total 
randomized.   
Treatment will be assigned in the following proportions: 2:1 to yield a total of up to 270 
randomized and treated subjects, or approximately 180 subjects randomized to treatment 
with RBX2660 and 90 subjects randomized to Placebo.  Subjects who are randomized 
but exit prior to the administration of the first blinded enema may be replaced with 
additional subjects which could cause overall number of subjects randomized to be 
greater than the 270 anticipated, however no more than 270 subjects will be treated with 


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a blinded enema.  The assignment is transmitted to the Rebiotix Operations Department, 
who will ship the correct IP to the site for each treatment.  Replacement subject will also 
be randomized to ensure proper blinding.  
 
6.7. Baseline Visit and Enema Administration 
The baseline visit is performed on the day of the blinded enema administration.  
Randomization and IP administration must occur within 21 days of the screening visit.  
Antibiotics for the control of CDI symptoms are prescribed or continued until 24-72 
hours before administration of the blinded study enema.  
The following study activities/data will be collected at the baseline visit prior to enema 
administration: 
• Inclusion/exclusion criteria are confirmed including the following: 
o The control of symptoms during antibiotic use is confirmed by 
review of the subject diary 
o Compliance to the washout period (discontinuation of the 
antibiotics for 24-72 hours prior to the baseline visit) is confirmed 
• Modified physical exam (no genitourinary exam unless medically 
indicated) is performed to establish baseline health status (findings that 
can be attributed to the subject’s medical history will not be documented 
as adverse events) 
• Central laboratory blood sample collection for CBC w/differential, HIV, 
Hepatitis A (IgG) Hepatitis B Anti-Hepatitis B surface antigen, Hepatitis 
C antibody, and Treponema Antibody. NOTE: the results are not needed 
before proceeding to treatment per the protocol 
• Central laboratory stool sample is collected. NOTE: the results are not 
needed before proceeding to treatment per the protocol 
• Onsite urine pregnancy test for subjects of child bearing potential 
• Assess vital signs: temperature, pulse, blood pressure, respiration 
• Changes to concomitant medications since the last visit 
• Review subject diary 
• Assess recurrence of CDI  
• Assess for adverse events since enrollment 
• Assess for protocol deviations  
 
Following successful completion of the baseline visit, investigational product is then 
administered via enema per the Instructions for Use and standard site procedure by the 
authorized IP Administrator; see Section 5.9.3.  Subjects remain at the site under 
supervision for at least one hour post-IP administration for vital sign assessment 
(temperature, heart rate, blood pressure, respiratory rate) about every 15 minutes and 


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observation.  If the subject’s vital signs do not return to the pre-administration values ± 
20%, assess the subject for a possible AE.  The subject is sent home after completion of 
study procedures and a review of the Subject Diary instructions, and is requested to call 
the study coordinator if symptoms recur or at the onset of an adverse event.  
 
Table 6-2:  Subject Baseline Stool and Blood Testing Conducted by Central Laboratory 
 
Test Name 
Material Tested 
Norovirus  
Stool 
Rotavirus  
Adenovirus  
Enteric pathogens (Shigella, Salmonella, 
Campylobacter, sorbitol-negative E. coli., 
Aeromonas, Plesiomonas, Yersinia, and shiga toxins) 
Giardia antigen 
Cryptosporidium antigen 
Acid-fast staining (Cyclospora, Isospora) 
Ova and parasites 
Vancomycin-resistant enterococci (VRE) 
Methicillin-resistant Staphylococcus aureus (MRSA) 
Vibrio 
Listeria 
Clostridium difficile 
Carbapenem-resistant Enterobacteriaceae Culture 
(CRE) 
CBC with Differential 
Blood 
HIV 
Hepatitis A (IgG) 
Hepatitis B Anti-Hepatitis B surface antigen 
Hepatitis C Antibody 
Treponema Antibody 
 
 
 
 
 
 
 


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6.8. In Office Visit (1-Week) 
Timely and complete follow-up visits will ensure the scientific integrity of the data is 
maintained and allow for continued oversight of subject safety.   
The following study activities/data will be collected at the 1-week follow-up visit: 
• Review of the subject diary for solicited events as defined in Section 7.4 
• Assess recurrence of CDI 
• Subject’s weight 
• Adverse event collection, including new onsets of obesity, metabolic 
syndrome, pre-diabetes, diabetes and/or autoimmune disorders 
• Cdiff32 health-related quality of life questionnaire 
• Changes to concomitant medications since the last visit 
• Assess for protocol deviations  
• For subjects who consented to the Optional Stool Sample collection, 
remind the subject to collect and ship a stool sample to Rebiotix 
 
6.9. In Office Visit (4-Week) 
The following study activities/data will be collected at the 4-week follow-up visit: 
• Assess recurrence of CDI 
• Subject’s weight 
• Adverse event collection, including new onsets of obesity, metabolic 
syndrome, pre-diabetes, diabetes and/or autoimmune disorders 
• Cdiff32 health-related quality of life questionnaire 
• Changes to concomitant medications since the last visit 
• Assess for protocol deviations  
• For subjects who consented to the Optional Stool Sample collection, 
remind the subject to collect and ship a stool sample to Rebiotix 
 
6.10. In Office Visit (8-Week) 
The 8-week follow-up visit is the primary efficacy endpoint timeline of the study. As 
such, every attempt should be made to ensure subjects understand the importance of this 
visit and are scheduled within the appropriate visit window.   
The following study activities/data will be collected at the 8-week follow-up visit: 
• Assess recurrence of CDI 
• Subject’s weight 
• Adverse event collection, including new onsets of obesity, metabolic 
syndrome, pre-diabetes, diabetes and/or autoimmune disorders 


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• Changes to concomitant medications since the last visit 
• Cdiff32 health-related quality of life questionnaire 
• Verify employment status 
• Assess changes in medical history, including Charlson Comorbidity Index 
variables.  Worsening of conditions are to be captured as an adverse event 
with appropriate source documentation.  An improvement in a condition 
may not have physician diagnosis or source documents to support 
improvement; subject self-reported improvement in a condition does not 
require medical documentation to support the improvement. 
• Assess for protocol deviations  
• For subjects who consented to the Optional Stool Sample collection, 
remind the subject to collect and ship a stool sample to Rebiotix 
 
6.11. Telephone Assessments (Weeks 2, 3 and 6)  
Qualified and trained site staff, as recorded on the Delegation of Authority Log, make 
the phone calls and may follow the script provided.  All data collected are to be recorded 
and reported as required in Section 6.15.  These phone calls must occur within the 
window specified in Table 6-1; phone calls out of window or missed phone calls are to 
be reported as protocol deviations.  
The following study activities/data will be collected at the telephone assessment calls: 
• Assess recurrence of CDI 
• Adverse event collection, including new onsets of obesity, metabolic 
syndrome, pre-diabetes, diabetes and/or autoimmune disorders 
• Changes to concomitant medications since the last visit 
• Assess for protocol deviations  
 
6.12. Telephone Assessments (3- and 6- Months) 
Qualified and trained site staff, as recorded on the Delegation of Authority Log, make 
the phone calls and may follow the script provided.  All data collected are to be recorded 
and reported as required in Section 6.15.  These phone calls must occur within the 
window specified in Table 6-1; calls out of window or missed calls are to be reported as 
protocol deviations.  
The following study activities/data will be collected at the telephone assessment calls: 
• Assess recurrence of CDI 
• Subject’s weight, self-reported 
• Adverse event collection, including new onsets of obesity, metabolic 
syndrome, pre-diabetes, diabetes and/or autoimmune disorders 


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• Changes to concomitant medications since the last visit 
• Cdiff32 health-related quality of life questionnaire 
• Verify employment status 
• Assess changes in medical history, including Charlson Comorbidity Index 
variables.  Worsening of conditions are to be captured as an adverse event 
with appropriate source documentation.  An improvement in a condition 
may not have physician diagnosis or source documents to support 
improvement; subject self-reported improvement in a condition does not 
require medical documentation to support the improvement 
• Assess for protocol deviations  
• For subjects who consented to the Optional Stool Sample collection, 
remind the subject to collect and ship a stool sample to Rebiotix 
 
The last phone call occurs 6 months after the date of the last administration of IP 
whether it was blinded or unblinded.   
 
6.13. Unscheduled Possible Recurrence Visit 
If CDI recurrence is suspected any time within 8 weeks of the last study enema due to 
documentation of three or more unformed/loose (i.e. Bristol Stool Scale type 6-7) stools 
within 24 or fewer consecutive hours for at least two consecutive days, possible 
treatment failure must be assessed.  An in-office visit is required and the subject must 
provide a fresh stool sample to test for the presence of C. difficile toxin as defined by the 
protocol.  
The following study activities/data will be collected at the Unscheduled Possible 
Recurrence visit: 
• Collect fresh stool sample to send to the central laboratory for analysis  
• Assess recurrence of CDI 
• Vital signs: temperature, pulse, blood pressure, respiration 
• Adverse event collection, including the new onsets of obesity, metabolic 
syndrome, pre-diabetes, diabetes and/or autoimmune disorders 
• Changes to concomitant medications since last visit 
• Cdiff32 health-related quality of life questionnaire 
• Assess changes in medical history, including Charlson Comorbidity Index 
variables.  Worsening of conditions are to be captured as an adverse event 
with appropriate source documentation.  An improvement in a condition 
may not have physician diagnosis or source documents to support 
improvement; subject self-reported improvement in a condition does not 
require medical documentation to support the improvement. 
• Assess for protocol deviations 


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• For subjects who consented to the Optional Stool Sample collection, 
remind the subject to collect and ship a stool sample to Rebiotix.     
 
Treatment failure (CDI recurrence) is defined as:  
• The presence of CDI diarrhea within 8 weeks of administration of a study 
enema, which includes a positive stool test for C. difficile toxin at the time 
of the diarrhea using the study required C. difficile testing algorithm. 
 
To ensure consistency across clinical sites for the determination of study failure, the 
central laboratory will perform the required C. DIFF QUIK CHEK COMPLETE® Test 
(QCC Test) rapid enzyme immunoassay test for glutamate dehydrogenase (GDH) and 
toxins A and B.  
In the case of suspected treatment failure where there is an immediate concern for 
subject safety, including the potential for serious adverse events, a C. DIFF QUIK 
CHEK COMPLETE® Test will be permitted to be conducted at the site or other 
laboratory known to the site in addition to sending a sample to the central laboratory for 
testing.   
If the C. DIFF QUIK CHEK COMPLETE® Test is not available by the site or other 
laboratory, a testing algorithm that directly assesses the presence of C. difficile toxin for 
laboratory confirmation of recurrent CDI may also be used to test a sample, in addition 
to sending a sample to the central laboratory for testing. 
Specifically, if the antigen and toxin are both positive per the QCC Test during the 
presence of diarrhea then the subject is considered a treatment failure.   
Treatment Failure:   
 
 
 
 
 
However, if the subject experiences diarrhea and the antigen and toxin are both negative 
per the QCC Test, then the subject is not considered a treatment failure as the presence 
of C. difficile was not established.  The study investigator will need to determine the 
appropriate treatment for the subject as to avoid the possibility of serious adverse events.   
Treatment Success: 
 
 
 
 
 


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Finally, if the antigen and toxin results per the QCC Test are inconclusive (one is 
positive and the other is negative), the stool sample will be sent to a central lab to 
perform a PCR test.  If the PCR test is negative, the subject will not be considered a 
treatment failure as the presence of C. difficile was not established.  If the PCR test is 
positive, then the subject will be considered a treatment failure. 
 
 
Inconclusive Test, send sample for PCR testing: 
 
 
 
 
 
 
Finally, if the QCC Test provides an invalid result such that the control line is not 
visible, the test will need to be repeated.  
Invalid result, repeat testing immediately with new QCC Test: 
 
 
 
 
6.14. Unblinded RBX2660  
Subjects with a documented treatment failure meeting the protocol definition in the 
blinded portion of the study may be scheduled for administration of an unblinded 
RBX2660 enema.  In order to receive the unblinded RBX2660 enema, a subject must 
have documentation of the following: 
• Meeting all treatment failure criteria as defined by the protocol 
• Compliance to the washout period (discontinuation of antibiotics for 24-72 
hours prior to administration visit) is confirmed  
• Negative urine-dipstick pregnancy test for subject of childbearing 
potential to confirm that they are not pregnant on the date of 
administration 
• Unblinded enema administration is to occur within 21 calendar days of 
failure determination 
 


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While subjects may choose to receive and understand they are receiving an unblinded 
RBX2660 enema; neither they nor the study site will be unblinded to their original 
treatment.    
If antibiotics are given to control symptoms, a 24-72hr washout period prior to 
administration of an unblinded RBX2660 enema is required, however the unblinded 
enema must still be administered within 21 calendar days of failure determination.   
If a subject receives an unblinded RBX2660 enema, the follow-up requirements will 
restart from the day of administration of the unblinded RBX2660 enema according to 
the same schedule as required for the blinded portion of the study.  This results in 
completion of a new subject diary from the day of unblinded RBX2660 enema 
administration until the day prior to the 1-week visit; in-office visits at 1-week (see 
Section 6.8), 4- week (see Section 6.9) and 8-week (see Section 6.10) as well as 
telephone calls at 2, 3 and 6 weeks (see Section 6.11) and at 3 and 6 months (see Section 
6.12) after the unblinded RBX2660 treatment. 
The study will be considered complete when all subjects have reached their 6-month 
follow-up after the last study enema received, including unblinded enemas, or have 
exited, whichever is earlier.   
6.15. 
Study Exit 
A Study Exit eCRF will be completed for each subject who is randomized. Data to be 
collected at this visit includes exit date and reason for study exit. This visit may occur in 
conjunction with the 6-month Follow-Up visit for subjects who complete the study and 
do not have ongoing adverse events. 
The following information will be collected on the Study Exit eCRF: 
• Exit date 
• Reason for study exit 
o Completed Study 
o Screen Failure 
o Withdrawal by subject 
o Study Terminated by Sponsor 
o Investigator withdrawal 
o Adverse Event 
o Death  
o Lost to Follow-Up 
o Other: (specify) 
Additional data will be collected when the following occur: 
• Medication changes 
• Adverse Events 


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6.16. 
Subject Withdrawal or Termination 
A subject’s study participation is considered complete once the 6-month telephone call 
is conducted after starting the last course of study drug or the subject exits the study, 
whichever comes first.  A subject may withdraw at any time for any reason or be 
withdrawn from the study prematurely for the following reasons: 
• Withdrawal of consent by subject 
• Lost to follow-up 
• Failure to comply to study requirements 
• Termination of study by the sponsor 
• Death 
• Other (to be specified). 
 
The reason for withdrawal is recorded on the Study Exit eCRF.  In the event that a 
subject requests to withdraw from the study, every attempt should be made to have the 
subject return for an early withdrawal visit and to complete the required visit 
questionnaires prior to withdrawal.  
For a subject who is suspected of being lost-to-follow-up, a minimum of three attempts 
must be made to contact subjects using two different contact methods (e.g., telephone, 
email, text, and letter); one attempt must be a registered postal letter or traceable courier 
notice (e.g., FedEx Express) to the subject’s last known address. The contact attempts 
and methods are to be recorded on the Study Exit eCRF.  The date the subject is 
regarded as lost to follow-up is the date of last actual encounter with the subject, such as 
the last phone call contact or visit.   
Completion of a Study Exit eCRF is required for all randomized subjects when they exit 
the study regardless of reason, including completion of all study requirements, visits and 
assessments.  Subjects who were randomized but were determined to be screen failures 
or otherwise exited prior to receiving a dose of the study drug should also be 
documented on a Study Exit eCRF. 
 
7. Assessment of Safety  
7.1. Adverse Event Management 
Adverse events, which include clinical laboratory test variables, will be monitored and 
documented from the time of informed consent. Subjects should be instructed to report 
any adverse event that they experience to the investigator.  Investigators should make an 
assessment for adverse events at each visit and record the event on the appropriate 
eCRF. 
Wherever possible, a specific disease or syndrome rather than individual associated 
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However, if an observed or reported sign or symptom is not considered a component of 
a specific disease or syndrome by the investigator, it should be recorded as a separate 
adverse event on the eCRF.  Additionally, the condition that led to a medical or surgical 
procedure (e.g., surgery, endoscopy, tooth extraction, or transfusion) should be recorded 
as an adverse event, not the procedure.  
Any medical condition already present at screening should not be reported as an adverse 
event unless the medical condition or signs or symptoms present at baseline changes in 
severity or seriousness at any time during the study.  In this case, it should be reported as 
an adverse event. 
Clinically significant abnormal laboratory or other examination (e.g. electrocardiogram) 
findings that are detected during the study or are present at screening and significantly 
worsen during the study should be reported as AEs.  The investigator will exercise his or 
her medical and scientific judgment in deciding whether an abnormal laboratory finding 
or other abnormal assessment is clinically significant. Clinically significant abnormal 
laboratory values occurring during the clinical study will be followed until repeat tests 
return to normal, stabilize, or are no longer clinically significant.  Any abnormal test that 
is determined to be an error does not require reporting as an AE. 
Recurrence of CDI is not to be reported as an AE unless hospitalization ≥ 24 hours is 
required to treat it.  If a hospitalization of ≥ 24 hours is not required, it is to be recorded 
on the Follow-up eCRF and applicable CDI Form which will be reviewed as a 
component of the efficacy assessment.  If hospitalization ≥ 24 hours is required, it is to 
be reported as an SAE. 
Adverse events should be followed until the event resolves or the subject exits the study.  
Events lasting > 3 months are considered to be chronic in nature.  
7.2. Definitions 

Adverse event:  an adverse event (AE) is any untoward medical occurrence in
a clinical investigation subject associated with the use of IP, which does not
necessarily have a causal relationship with the treatment.  An adverse event
can therefore be any unfavorable and/or unintended sign (including an
abnormal laboratory finding), symptom, or disease temporally associated with
the use of IP.
o
All adverse events, including observed or volunteered problems,
complaints, or symptoms are to be recorded on the eCRF within 10
days of site awareness.

Adverse reaction:   All noxious and unintended responses to IP should be
considered an adverse reaction. “Responses” to IP means that a causal


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relationship between IP and an adverse event is at least a reasonable 
possibility, i.e. the relationship cannot be ruled out.   

Serious adverse event (SAE): An adverse event or adverse reaction is
considered serious if, in the view of either the investigator, Medical Monitor
or sponsor, it results in any of the following outcomes:
o
Death
o
Life-threatening adverse event
NOTE: An adverse event or adverse reaction is considered “life-
threatening” if, in view of either the investigator or Sponsor, its
occurrence places the subject at immediate risk of death.  It does not
include an event that, had it occurred in a more severe form, might have
caused death.
o
Hospitalization ≥ 24 hours or prolongation of an existing hospitalization
NOTE: Any hospital admission with at least one overnight stay will be
considered an inpatient hospitalization.  An emergency room visit without
hospital admission will not be recorded as a serious adverse event (SAE)
under this criterion, nor will hospitalization for a procedure scheduled or
planned before signing of informed consent.  However, unexpected
complications and/or prolongation of hospitalization that occur during
elective surgery should be recorded as AEs and assessed for seriousness.
Admission to the hospital for social or situational reasons (i.e., no place to
stay, live too far away to come for hospital visits) will not be considered
inpatient hospitalizations.
o
Persistent or significant incapacity or substantial disruption of the ability
to conduct normal life functions
o
Congenital anomaly/birth defect.
o
Important medical event
NOTE: Important medical events that may not result in death, be life-
threatening, or do require hospitalization may be considered serious
when, based upon appropriate medical judgment, they may jeopardize the
subject and may require medical or surgical intervention to prevent one of
the outcomes above.  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
hospitalizations, or the development of drug dependency.

Unexpected Adverse Reaction: An adverse reaction, the nature or severity of
which is not consistent with the applicable product information.

Unexpected adverse event or unexpected suspected adverse reaction: An
adverse event or suspected adverse reaction is considered “unexpected” if it is
not listed in the Investigator’s Brochure or is not listed at the specificity or
severity that has been observed.
7.3. Anticipated Adverse Events 


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The following is list of anticipated adverse events that may or may not be causally 
related to IP, the enema procedure, or CDI:  
 gas (flatulence) 
 belching 
 abdominal distension or bloating 
 increased diarrhea 
 abdominal cramping or pain 
 constipation 
 colitis 
 fever ≥ 37.8° C (100.0°F) 
 fatigue 
 chills 
 transmission of disease from the donor to recipient 
 rectal irritation or pain 
 rectal bleeding 
 nausea 
 vomiting 
 hypotension 
 puncture of the intestine 
7.4. Solicited Events 
The following list of anticipated events are solicited from subjects via the Subject Diary:   
 gas (flatulence) 
 abdominal distension or bloating 
 rectal irritation or pain 
 chills/severe shivering 
 abdominal pain or cramping 
 increased diarrhea 
 constipation 
 rectal bleeding 
 nausea 
 vomiting 
 fever ≥ 37.8° C (100.0°F) 
Through the completion of the Subject Diary, subjects are asked specific questions 
regarding frequency and severity of the solicited AEs.   
 
7.5. Preexisting Condition 
A preexisting condition, including abnormal physical exam findings, is one that is 
present at the start of the study and is to be recorded at the time of the screening visit.  A 
preexisting condition is only to be recorded as an adverse event if the frequency, 
intensity or the character of the condition worsens during study participation. 
 


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7.6. Serious Adverse Event Reporting – Procedures for Investigators 
7.6.1. Initial Reports 
All SAEs occurring from the time of informed consent until 6 months after 
administration of the last study enema of IP must be reported to Medpace Clinical Safety 
within 24 hours of the knowledge of the occurrence (this refers to any adverse event 
that meets any of the aforementioned serious criteria).  All serious adverse events that 
the investigator considers related to study drug occurring after 6 months after 
administration of the last enema of IP must be reported to the Sponsor. 
To report the SAE, complete the SAE form electronically in the electronic data capture 
(EDC) system for the study.  When the form is completed, Medpace Safety personnel 
will be notified electronically and will retrieve the form.  If the event meets serious 
criteria and it is not possible to access the EDC system, send an email to Medpace 
Safety at medpace-safetynotification@medpace.com or call the Medpace SAE hotline 
(phone number listed below), and fax the completed paper SAE form to Medpace (fax 
number listed below) within 24 hours of awareness.  When the EDC system becomes 
available, the SAE information must be entered within 24 hours of the system becoming 
available. 
 
Safety Contact Information: 
Medpace Clinical Safety 
 
Medpace SAE hotline – USA: 
 
 
 
 
7.6.2. Follow-Up Reports 
The investigator must continue to follow the subject until the SAE has subsided or until 
the condition becomes chronic in nature, stabilizes (in the case of persistent impairment) 
or the subject dies.  Chronic is defined as a condition lasting >3 months.  
Within 24 hours of receipt of follow-up information, the investigator must update the 
SAE form electronically in the EDC system for the study and submit any supporting 
documentation (e.g., subject discharge summary or autopsy reports) to Medpace Clinical 
Safety via fax or e-mail.  If it is not possible to access the EDC system, refer to the 
procedures outlined above for initial reporting of SAEs.  
 


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7.7. Grading Event Severity 
Identified, subject reported and unsolicited adverse events are to be graded, according to 
severity, by the site investigator.  For this classification of events, severity is not the 
same as seriousness, which is defined in Section 7.1.  Severity is an indication of the 
intensity or a specific event (e.g., mild, moderate or severe).  Classification of an event 
as serious relates to an event’s outcome or intervention criteria and is usually associated 
with events that pose a threat to a subject’s life or functioning.  An event can be severe 
but not serious, such as a migraine.   
Using the definitions in Table 7-1, the site investigator will categorize the severity of a 
solicited or unsolicited event on the AE eCRF.  If a particular event is not listed on this 
table, the NIH/NCI Common Terminology Criteria for Adverse Events found at online at 
the following address may be utilized.  
https://ctep.cancer.gov/protocolDevelopment/electronic_applications/docs/CTCAE_v5_
Quick_Reference_8.5x11.pdf  
Table 7-1:  Severity Grading Table* 
 
Parameter 
Grade1: Mild 
Grade 2: 
Moderate 
Grade 3: Severe 
Grade 4: 
Potentially 
Life-
threatening 
Flatulence 
(gas) 
Symptoms causing 
no or minimal 
interference with 
usual social and 
functional activities  
Symptoms causing 
greater than 
minimal 
interference with 
usual social and 
functional activities  
NA 
 
NA 
Belching 
(burping) 
Symptoms causing 
no or minimal 
interference with 
usual social and 
functional activities  
Symptoms causing 
greater than 
minimal 
interference with 
usual social and 
functional activities  
 
NA 
 
NA 
Abdominal 
distension or 
bloating 
Symptoms causing 
no or minimal 
interference with 
usual social and 
functional activities  
Symptoms causing 
greater than 
minimal 
interference with 
usual social and 
functional activities  
 
Symptoms causing 
inability to perform 
usual social and 
functional activities  
 
NA 
Increased 
diarrhea 
Increase of ≤ 3 
stools over baseline 
per 24-hour period  
 
Increase of 4 – 6 
stools over baseline 
per 24-hour period  
 
Bloody diarrhea if 
not present at 
baseline OR increase 
of ≥ 7 stools over 
baseline per 24-hour 
Life-threatening 
consequences, 
e.g., hypotensive 
shock 
 


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Parameter 
Grade1: Mild 
Grade 2: 
Moderate 
Grade 3: Severe 
Grade 4: 
Potentially 
Life-
threatening 
period OR IV fluid 
replacement 
indicated if not 
indicated at baseline 
Abdominal 
cramping/pain 
Discomfort/pain 
causing no or 
minimal interference 
with usual social 
and functional 
activities  
 
Discomfort/pain 
causing greater 
than minimal 
interference with 
usual social and 
functional activities  
Discomfort/pain 
causing inability to 
perform usual social 
and functional 
activities  
 
Disabling pain 
causing inability 
to perform basic 
self-care OR 
inpatient 
hospitalization ≥ 
24 hours 
Constipation 
Occasional or 
intermittent 
symptoms, 
occasional use of 
stool softeners, 
laxatives, dietary 
modifications or 
enema 
Persistent 
symptoms with 
regular use of 
laxatives or enemas 
indicated 
Symptoms causing 
inability to perform 
usual social and/or 
functional activities 
Life-threatening 
consequences, 
e.g., obstruction, 
toxic megacolon 
Colitis 
No symptoms, 
regardless of 
pathologic or 
radiographic 
evidence of 
inflammation 
Abdominal pain, 
mucus or blood in 
the stool 
Abdominal pain, 
fever, change in 
bowel habits with 
ileus; peritoneal 
signs 
Life-threatening 
consequences, 
e.g., perforation, 
bleeding, 
ischemia, 
necrosis, toxic 
megacolon 
Fever 
37.8 – 38.6°C 
(100.0 – 101.5° F)  
38.7 – 39.3°C 
(101.6 – 102.8° F)   
39.4 – 40.5°C 
(102.9 – 104.9° F)  
> 40.5°C 
(104.9° F)  
Fatigue/malaise 
Symptoms causing 
no or minimal 
interference with 
usual social and 
functional activities  
Symptoms causing 
greater than 
minimal 
interference with 
usual social and 
functional activities  
Symptoms causing 
inability to perform 
usual social and 
functional activities  
Incapacitating 
fatigue/malaise 
symptoms causing 
inability to 
perform basic 
self-care functions 
Chills 
Symptoms causing 
no or minimal 
interference with 
usual social and 
functional activities  
Symptoms causing 
greater than 
minimal 
interference with 
usual social and 
functional activities  
Symptoms causing 
inability to perform 
usual social and 
functional activities  
NA 
Rectal 
discomfort or 
irritation 
No symptoms or 
symptoms not 
Symptomatic with 
medical 
intervention 
Symptoms causing 
inability to perform 
usual social and 
NA 


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Parameter 
Grade1: Mild 
Grade 2: 
Moderate 
Grade 3: Severe 
Grade 4: 
Potentially 
Life-
threatening 
requiring medical 
intervention 
(topical 
medications / 
treatments) 
indicated 
functional activities 
or requiring medical 
intervention other 
than topical 
medications / 
treatments 
Rectal bleeding 
Mild or intermittent 
without transfusion 
Persistent without 
transfusion 
Requires transfusion 
Life-threatening 
consequences 
Nausea 
Transient (≤ 24 
hours) or 
intermittent nausea 
with nor or minimal 
interference with 
oral intake 
Persistent nausea 
resulting in 
decreased intake 
for 24-48 hours 
Persistent nausea 
resulting in 
decreased intake > 
48 hours OR 
aggressive 
rehydration 
indicated, e.g., IV 
fluids 
Life-threatening 
consequence, e.g., 
hypotensive shock 
Vomiting 
Transient (≤ 24 
hours) or 
intermittent 
vomiting with nor or 
minimal interference 
with oral intake 
Frequent episodes 
of vomiting with 
no or mild 
dehydration 
Persistent vomiting 
resulting in 
orthostatic 
hypotension OR 
aggressive 
rehydration 
indicated, e.g., IV 
fluids  
Life-threatening 
consequence, e.g., 
hypotensive shock 
Hypotension 
NA 
Symptomatic, 
corrected with oral 
fluid replacement 
 
Symptomatic, IV 
fluids indicated 
 
Shock requiring 
use of 
vasopressors or 
mechanical 
assistance to 
maintain blood 
pressure 
 
Adverse event 
not identified 
elsewhere in 
this table 
Symptoms causing 
no or minimal 
interference with 
usual social and 
functional activities  
Symptoms causing 
greater than 
minimal 
interference with 
usual social and 
functional activities  
Symptoms causing 
inability to perform 
usual social and 
functional activities  
Symptoms 
causing inability 
to perform basic 
self-care functions 
OR medical or 
operative 
intervention 
indicated to 
prevent permanent 
impairment, 


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Parameter 
Grade1: Mild 
Grade 2: 
Moderate 
Grade 3: Severe 
Grade 4: 
Potentially 
Life-
threatening 
persistent 
disability, or death  
*Adapted from Division of AIDS Table for Grading of Severity of Adult and 
Pediatric Adverse Events and Addendum 3: Rectal Grading Table for Use in 
Microbicide Studies; May 2012. 
7.8. Causality 
For all adverse events, the investigator must pursue and obtain adequate information to 
determine the outcome of the adverse event and to assess whether the AE meets the 
criteria for classification as an SAE, serious suspected adverse reaction, suspected 
adverse reaction, unexpected adverse event, or unexpected suspected adverse reaction.  
For all adverse events, sufficient information should be obtained by the investigator to 
determine the causality of the adverse event.  For adverse events with a causal 
relationship to the IP or the enema procedure, follow-up by the investigator is required 
until the event or its sequelae resolve or stabilize at a level acceptable to the investigator. 
The Investigator will make a causality assessment for all AEs and decide whether there 
is a reasonable possibility that the AE may have been caused by the study product or 
procedure, including an assessment of biologic plausibility, presence or absence of 
alternative causal explanations (such as continuation or exacerbation of the subject’s 
recurrent CDI symptoms), and temporal relationship to product administration and/or 
the procedure.  Relatedness to RBX2660 or the enema procedure is defined as: 
 
Unrelated: The event is due to an underlying or concurrent illness or effect of 
concomitant therapy and is not related to the study product or procedure (e.g., 
has no temporal relationship to study product or procedure, or has a much more 
likely alternative etiology) 
Possible: There is some temporal relationship between the event and the 
administration of the study product or procedure, though the event could also be 
explained by the subject’s medical condition or other therapies. 
Probable: The temporal relationship between the event and administration of the 
study product or procedure is suggestive, and the event is unlikely to be 
explained by the subjects’ medical condition or other therapies alone.  
Definite: The event follows a reasonable temporal sequence from administration 
of the study product or procedure, follows a known or suspected response pattern 
to the study product and/or procedure, improves upon stopping the study 
product, and reappears upon repeated exposure, if that occurs. 
 


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If an investigator is unsure about whether to report a finding as an adverse event, s/he is 
encouraged to enter the finding as an AE into the study database.   
 
7.9. Pregnancy Reporting 
If the subject or partner of a subject participating in the study becomes pregnant during 
the study after IP administration, the investigator should report the pregnancy to 
Medpace Clinical Safety within 24 hours of being notified.  Medpace Clinical Safety 
will then forward the Exposure In Utero form to the investigator for completion.  
The subject or partner should be followed by the investigator until completion of the 
pregnancy.  If the pregnancy ends for any reason before the anticipated date, the 
investigator should notify Medpace Clinical Safety.  At the completion of the pregnancy, 
the investigator will document the outcome of the pregnancy.  If the outcome of the 
pregnancy meets the criteria for immediate classification as an SAE (i.e., postpartum 
complication, spontaneous abortion, stillbirth, neonatal death, or congenital anomaly), 
the investigator should follow the procedures for reporting an SAE. 
 
7.10. Medical Monitor 
An independent Medical Monitor reviews all serious adverse events and other events 
reported by the site as related to the investigational product or enema procedure to 
provide an objective, qualified judgment of the events.  The Medical Monitor is a 
physician not participating as an investigator in this study.  The Medical Monitor has the 
responsibility to review and evaluate the information relevant to product safety 
throughout the development and implementation of the protocol.  The Medical Monitor 
will remain blinded throughout the study.  The Medical Monitor performs the following 
functions: 
 Reviews SAEs and other adverse events as requested; 
 Reviews the study protocol and Investigator’s Brochure for adequacy of 
safety oversight; 
 Confers with site investigators, DSMB and the Rebiotix Clinical 
Department as applicable regarding potential safety concerns; 
 Provides medical surveillance and provides appropriate recommendations 
for the conduct of the study as needed. 
 
Details of the Medical monitor activities and expectations will be documented in a 
Safety Management Plan. 
 
 


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7.11. Data and Safety Monitoring Board (DSMB) 
The DSMB consists of three physicians specializing in infectious diseases or 
gastroenterology who have experience managing subjects with recurrent CDI and are not 
investigators in the study. A biostatistician who is not involved with study design or 
analyses may assist the DSMB.  The DSMB will operate in accordance with applicable 
sections of the FDA Guidance for Clinical Trial Sponsors Establishment and Operation 
of Clinical Trial Data Monitoring Committees.   
Details of the DSMB logistics, activity and responsibilities will be included in a DSMB 
charter.  General responsibilities include oversight of subject safety through data review 
and assessment of trends.  This includes the ability to stop the study based on pre-
defined stopping rules.  Additionally, the DSMB will provide an independent review of 
the efficacy data to ensure the benefits of providing this treatment to subjects outweighs 
the risks.   
 
7.12. Study Stopping Rules for Safety 
Medpace Safety personnel or designee will review all adverse events reported during the 
study to observe for trends and unanticipated events, either in severity, seriousness or 
incidence, and to assess if a study stopping rule may be triggered.  Adverse event review 
includes the event description, onset date in relation to the treatment date, and the 
investigator’s determination of causal relatedness to IP, the enema procedure, and pre-
existing condition, as well as rating the event’s severity and seriousness.  Additionally, 
the Medical Monitor assessment of seriousness and causality will be provided and 
reviewed as it may be different from the site investigator report of the event.  If 
necessary, the DSMB may be unblinded to adjudicate specific adverse events as per the 
study stopping rules, below.  Anticipated AEs may trigger the study stopping rules. 
The DSMB will determine whether enrollment should be paused, the study terminated, 
or other actions taken based on their assessment that: 
a) There is probable cause that IP or enema procedure (e.g., due to transfer 
from an RBX2660 donor) contributed to a pathogenic intestinal infection in 
the stool of any subject, or 
 
b) Any series of events of major significance such as death or other serious 
outcome for which a causal connection with the IP is plausible represents 
an excess of the important adverse event(s) in one of the study arms. 
 
To support the DSMB assessment, the Rebiotix Operations Department may be asked to 
review the donor history and batch processing/release records for the product unit(s) 
administered in these cases, and forward its report to the DSMB Chair for review.  The 
DSMB Chair will review the Operations Department report and may be unblinded to the 


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randomization assignment.  If probable cause is suspected, the DSMB Chair will 
convene the entire DSMB for event review.   
Upon the DSMB’s determination of action, the Chair will notify the Rebiotix Clinical 
Department, who will notify the study sites.  
If enrollment is stopped, Rebiotix and the study sites will assess for the occurrence of 
similar events, and evaluate IP and study records for possible root cause(s).  Enrollment 
may be re-started upon further review and approval from the DSMB.  As appropriate, 
the DSMB may recommend study termination or measures short of termination with the 
objective of reducing the risk of adverse events.   
 
7.13. Sponsor Reporting of Adverse Events 
The sponsor will report all relevant information about suspected unexpected serious 
adverse reactions that are fatal or life-threatening as soon as possible to the FDA & 
Health Canada, and in any case no later than seven days after knowledge by the sponsor 
of such a case, and that relevant follow-up information will subsequently be 
communicated within an additional eight days.  
All other suspected unexpected serious adverse reactions will be reported to the FDA as 
soon as possible but within a maximum of 15 days of first knowledge by the sponsor.  
The sponsor will also inform all investigators as required.  
 
7.13.1. Timeline for Reporting Requirements 
The following describes the expedited safety reporting requirements by timeline for 
reporting and associated type of event: 
 
 Within 7 calendar days of event discovery by Rebiotix 
Rebiotix will notify FDA and Health Canada of any unexpected fatal or life-
threatening suspected adverse reaction as soon as possible but in no case later 
than seven calendar days after the sponsor's initial receipt of the information.  
 
 Within 15 calendar days of event discovery by Rebiotix  
The AE must meet all three of these criteria for expedited reporting. 
Any study event that is: 
o suspected adverse reaction 
o serious 
o unexpected 
 


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7.13.2. Additional Reporting Requirements 
The sponsor will identify in IND safety reports all previous reports concerning similar 
adverse events and analyze the significance of the current event in light of the previous 
reports. 
 
7.13.3. Reporting AEs to FDA and Health Canada 
Rebiotix will report adverse events to FDA and Health Canada as required by applicable 
regulations.  All adverse events will be reported in the annual and final clinical study 
reports.   
 
 
 
 
 
8. Sample Size and Statistical Considerations 
This study has a Statistical Analysis Plan which contains complete details of the 
statistical methodology and analyses that will be employed for this study.  
 
8.1. Assessment of Efficacy 
The primary efficacy parameter is treatment success, defined as the absence of CDI 
diarrhea through 8 weeks after completing a study treatment.  Efficacy assessments will 
occur at the 1-, 4- and 8-week office visits. 
Recurrence of CDI after 8 weeks from completion of the assigned study treatment is 
considered a new C. difficile infection, as defined by the Centers for Disease Control 
(CDC website http://www.cdc.gov/hai/eip/cdiff_techinfo.html; published August 30, 
2012; accessed October 02, 2016).   
Primary Efficacy Endpoint:  Recurrence of CDI within 8 weeks of blinded treatment. 
 
The primary efficacy analysis will be performed on all pre-defined analysis populations 
comparing the RBX2660 arm to the Placebo arm, Intent-to-Treat (ITT), modified Intent-
to-Treat (mITT) and Per-Protocol (PP).  However, the determination of meeting the pre-
defined primary efficacy endpoint will utilize the mITT population results.  The primary 
analysis of the study will be a Bayesian hierarchical model, which formally incorporates 
data from a previous randomized Phase 2b study (Protocol 2014-01) of RBX2660. This 
analysis will demonstrate the efficacy of RBX2660 by showing a sufficiently high 
posterior probability that the rate of success in the treatment group is superior to the rate 
of success in the control group.   Secondary efficacy endpoints will also be completed on 
all analysis populations.  Finally, all safety analyses will be performed on the Safety 


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population (SP) which includes all subjects in whom a blinded enema was at least 
opened and attempted.   
A primary efficacy and safety analysis for primary efficacy and safety will be conducted 
when 100% of the applicable subjects in each group have completed their 8-week 
follow-up assessment after receiving the blinded treatment.  Safety analysis will be 
completed for safety endpoints defined at or prior to 8 weeks following blinded 
treatment along with any longer term safety data available at the time of analysis.  The 
analyses will be submitted to FDA for review. One or more interim analyses will be 
conducted to evaluate the primary efficacy endpoint for possible early study stopping for 
success or futility.  The first interim analysis will occur after a minimum of 160 subjects 
have been treated and evaluated for efficacy.  A second interim analysis could occur 
when 220 subjects have been treated and evaluated for efficacy if success is not 
achieved at the first interim analysis.  The details of the planned interim analyses are 
included in the statistical analysis plan.   
Final long-term safety results as well as unblinded RBX2660 efficacy data will be 
analyzed and reported in a final report after the last subject has completed the 6-month 
follow-up visit for their study treatment. This includes 6-month follow-up after an 
unblinded RBX2660 study treatment. At that time the study will be considered 
complete.   
To demonstrate a 69% success with one dose of the RBX2660 treatment group vs. 47% 
success in the Placebo control group in a 2:1 randomization ratio, 240 subjects are 
required (power ≥90% with a nominal 2.5% type I error rate).  Up to an additional 30 
subjects will be enrolled to allow for a 10% loss-to-follow up rate, for a total of up to 
270 subjects.  Considering the 2:1 randomization, this results in approximately 180 
subjects in the RBX2660 arm and 90 subjects in the Placebo arm.  Potential subjects 
who have signed consent but do not meet all inclusion and/or exclusion criteria will be 
considered screen failures.  Randomized subjects who withdraw for any reason prior to 
administration of the blinded enema will be replaced without counting toward the 
sample size cap which may result in more than 270 subjects being randomized, however 
no more than 270 subjects will be treated with a blinded enema.  Replacement subjects 
will also be randomized to ensure proper blinding.  
Subjects who are exited prior to randomization or randomized but exited prior to 
treatment may be re-enrolled and thus randomized under different subject identification 
numbers (re-randomized).  If this occurs, they will be included in the analysis using their 
second randomization assignment and subsequent assessments only and will only be 
counted once towards the total randomized. 
 


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9. Study Administration 
9.1. Institutional Review Board / Research Ethics Board Approval 
The protocol, Informed Consent Form/HIPAA Form must be reviewed and approved by 
the respective IRB/REB and Rebiotix before subject recruitment and enrollment begins.   
Prior to subject enrollment, a signed copy of the IRB/REB approval letter addressed to 
the investigator and a full copy of the IRB/REB-approved Informed Consent 
Form/HIPAA Form must be submitted to Rebiotix, certifying trial approval. 
Investigators are responsible for submitting and obtaining initial approval and 
continuing approval from the IRB/REC and forwarding copies of the approval letters to 
Rebiotix.  The approval letters are to be kept in the Trial Master File (TMF) designated 
for this study.   
US Sites 
Investigators are responsible for submitting and obtaining initial approval and 
continuing approval from the IRB and forwarding copies of the approval letters to 
Rebiotix.  The approval letters are to be kept in the Trial Master File (TMF) designated 
for this study.   
The investigator will notify the Rebiotix study manager within five (5) business days of 
withdrawal of IRB approval.  
Institutional Review Boards will operate in accordance to 21 CFR 56 and their own 
standard operating procedures. 
Canadian Sites 
Prior to subject enrollment, a signed copy of the REB approval letter addressed to the 
investigator, a full copy of the REB-approved Informed Consent Form, and completed 
Research Ethics Board Attestation must be submitted to Mapi Group, the Agent for 
Rebiotix in Canada, and to Rebiotix.  Investigators are responsible for submitting and 
obtaining initial approval and continuing approval from the REB and forwarding copies 
of the approval letters to Mapi Group and Rebiotix.  The approval letters are to be kept 
in the Trial Master File (TMF) designated for this study.   
The investigator will notify Mapi Group and Rebiotix within five (5) business days of 
withdrawal of REB approval.  
Research Ethics Boards will operate in compliance with Health Canada regulations and 
their own standard operating procedures. 
 
9.2. Form 1572 and Financial Disclosure 
The principal investigator at each site (US and Canada) will complete and return a 
study-specific Form 1572 to the sponsor before beginning the study, as required by 


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federal regulations.  In the event of a change in study personnel, the site will complete 
and submit a new Form 1572 to Rebiotix within 60 days of the change. The investigator 
agrees to be responsible for conducting the investigational study in accordance with the 
protocol, applicable FDA regulations including reporting and record-keeping 
requirements, GCPs, local IRB/REB requirements, and controlling dispensation and 
administration of RBX2660 and placebo.  In addition, the investigator is responsible for 
ensuring that informed consent is obtained from each subject prior to participating in the 
study, as well as protecting the rights, safety and welfare of participating subjects. 
All investigators will be required to sign a Financial Disclosure form, which certifies the 
investigator’s and his/her immediate family’s financial interest in Rebiotix and study 
outcomes.  Investigators must inform Rebiotix of any changes to the information 
documented on the Financial Disclosure form throughout the course of the study and for 
a period of one year following completion of the study. 
 
9.2.1. Canadian Sites:  Qualified Investigator Undertaking Form 
The principal investigator(s) at each Canada site will complete and return the Qualified 
Investigator Undertaking Form to Mapi Group before beginning the study, as required 
Health Canada.  The investigator agrees to be responsible for conducting the 
investigational study in accordance with the protocol, applicable Health Canada 
regulations including reporting and record-keeping requirements, GCPs, local REB 
requirements, and controlling dispensation and administration of RBX2660 and placebo.  
In addition, the investigator is responsible for ensuring that informed consent is obtained 
from each subject prior to participating in the study, as well as protecting the rights, 
safety and welfare of participating subjects. 
 
9.3. Subject Confidentiality 
All information and data sent to Rebiotix, and/or its designees concerning subjects and 
their participation in this study are considered confidential by Rebiotix and its designees 
(subcontractors or contract research organization).  Only authorized Rebiotix personnel 
or approved contracted agents of Rebiotix have access to some portions of these 
confidential files and will act in accordance with applicable regulations.  The IRBs, 
REBs, FDA and Health Canada also have the right to inspect and copy all records 
pertinent to this study.  All data used in the reporting of the study will eliminate 
identifiable references to the subjects as much as possible.   
 
9.4. Study Oversight 
Clinical personnel at Rebiotix have knowledge of Good Clinical Practices, pertinent 
laws and regulations, and documented training in standard operating procedures 


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pertaining to study management and monitoring and will provide study oversight.  
Rebiotix can be contacted at; 
 
Rebiotix Inc 
2660 Patton Road 
Roseville, MN 55113 
Phone: 651-705-8770 
 
9.5. Study Site Qualification 
Investigational center qualification visits or phone calls will be conducted by Rebiotix 
prior to acceptance of a site into this study.  The site qualification visit or phone call will 
be scheduled to include time with the study investigator, co-investigators, study 
coordinator and other study personnel.  Areas of discussion include a review of 
personnel training, investigator qualifications, IP Administrator qualifications, adequacy 
of potential subject pool, FDA- or Health Canada-regulated study experience, this 
study’s specific requirements for procedures and equipment, and a review of staffing 
availability and appropriateness.  A written report of the qualification call/visit will be 
generated by the sponsor representative who conducts the call/visit.  Resolution of any 
concerns and/or completion of any appropriate study activities identified during the 
qualification process will be documented and submitted to the study investigator.   
 
9.6. Investigator / Site Training 
The sponsor will provide appropriate training to each investigator, and IP Administrator, 
study coordinator(s), and other site personnel who will be involved in the study prior to 
study initiation.  Training will address topics including ordering, secure storage and 
administration procedures, Subject Diary instructions, follow-up visit and phone call 
requirements, adverse event reporting, and accurate data collection. Training will 
include a detailed review of the protocol, eCRF completion, study-specific procedures, 
monitoring logistics, and regulatory requirements.  
 
9.7. Data Management 
Electronic case report forms specifically created for this study will be used to collect 
study data.  The study investigator or his/her designee at each site is responsible for 
recording all data onto the study eCRFs as noted on the Delegation of Authority Log.  
The investigator must review and electronically sign all eCRFs as instructed; these 
responsibilities cannot be delegated to another person.  Ongoing data review will be 
performed according to the study-specific Data Management Plan. 
 


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9.8. Monitoring 
This study is monitored according to the study-specific Clinical Monitoring Plan.  The 
investigator must allocate adequate time for such monitoring activities.  The investigator 
must also ensure that the monitor or other compliance or quality assurance reviewers are 
given access to all study-related documents and study-related facilities (e.g. pharmacy, 
diagnostic laboratory, etc.), and has adequate space to conduct the monitoring visit.  
Study monitors and their activities are managed by the study manager or qualified 
designee; see Section 9.4.  Study monitors will follow requirements as documented in 
monitoring procedures and the study-specific Monitoring Plan. 
 
9.9. Direct Access to Source Data/Documents 
The investigator is expected to facilitate study-related monitoring, audits, and 
inspections by the IRB/REB, sponsor and sponsor representatives, FDA, and Health 
Canada of all study-related documents including direct access to original source 
documents such as medical records and lab results, regulatory documents, study data, 
etc.  The investigator will ensure access for the inspection of applicable study-related 
facilities, e.g., pharmacy, laboratory, exam rooms, etc. 
Participation as a study investigator and study site in this study implies acceptance and 
support of inspections by FDA, Health Canada and/or Rebiotix or its designee(s). 
 
9.10. Investigator Responsibilities 
The investigator is responsible for ensuring that the study is conducted according to the 
protocol, applicable FDA or Health Canada regulations for investigational new drugs, 
HIPAA (US only), GCPs and local IRB/REB requirements.  Specific responsibilities are 
listed in this protocol.  
Site study records and reports are kept in the Investigator Study File (ISF).  Records and 
reports will remain on file for a minimum of two years (US sites) or 25 years (Canada 
sites) after either the completion/termination of this study or the date RBX2660 receives 
market approval for the indication being studied, whichever is later.  The study 
investigator must contact Rebiotix before destroying any records and reports pertaining 
to the study to ensure that they no longer need to be retained.  Rebiotix must be 
contacted if the study investigator plans to leave the site to ensure that arrangements for 
a new investigator or records transfer are made prior to investigator departure.  
The investigator will promptly report to the IRB/REB all changes in the research activity 
and all unanticipated problems involving risk to subjects or others, and that he/she will 
not make any changes in the research without prior written approval from the sponsor 
and IRB.  The investigator will adhere to all IRB/REB requirements imposed on this 
study. 


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9.11. Investigator Records  
Records to be maintained by the investigator in the ISF include: 
 Protocol and all amendments 
 Signed Form 1572  
 Signed Financial Disclosure Form(s)  
 IRB approval letter including consent and HIPAA (US sites) 
 IRB Membership list or Letter of Assurance (US sites) 
 Signed Qualified Investigator Undertaking Form (Canadian sites) 
 REB approval letter including the Informed Consent Form (Canadian sites) 
 REB Attestation (Canadian sites) 
 REB membership list or Letter of Assurance (Canadian sites) 
 All correspondence relating to the study between the 
site/investigator/coordinator and the IRB/REB, sponsor, CRO, and Mapi 
Group, as applicable. 
 CVs and professional licenses for all investigators and key study personnel 
 Delegation of Authority Log/Site Signature Log 
 Product Accountability Log (or electronic pharmacy equivalent) 
 Study Visitor Sign-in Log 
 Subject Screening/Enrollment log 
 Reports submitted to Rebiotix and/or the IRB/REB 
 
The following records must be maintained for each subject enrolled in the study: 
 Signed Informed Consent/local privacy authorization as applicable 
 Compete, accurate and current eCRFs  
 Adverse event reports and any supporting documentation 
 Protocol deviations 
 Complete medical records, including procedure reports, lab reports, 
professional notes, etc. 
 Records pertain to subject death during the investigation (including death 
records, death certificate, and autopsy report if performed). 
 
Rebiotix reserves the right to secure data clarification and additional medical 
documentation on subjects enrolled in this study at any time.  
 
9.12. Investigator Reports 
Study investigators are required to submit the following reports at the times specified 
below: 
 Adverse Events:  report all AEs to the sponsor via eCRF within ten (10) 
business days of discovery. 
 Serious Adverse Events and suspected adverse reactions:  report to the sponsor 
via eCRF within 24 hours of discovery. 


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 Progress and Final reports:  the investigator will submit annual study progress 
reports and a final study report when the study is terminated at the site to 
Rebiotix and the IRB/REB. 
9.13. Study Site Termination 
Rebiotix reserves the right to terminate a study site for any of the following reasons: 
 Failure to properly secure subject informed consent or HIPAA Authorization 
prior to study enrollment or the conduct of any study-required 
procedures/assessments. 
 Failure to report adverse events as required in Section 6.15. 
 Protocol deviations. 
 Repeated failure to appropriately and accurately complete eCRFs. 
 Failure to enroll an adequate number of subjects. 
 Loss of or unaccounted for product inventory. 
 Administrative decision by the company. 
 
9.14. Protocol Amendments 
Neither Rebiotix, its designees (subcontractors or contract research organization) nor the 
study investigators may modify this protocol without obtaining written approval of the 
FDA, Health Canada, and/or IRB/REBs as required.  No modifications may be made 
without prior written approval of Rebiotix. 
 
9.15. Protocol Deviations 
Any deviations from this protocol undertaken to protect the life or physical well-being 
of a subject in an emergency situation must be reported to Rebiotix within 48 hours of 
occurrence and to the respective IRB/REB as soon as possible, but in no event no later 
than five calendar days after the emergency occurs.  Protocol deviations of any kind 
must be avoided as much as possible and those that do occur will be tracked in the 
clinical study database. 
 
9.16. Inspection/Auditing 
The investigator will permit study-related inspection/auditing by the IRB/REC, sponsor, 
FDA and/or Health Canada to ensure compliance to applicable regulations, GCPs, and 
the study protocol. 
 
 
 
 
 


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10. Publication Plan 
 
Rebiotix has unrestricted publication rights of the study data.  The study site and the 
investigator are not entitled to make any publication or release any information pertaining 
to this study or its results without the prior written consent of Rebiotix.  The decision as 
to whether to provide such consent shall be made once Rebiotix has had an opportunity to 
review the contents of any proposed publication or release regarding the investigation 
and, if necessary, to delay any publication or release in order to protect the confidential or 
proprietary nature of any information contained therein.  Merely the fact that the 
proposed publication or release contains statements unfavorable to Rebiotix shall not 
constitute grounds for prohibiting publication; however, all unfavorable statements must 
be based on adequate scientific evidence.  Rebiotix reserves the right to give the data to 
third parties for publication or release and to name co-authors.  Rebiotix retains the right 
to review and edit all proposed manuscripts, abstracts, publications, and presentations 
based upon this study or its results prior to submission to any organization, business, 
agency, person, publisher, society, or other entity. 
 
 
 
11. References 
 
Centers for Disease Control.  http://www.cdc.gov/hai/eip/cdiff_techinfo.html; published 
August 30, 2012; accessed October 02, 2016. 
Bakken JS. Feces transplantation for recurrent Clostridium difficile infection:  US 
experience and recommendations.  Microb Ecol Health Dis. 2015;26:27657. doi: 
10.3402/mehd.v26.27657. 
Department of Health and Human Services.  http://www.hhs.gov/ash/initiatives/mcc; 
accessed October 02, 2016. 
Eiseman B, Silen W, Bascom GS, Kauvar AJ. Fecal enema as an adjunct in the treatment 
of pseudomembranous enterocolitis. Surgery. 1958;44:854 – 9. 
Miller MA, Louie T, Mullane K, Weiss K, Lentnek A, Golan Y, et al. Derivation and 
validation of a simple clinical bedside score (ATLAS) for Clostridium difficile infection 
which predicts response to therapy. BMC Infect Dis. 2013;13:148. 
 

SAP_001.pdf: 
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Protocol 2017-01 
Statistical Analysis Plan 
25 September 2020 
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Version 8.0 
CONFIDENTIAL 
Protocol 2017-01 
A Phase 3 Prospective, Randomized, Double-blinded, Placebo-controlled Clinical Study to 
Evaluate the Efficacy and Safety of Rebiotix RBX2660 (microbiota suspension) for the 
Prevention of Recurrent Clostridium difficile Infection 
NCT03244644 
Statistical Analysis Plan 
 Version: 8.0  
Date: 25 September 2020 
This document is the confidential information of Rebiotix Inc.  It may not be disclosed to parties 
not associated with the clinical investigation or used for any purpose without the prior written 
consent of Rebiotix Inc. 


Rebiotix Inc. 
Protocol 2017-01 
 
Statistical Analysis Plan 
 
 
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Version 8.0 
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Table of Contents 
1 
LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS ................................................................... 5 
2 
INTRODUCTION ............................................................................................................................................... 6 
3 
STUDY OBJECTIVES AND ENDPOINTS ...................................................................................................... 6 
3.1 
PRIMARY OBJECTIVE ...................................................................................................................................... 6 
3.2 
SECONDARY OBJECTIVE ................................................................................................................................. 6 
3.3 
OTHER OBJECTIVES ........................................................................................................................................ 6 
4 
STUDY DESIGN ................................................................................................................................................. 7 
4.1 
GENERAL STUDY DESIGN ............................................................................................................................... 7 
4.1.1 
Treatment Success ............................................................................................................................... 10 
4.1.2 
Sustained Clinical Response ................................................................................................................ 10 
4.1.3 
Treatment Failure ................................................................................................................................ 10 
4.1.4 
Determination of Treatment Failures .................................................................................................. 11 
4.2 
BLINDING ..................................................................................................................................................... 11 
4.3 
METHOD OF ASSIGNMENT OF SUBJECTS TO TREATMENT ARMS ................................................................... 12 
4.4 
DETERMINATION OF SAMPLE SIZE ................................................................................................................ 13 
4.5 
INTERIM SAMPLE SIZE RE-ESTIMATION ........................................................................................................ 13 
4.6 
RANDOMIZED TREATMENT ARMS ................................................................................................................ 14 
5 
CHANGES IN THE CONDUCT OF THE STUDY OR PLANNED ANALYSES ...................................... 14 
5.1 
CHANGES IN THE CONDUCT OF THE STUDY .................................................................................................. 14 
6 
BASELINE, EFFICACY AND SAFETY EVALUATIONS .......................................................................... 15 
6.1 
SCHEDULE OF EVENTS .................................................................................................................................. 15 
6.2 
TIME POINT ALGORITHMS ............................................................................................................................ 17 
6.2.1 
Relative Day......................................................................................................................................... 17 
6.2.2 
Windows ............................................................................................................................................... 17 
6.3 
ANALYSIS POPULATIONS .............................................................................................................................. 18 
6.3.1 
Intent-to-Treat Population ................................................................................................................... 18 
6.3.2 
Modified Intent-to-Treat Population .................................................................................................... 19 
6.3.3 
Per-Protocol Population ...................................................................................................................... 20 
6.3.4 
Safety Population ................................................................................................................................. 20 
6.4 
EFFICACY VARIABLES .................................................................................................................................. 20 
6.4.1 
Primary Efficacy Endpoint .................................................................................................................. 20 
6.4.2 
Secondary Efficacy Endpoint ............................................................................................................... 20 
6.4.3 
Other Efficacy Endpoints ..................................................................................................................... 21 
6.5 
SAFETY VARIABLES ..................................................................................................................................... 22 
6.5.1 
Safety Endpoints .................................................................................................................................. 22 
6.5.2 
Extent of Exposure to Study Treatment ................................................................................................ 22 
6.5.3 
Adverse Events ..................................................................................................................................... 22 
6.5.4 
Other Observations Related to Safety .................................................................................................. 24 
7 
STATISTICAL ANALYSIS ............................................................................................................................. 25 
7.1 
DISPOSITION OF SUBJECTS ............................................................................................................................ 25 
7.2 
PROTOCOL DEVIATIONS ............................................................................................................................... 26 
7.3 
BASELINE ASSESSMENTS .............................................................................................................................. 26 


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7.4 
DEMOGRAPHIC AND OTHER BASELINE CHARACTERISTICS ........................................................................... 26 
7.5 
PRIOR AND CONCOMITANT THERAPY ........................................................................................................... 27 
7.6 
ANALYSIS OF EFFICACY ............................................................................................................................... 27 
7.6.1 
Analyses Supporting the Primary Efficacy Endpoint ........................................................................... 27 
7.6.2 
Analysis Secondary Efficacy Endpoint ................................................................................................ 30 
7.6.3 
Other Endpoint Analysis ...................................................................................................................... 32 
7.7 
SAFETY ANALYSIS ........................................................................................................................................ 35 
7.7.1 
Adverse Events ..................................................................................................................................... 35 
7.7.2 
Other Analyses Related to Safety ......................................................................................................... 37 
8 
STATISTICAL METHODS ............................................................................................................................. 39 
8.1 
GENERAL METHODOLOGY............................................................................................................................ 39 
8.2 
ADJUSTMENTS FOR COVARIATES.................................................................................................................. 40 
8.3 
HANDLING OF DROPOUTS OR MISSING DATA ............................................................................................... 40 
8.4 
MEDICAL MONITOR ADJUDICATION ............................................................................................................. 40 
8.5 
EFFICACY ANALYSES AND DATA MONITORING ........................................................................................... 41 
8.5.1 
Study Stopping Rules ........................................................................................................................... 41 
8.6 
MULTI-CENTER STUDIES AND POOLING OF CENTERS ................................................................................... 42 
8.7 
MULTIPLE COMPARISONS/MULTIPLICITY ..................................................................................................... 42 
8.8 
EXAMINATION OF SUBGROUPS ..................................................................................................................... 42 
9 
COMPUTER SOFTWARE .............................................................................................................................. 43 
10 APPENDIX: BAYESIAN ANALYSIS PLAN ................................................................................................. 44 
 


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1 
LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS 
Abbreviation 
Definition 
AIDS 
Acquired Immunodeficiency Syndrome 
AE 
adverse event(s) 
ATC 
anatomical therapeutic chemical 
CI 
confidence interval 
CDAD 
Clostridium difficile-associated diarrhea 
CDI 
Clostridium difficile infection 
Cdiff32 
CDI-specific health-related quality of life survey instrument 
CSR 
Clinical Study Report 
DSMB 
Data Safety Monitoring Board 
eCRF 
electronic case report form 
EAC 
Endpoint Adjudication Committee 
FDA 
Food and Drug Administration 
FT 
fecal transplant 
HIV 
human immunodeficiency virus 
HIPAA 
Health Insurance Portability and Accountability Act of 1996 
IBD 
inflammatory bowel disease 
IBS 
irritable bowel syndrome 
ID 
study identification number 
ITT 
intent-to-treat 
IP 
investigational product (RBX2660 or placebo) 
ICU 
intensive care unit 
IVIG 
intravenous immunoglobulin 
MedDRA 
Medical Dictionary for Regulatory Activities 
MITT 
modified intent-to-treat 
mL 
milliliters 
N 
number of subjects 
PP 
per-protocol 
PT 
preferred term 
RBX2660 
investigational drug product being evaluated 
SAE 
serious adverse event 
SAP 
Statistical Analysis Plan 
SD 
standard deviation 
SOC 
system organ class 
TEAE 
treatment-emergent adverse event 
US 
United States 
WHO 
World Health Organization 
 
 
 


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2 
INTRODUCTION 
RBX2660 (microbiota suspension) is being studied for the prevention of recurrent Clostridium 
difficile infection (CDI) in adult subjects. The purpose of this study is to confirm the efficacy and 
safety of RBX2660 for the prevention of recurrent CDI in subjects who have had prior recurrent 
Clostridium difficile infection that was resolved with antibiotic treatment. 
 
3 
STUDY OBJECTIVES AND ENDPOINTS 
3.1 
Primary Objective 
To confirm the efficacy of RBX2660 as compared to a Placebo in preventing recurrent episodes 
of CDI through 8 weeks. 
 
3.2 
Secondary Objective 
The secondary objective of this study is: 
To evaluate the sustained clinical response rate of RBX2660 as compared to Placebo after 
blinded treatment.  
 
3.3 
Other Objectives 
1. To confirm the safety and tolerability of RBX2660. 
2. To identify baseline characteristics predictive of efficacy outcomes. 
3. To characterize the changes from baseline fecal microbial composition in 
subjects treated with RBX2660 as compared to Placebo. 
4. To characterize the changes from baseline comorbidities in subjects treated 
with RBX2660 as compared to Placebo. 
5. To evaluate health-related quality of life for CDI as measured by the Cdiff32 
questionnaire. 
6. To characterize the baseline severity of CDI in subjects with documented CDI 
recurrence. 


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7. Evaluate treatment success of RBX2660 in Placebo subjects who are 
documented study treatment failures then went on to receive RBX2660. 
8. Assess the ability of more than one dose of RBX2660 to prevent CDI 
recurrence. 
9. Assess the combined treatment success of all subjects receiving a single dose 
of RBX2660 during the study both to prevent recurrent CDI as well as prevent 
new CDI episodes.  
10. Assess the clearance rate of vancomycin resistant enterococcus in subjects 
who are carriers at baseline. 
11. Assess the clearance of C. difficile following enema treatment at 4 and 8-
weeks and 3 and 6 months after blinded study treatment in subjects receiving 
RBX2660 and those receiving Placebo. 
 
4 
STUDY DESIGN 
4.1 
General Study Design 
This is a prospective, multicenter, randomized, double-blinded, placebo-controlled Phase 3 study 
to confirm the efficacy and safety of RBX2660 for the prevention of recurrent CDI.   
 
Up to 270 subjects may be randomized and treated in this study.  Randomization will be at a 2:1 
ratio (RBX2660:Placebo) which means approximately 180 subjects randomized to treatment 
with RBX2660 and 90 subjects randomized to Placebo.  Consented subjects who meet all 
inclusion and none of the exclusion criteria will be randomized.  Potential subjects who are 
consented but do not meet the inclusion and/or exclusion criteria will be considered screen 
failures and will not count towards total randomized.  Subjects who are randomized but exit 
prior to the administration of the first blinded enema may be replaced with additional subjects 
which could cause overall number of subjects randomized to be greater than the 270 anticipated, 
however no more than 270 subjects will be treated with a blinded enema.  Replacement subjects 
will also be randomized in order to ensure proper blinding.  Subjects will be randomized to one 
of the two following study arms: 
 


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Placebo Arm – Group A 
• Placebo dosing consists of one enema of placebo given in a blinded manner.   
 
RBX2660 Arm – Group B 
• Active treatment consists of one enema of RBX2660 given in a blinded manner.   
 
The target population is adults (≥ 18 years old) with recurrent CDI who have had at least one 
recurrence after a primary episode (i.e., at least two episodes) and have completed at least one 
round of standard oral antibiotic therapy. 
 
The definition of recurrent CDI for study entry is a documented diagnosis of: 
1. CDI diarrhea that began within 8 weeks after completion of previous CDI treatment; 
2. and at least one positive stool test for the presence of toxigenic C. difficile; which must be 
within 30 days prior to or on the date of enrollment (informed consent).   
 
To be considered for enrollment, a subject must have medical record documentation of recurrent 
CDI per the above study definition that includes either:  
a) at least one recurrence after a primary episode and has completed at least one round of 
standard-of-care oral antibiotic therapy or;  
b) had at least two episodes of severe CDI resulting in hospitalization within the last year. 
 
The definition of CDI diarrhea for use in enrolled subjects throughout the study includes: 
1. The passage of three or more unformed/loose stools (i.e. Bristol Stool Scale type 6-7) in 
24 or fewer consecutive hours for at least two consecutive days; 
2. and a positive stool test for the presence of C. difficile toxin; documented at the time of 
the diarrhea. 
 


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Potential subjects are expected to already be taking or have just been prescribed antibiotics to 
control recurrent CDI symptoms at the time of enrollment per the investigator’s standard of care.  
This means patients who have already completed their prescribed course of antibiotics to treat 
recurrent CDI, cannot be considered for enrollment unless they have another recurrence.   
 
Once enrolled (informed consent obtained), in order to be randomized and treated;  
1. Subject must meet all the inclusion and none of the exclusion criteria; 
2. Antibiotics will have been administered for a minimum of 10 consecutive days prior to 
the washout period; 
3. CDI symptoms must be under control leading into the antibiotic washout period.  
 
Control of symptoms is defined as no longer meeting the symptomatic criteria for CDI diarrhea 
which is the passage of three or more unformed/loose stools (i.e. Bristol Stool Scale type 6-7) in 
24 or fewer consecutive hours for at least two consecutive days while taking antibiotics.  Thus, 
at minimum the Subject Diary (completed from time of enrollment until day of treatment) must 
show that symptoms are under control the two days prior to the washout period in order for the 
subject to be treated.   
 
Once screening is completed and all inclusion and exclusion verified, the subject can be 
randomized.  The assigned study treatment will be scheduled to consist of a single blinded study 
enema according to the randomization assignment.  Treatment is to be completed as soon as 
possible, but no more than 14 calendar days following randomization.  A minimum of 24hr to 
maximum of 72hr antibiotic washout period is required prior to administration of the assigned 
study treatment.    
 
In-office study follow-up visits occur at weeks 1, 4 and 8 after completing the blinded study 
treatment.  Telephone assessments for adverse events occur during weeks 2, 3 and 6 after the 
study enema and at months 3 and 6.  Subjects are required to keep a detailed diary to assess for 


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solicited events from the date of enrollment to the 1-week follow-up visit after receiving the 
blinded study enema.  The diary will be collected and reviewed at the baseline visit prior to 
blinded enema administration, and then collected and reviewed at the 1-week follow-up visit 
following enema administration.  
 
Subjects who are deemed failures following the blinded treatment per the pre-specified treatment 
failure definition may elect to receive an unblinded RBX2660 enema.  The unblinded enema is 
to be administered within 21 calendar days of failure determination. If a subject has a CDI 
episode on-study and the treatment plan is to pursue a treatment other than that specified in the 
protocol, the subject will be exited prior to receipt of the non-study treatment (ex. fecal 
microbiota transplant).  
 
4.1.1 Treatment Success 
Treatment success is defined as:  
• The absence of CDI diarrhea for 8 weeks after completing a study treatment.  
  
4.1.2 Sustained Clinical Response 
 
Sustained clinical response is defined as: 
• Treatment success of the presenting CDI recurrence and no new CDI episodes for greater 
than 8 weeks after completing a study treatment.        
  
4.1.3 Treatment Failure 
Treatment failure (CDI recurrence) is defined as:  
• The presence of CDI diarrhea within 8 weeks of administration of a study enema; which 
includes a positive stool test for C. difficile toxin at the time of the diarrhea. 
 
To ensure consistency across clinical sites for the determination of study failures, the central 
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immunoassay for glutamate dehydrogenase (GDH) and toxins A and B. If the test is 
inconclusive, the central laboratory will conduct a second confirmatory test by PCR.  
In the case of suspected treatment failure where there is an immediate concern for subject safety, 
including the potential for serious adverse events, a C. DIFF QUIK CHEK COMPLETE® Test 
will be permitted to be conducted at the site or other laboratory known to the site in addition to 
sending a sample to the central laboratory testing. If the C. DIFF QUIK CHEK COMPLETE® 
Test is not available, a testing algorithm that directly assesses the presence of C. difficile toxin 
for laboratory confirmation of recurrent CDI should be used. In the event, the test results from 
the central laboratory differ from that of a local laboratory, the results from the central laboratory 
will be used in determination of treatment failure. For subjects who are suspected of treatment 
failure, but for whom there is not a test result from the central laboratory for the suspected 
episode, a deviation will be entered to the database, and due documentation of the subject’s 
symptoms and all available treatment and outcome data will be sent to the Endpoint Adjudication 
Committee (EAC) for blinded, independent adjudication of treatment outcome.   
4.1.4 Determination of Treatment Failures 
The site investigator first makes the initial determination of treatment success or failure based on 
the pre-defined study definitions.  The site investigator’s assessment will be provided to the EAC 
for blinded, independent adjudication of treatment outcome that will be utilized for study 
analysis and reporting purposes. This may be done on an on-going basis or a single meeting as 
deemed appropriate.   
 
4.2 
Blinding 
Subjects, site personnel, the Data and Safety Monitoring Board (DSMB), EAC, and the Medical 
Monitor are initially blinded to the randomization assignment.  Blinding will be maintained 
through the data cutoff for the primary efficacy analysis after which the blind will be lifted for 
the DSMB.  The DSMB may be unblinded to individual assignments earlier as needed to 
adjudicate certain adverse events.  Individual randomization assignments will not be revealed to 


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the subjects, site personnel, EAC or the Medical Monitor until the study is completed.  Sites will 
be provided an emergency unblinding code if it becomes medically necessary to know the 
treatment group assigned for a particular subject. 
 
Subjects will be randomized to receive either RBX2660 or placebo through an Interactive 
Response Technology (IRT) System prior to receiving the first assigned blinded study treatment.    
Randomization assignment is revealed to Rebiotix Operations Department personnel for product 
preparation and shipment.  The Rebiotix Kit is shipped to the site in a sealed carton that is only 
opened by the IP administrator at the time of administration, out of view of the subject and other 
site personnel.  While the IP administrator will not have access to the actual randomization 
assignment, it may be possible to guess through visualization at the time of enema preparation 
and administration.  Additionally, subjects will be instructed to disregard any product 
characteristics that may become evident during or after administration such as the presence or 
absence of color or odor.  To limit study bias, the IP administrator is not allowed to perform any 
of the subject study assessments. 
 
4.3 
Method of Assignment of Subjects to Treatment Arms 
After enrollment and confirmation of inclusion/exclusion criteria compliance, randomization 
occurs via the study database with blinded treatment occurring within 14 calendar days of 
randomization.  The randomization assignment is transmitted only to the Rebiotix Operations 
Department, who will ship the correct IP to the site for each treatment.  Subjects are considered 
enrolled once they sign the informed consent form. 
 
Trained and authorized study personnel will access the centralized randomization system to 
randomize the subject to one of the available treatment arms.   The randomization schedule will 
be created using randomized blocks within four strata based on antibiotics used at screening 
(Vancomycin alone, Vancomycin in combination, Fidaxomicin or Other), and randomized 
subjects will be assigned one of two treatments (Group A or Group B) within the appropriate 


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stratum.  The randomization will not be stratified by site, so each site will draw from the same 
set of blocks.  A subject will be assigned the next available treatment allocation from the 
randomization list.  The randomization code and the date and time of randomization will be 
captured in the clinical database.  Randomization will be in a 2:1 ratio with approximately 2 
subjects randomized to active treatment for every 1 subject randomized to placebo.  
 
Enrolled subjects who are exited prior to randomization or are randomized but exited prior to 
attempting the blinded treatment may be re-enrolled and thus randomized under a different 
subject identification numbers (re-randomized).  If this occurs, they will be included in the 
analysis using their second randomization assignment and subsequent assessments and will only 
be counted once towards the total number randomized.   
 
4.4 
Determination of Sample Size 
To demonstrate a 69% success with one dose of the RBX2660 treatment group vs. 47% success 
in the Placebo control group, 240 subjects are required (power > 90%, nominal 2.5% type I error 
rate).  Up to an additional 30 subjects will be enrolled to allow for a 10% loss-to-follow up rate, 
for a total of approximately 270 subjects.  Considering the 2:1 randomization, this results in 
approximately 180 subjects in the RBX2660 arm and 90 subjects in the Placebo arm.  Enrolled 
subjects who have signed consent and withdraw for any reason prior to administration of the first 
blinded enema will be replaced without counting toward the sample size cap.  Replacement 
subjects will be randomized to ensure proper blinding.  
 
4.5 
Interim Sample Size Re-estimation 
There are no plans for an interim sample size re-estimation being completed for this study.  
 


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4.6 
Randomized Treatment Arms 
Placebo Arm – Group A 
• Placebo dosing consists of one enema of placebo given in a blinded manner.   
RBX2660 Arm – Group B 
• Active treatment consists of one enema of RBX2660 given in a blinded manner. 
 
5 
CHANGES IN THE CONDUCT OF THE STUDY OR PLANNED ANALYSES 
5.1 
Changes in the Conduct of the Study 
There have been no changes to the conduct of the study or planned analysis. 


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6 
BASELINE, EFFICACY AND SAFETY EVALUATIONS 
6.1 
Schedule of Events 
Activity 
Screening 
(Enrollment) 
Baseline / 
Enema 
Administration 
(≤21 days from 
screening) 
Follow-up Visits 
1-,  4- and 8-
Week (± 3 days) 
Assessments1  
Weekly Phone 
Assessment 
(Weeks 2, 3 and 
6 , (± 3 days) 
Unscheduled 
Possible 
Recurrence Visit 
Unblinded 
Enema 
Administration1 
(≤10 calendar 
days from Tx 
Failure) 
Phone 
Assessment at 3 
and 6 months  
(± 14 days) 
Informed consent obtained 
X 
 
 
 
 
 
 
Demographics, medical history 
X 
 
 
 
 
 
 
Prescribe/continue antibiotics for 
CDI symptom control 
X 
 
 
 
 
 
 
Modified physical 
exam conducted 
 
X 
 
 
 
 
 
Stool sent to Rebiotix by subjects for 
testing and archiving (optional) 
X 
 
X 
 
X 
 
X 
Central Lab CBC w/differential 
testing3 
X3 
X 
 
 
 
 
 
Central Lab CMP & CRP testing 
X 
 
 
 
 
 
 
C. difficile testing4 
X4 
 
 
 
X4 
 
 
Central Lab stool and blood testing  
 
X 
 
 
 
 
 
Urine pregnancy testing performed 
at site (if applicable) 
X 
X 
 
 
 
X 
 
Cdiff32 Questionnaire 
X 
 
X 
 
X 
 
X 
Inclusion/exclusion criteria 
confirmed 
X 
X 
 
 
 
 
 
Randomization assignment 
X5 
 
 
 
 
 
 
24-72hr washout period confirmed 
 
X 
 
 
 
X 
 
Enema administered 
 
X 
 
 
 
X 
 
Product complaint (if applicable) 
 
X 
 
 
 
X 
 
Recurrence or New CDI symptoms 
assessed 
 
X 
X 
X 
X 
X 
X 
Vital signs assessed 
X 
X 
 
 
X 
X 
 
Subject Diary discussed/reviewed 
X 
X 
X6 
 
 
X 
 
Employment status assessed 
X 
 
X7 
 
 
 
X 
Concomitant medications 
X 
X 
X 
X 
X 
X 
X 
Medical History Assessment 
X 
 
X7 
 
X 
 
X 
Adverse events assessed 
 
X 
X 
X 
X 
X 
X 
Solicited events assessed2 
 
X 
X2 
 
 
X2 
 
Protocol deviations (if applicable) 
X 
X 
X 
X 
X 
X 
X 


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1 Documented treatment failures may receive an unblinded RBX2660 enema. If an unblinded enema is administered, the follow-up visit requirement re-start based on the date of last 
enema administration.  
2 Solicited events are collected in the Subject Diary from the day of administration of any study enema (blinded or unblinded) until the day prior to the 1-week visit. The Subject Diary 
is collected and reviewed at the 1-week visit.  Solicited events that increase in severity from screening should be assessed for a possible adverse event. 
3 Exclusion criteria for absolute neutrophil count should be assessed based on the CBC collected at the screening visit. 
4 Perform within 30 days prior to or at enrollment and if CDI recurrence is suspected. 
5 Randomization should occur after screening criteria has been assessed and eligibility confirmed (4-14 calendar days from randomization to blinded study treatment) 
6 Subject Diary is reviewed only at the 1 week follow-up visit 
7 Collected at the 8 week follow-up visit only. 


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6.2 
Time Point Algorithms 
6.2.1 Relative Day 
The date of study enema administration will be considered relative day 1, and the day before study 
enema administration will be relative day -1.  Relative days will be calculated as follows only 
when the full assessment date is known (i.e., partial dates will have missing relative days, unless 
imputation methods have been specified): 
 
For days before first enema: 
Date of Assessment – Date of first enema administration 
 
For days on or after first enema: 
Date of Assessment – Date of first enema administration +1 
 
6.2.2 Windows 
For the purpose of statistical analysis, the analysis visit windows will be calculated in terms of 
study days since the day of the study enema administration, as illustrated in the following tables: 
Analysis Windows for Clinical Visits 
Visit 
Scheduled Study 
Day 
Visit Window in 
Protocol (Days) 
Visit Window for 
Analysis (Days) 
Screening Visit 
-30 through -1 
-30 through -1 
-30 through -10 
Baseline/1st Enema 
1 
1 
1 
Week 1 Office 
8 
5 - 11 
5 - 11 
Week 4 Office 
29 
26 - 32 
26 - 32 
Week 8 Office 
57 
54 - 60 
54 - 60 
 
 
 


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Analysis Windows for Phone Assessments  
Visit 
Scheduled Study 
Day 
Visit Window in 
Protocol (Days) 
Visit Window for 
Analysis (Days) 
Week 2 - Phone 
15 
12 - 18 
12 - 18 
Week 3 - Phone 
22 
19 - 25 
19 - 25 
Week 6 - Phone 
43 
40 - 46 
40 - 46 
3 month - Phone 
90 
76 – 104 
76 – 104 
6 month - Phone  
180 
166 – 194 
166 – 194 
 
If a subject has more than one assessment occurring in the same visit window, the data from the 
visit closest to the scheduled study day will be used for analysis.  If two visits have the same 
distance from the scheduled study day, i.e., one visit occurs -1 day from study day, and the other 
visit occurs +1 day from study day, the data from the visit after the scheduled study day will be 
used for analysis. 
 
Analysis Windows for Adverse Event Reporting by Onset Interval 
Onset Interval Name 
Onset Interval Analysis Window 
Window for Analysis 
(Days) 
Baseline  
Screening - < Baseline 
≤ -1 
1-week  
Baseline - 1-week  
1 through 8 
4-week  
> 1-week - 4-week 
9 through 29 
8-week  
> 4-week - 8-week  
30 through 57 
4-month 
> 8-week - 4-month 
58 through 121 
6-month 
> 4-month - 6-month 
122 through 180+ 
 
 
6.3 
Analysis Populations 
The modified Intent-to-Treat will be analysis of import for the primary endpoint. 
6.3.1 Intent-to-Treat Population 
Analysis of the primary efficacy endpoint will be done using the Intent-to-Treat (ITT) population 
as a sensitivity analysis. The ITT population is defined as all randomized subjects.  Subjects will 
be analyzed according to the randomized treatment rather than the actual treatment received 
regardless of treatment misallocations.  Re-randomized subjects will be summarized using their 
second treatment assignment (see Section 4.3 for details).  Randomized subjects who exited prior 


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to receiving blinded treatment will not be included in the analysis.  Randomized subjects in 
whom blinded treatment was attempted but delivery of the enema was not successful will be 
counted as treatment failures.  Randomized and treated subjects who exited prior to their 8-week 
efficacy assessment will be counted as treatment failures even if protocol required treatment 
failure documentation is not on file.  Additionally, the secondary efficacy endpoints may be 
evaluated using the ITT population. 
 
6.3.2 Modified Intent-to-Treat Population 
For the Modified Intent-to-Treat (mITT) population, subjects will be analyzed according to the 
randomized treatment assignment despite any treatment misallocations.  
 
Additionally, the mITT population will be defined as all randomized subjects who successfully 
received blinded treatment but excluding;  
• subjects who withdrew prior to treatment; 
• subjects in whom treatment was attempted but not completed and;  
• subjects who discontinue from the study prior to evaluation of treatment failure/success for 
the primary endpoint if the reason for exit is not related to CDI symptoms.  Reason for exit 
will be captured on the exit form to allow for identification of such subjects.  
Examples of reasons unrelated to CDI symptoms may include; 
• Withdrawal of consent 
• Death unrelated to CDI 
 
Subjects who were exited prior to the 8-week efficacy assessment due to CDI related symptoms 
will be counted as treatment failures even if documentation as required per the protocol is not on 
file for the treatment failure.  
 
The mITT population will be used for the primary efficacy reporting purposes. The mITT 
population will also be used for secondary and exploratory endpoints evaluation. The analysis of 
the ITT and PP populations may also be provided in addition to the mITT for purposes of 
regulatory evaluation.   


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6.3.3 Per-Protocol Population 
The per-protocol (PP) population will consist of all subjects who successfully received blinded 
treatment analyzed according to the treatment they received, excluding; 
• Subjects who have documented deviations to inclusion or exclusion criteria. 
• Subjects who exited prior to the 8-week efficacy evaluation if the reason for exit was not 
related to CDI symptoms in the same manner as the mITT population.  
The primary, secondary and exploratory endpoints will be evaluated using the PP population.    
 
6.3.4 Safety Population 
The safety population (SP) will be defined as the population of randomized subjects who had any 
blinded treatment attempted or completed.  Subjects will be analyzed according to the treatment 
they actually received should misallocations occur.  The safety population will be used in 
analysis of all safety endpoints. 
 
6.4 
Efficacy Variables 
6.4.1 Primary Efficacy Endpoint 
The primary efficacy endpoint is the recurrence of CDI within 8 weeks of blinded treatment. The 
primary analysis of the study will be a Bayesian hierarchical model, which formally incorporates 
data from a previous randomized Phase 2B study (Protocol 2014-01) of RBX2660. This analysis 
will demonstrate the efficacy of RBX2660 by showing a sufficiently high posterior probability 
that the rate of success in the treatment group is superior to the rate of success in the control 
group.  
 
6.4.2 Secondary Efficacy Endpoint 
The secondary efficacy endpoint is the recurrence of CDI through 6 months after blinded 
treatment. This will be analyzed as the difference in time to CDI event between RBX2660 and 
Placebo following blinded treatment through 6 months of follow-up. This analysis will only be 


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analyzed if statistical significance (p<0.05) is demonstrated for the primary efficacy endpoint.  
These endpoints will be tested at a two-sided 0.05 significance level.   
 
The null hypothesis of interest is that there is no difference between RBX2660 and Placebo 
treated subjects in the probability of CDI at any time point during the 6 months of follow-up. The 
alternative is that there is a difference in the probability of CDI at any time point during 6 
months of follow-up.     
 
The power to detect the difference between RBX2660 and Placebo treatment at 6 months was 
estimated by 10,000 simulations of time to event data, with 104 subjects per treatment 
group.  The data was simulated assuming the exponential distribution with the rate parameter 
lambda estimated using the 8-week success rates of 69% and 47% for RBX2660 and Placebo, 
respectively. Using the log-rank test, the power to detect the difference between the two groups 
at 6 months was 99.5%. 
 
6.4.3 Other Efficacy Endpoints 
Other efficacy endpoints include the following: 
• Baseline characteristics  
• Subject fecal microbial composition at Screening, and 4-weeks, 8-weeks, 3 months and 6 
months after blinded study treatment. 
• Charlson Comorbidity Index at Screening, 8-week, 3-, and 6-month phone assessments.  
• Cdiff32 questionnaire at Screening, Week 1, 4, 8, month 3 and 6 
• ATLAS score for CDI severity of qualifying CDI event. 
• Recurrence of CDI within 8 weeks of unblinded (open-label) RBX2660 treatment in Placebo 
subjects who were documented blinded study treatment failures. 
• Recurrence of CDI within 8 weeks of unblinded (open-label) RBX2660 treatment in subjects 
who received a blinded dose of RBX2660. 
• Occurrence of CDI through 6 months in all subjects receiving a single dose of RBX2660. 
• Concentration of vancomycin resistant enterococcus in stool samples for subjects who were 
carriers at baseline. 
• Presence of C. difficile in stool samples at Screening, and 4-weeks, 8-weeks, 3 months and 6 
months after blinded study treatment.  


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6.5 
Safety Variables 
All the safety analyses will be based on the safety population; see Section 6.3.4 in this document. 
 
6.5.1 Safety Endpoints 
• Number of adverse events per subject 
• Timing of attributable adverse event post-treatment exposure (treatment emergent adverse 
event TEAE) 
• Duration of TEAE 
• Relatedness of TEAE 
• Severity of TEAE 
• Causality of TEAE to IP, enema, C. difficile or prior condition Number of each of the following 
through 8 weeks post blinded treatment: death, septic shock, toxic megacolon, colonic 
perforation, emergency colectomy, and ICU admission. 
• Onset of new chronic conditions relative to blinded treatment administration. 
 
6.5.2 Extent of Exposure to Study Treatment 
The number and proportion (RBX2660 vs Placebo) of enemas administered for each subject will 
be summarized using descriptive statistics by treatment arm at each study enema administration 
(blinded and unblinded).  Time between study enema administrations will be summarized using 
descriptive statistics for both blind and unblinded enemas.  Additional analysis will be done for 
those subjects who received both Placebo and RBX2660 enemas. 
 
6.5.3 Adverse Events 
An adverse event (AE) is any untoward medical occurrence associated with the use of IP, 
whether or not the event is considered product-related.  Preexisting conditions that worsen in 
frequency, intensity or character of the condition during study participation will be recorded as 
an AE.  AEs will be recorded for each subject from the day of enrollment through the 6-month 


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telephone assessment. Serious adverse events (SAEs) will be recorded for each subject from the 
day of enrollment through the duration of the study. 
 
6.5.3.1 Adverse Event Dictionary 
AEs and SAEs will be classified by the current Medical Dictionary for Regulatory Activities 
(MedDRA) version.  The verbatim term recorded by the investigator will be mapped in 
MedDRA and system organ class (SOC), high level group term (HLGT), high level term (HLT), 
preferred term (PT), and lower level term (LLT) will be attached to the clinical database.  Tables 
and listings will present data at the SOC and PT level. 
 
6.5.3.2 Adverse Event Severity 
Severity of AEs will be graded as mild, moderate, severe, or potentially life-threatening.  The 
severity grade of events for which the investigator did not record severity will be categorized as 
“Unknown” for tabular summaries and data listings, and will be considered the least severe for 
the purposes of sorting for data presentation.  For summaries by severity grade, if a subject has 
multiple events occurring in the same SOC or PT, then the most severe event will be selected. 
 
6.5.3.3 Relationship of Adverse Events to Study Treatment 
Relationship of an AE to the Investigational Product (IP), the enema procedure, C. difficile 
disease, and a preexisting condition will be classified by the investigator as definite, probable, 
possible, or unrelated.  Related events will include AEs that are classified as definitely, probably, 
or possibly related.  IP includes both the Placebo and RBX2660 enemas.  
 
6.5.3.4 Treatment-Emergent Adverse Events 
Adverse events will be considered treatment-emergent adverse events (TEAE) according to the 
following algorithm: 
• If the complete onset date of an AE is known, then: 
o If AE onset date is prior to initial treatment date, then the AE will not be considered 
treatment-emergent. 


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o If AE onset date occurs on or after initial treatment date, then the AE will be 
considered treatment-emergent. 
• If AE onset date is partially known, then: 
o If day is unknown, and month/year occurs on or after first enema month/year, then 
AE will be considered treatment-emergent. Otherwise, AE will not be considered 
treatment-emergent. 
o If month/day is unknown, and year occurs on or after first enema year, then AE will 
be considered treatment-emergent.  Otherwise, AE will not be considered treatment-
emergent. 
 
6.5.4 Other Observations Related to Safety 
6.5.4.1 Solicited Adverse Events 
The following list of anticipated adverse events are solicited from subjects via the Subject Diary 
from the date of enrollment through the 7th day after receiving the randomized study treatment. 
For those subjects who receive an unblinded RBX2660 enema, these events are again collected 
in a new post-treatment Subject Diary from the day of the unblinded RBX2660 enema to the 7th 
day after receiving the unblinded RBX2660 enema: 
• gas (flatulence) 
• abdominal distension or bloating 
• rectal irritation or pain 
• chills/severe shivering 
• abdominal pain or cramping 
• increased diarrhea 
• constipation 
• rectal bleeding 
• nausea 
• vomiting 
• fever ≥ 37.8° C (>100.0°F) 
Frequency and severity of the solicited AEs will also be captured in the Subject Diary. 


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6.5.4.2 Major Complications of CDI Events 
Frequencies of major complications of CDI including death, septic shock, toxic megacolon, 
colonic perforation, emergency colectomy, or ICU admission will be collected through the 6-
month telephone assessment. 
 
6.5.4.3 Hospitalizations for Recurrent CDI 
Details regarding hospitalizations due to CDI, including admission and discharge date/time, 
reason for admission, whether the subject was admitted to an ICU, and ICU admission and 
discharge date/time (if applicable), will be collected following both the blinded and unblinded 
study enemas. 
 
7 
STATISTICAL ANALYSIS 
7.1 
Disposition of Subjects 
A summary table will present subject disposition for all subjects enrolled.  The table will show 
the number of subjects enrolled (consented), the number of enrolled subjects not randomized, 
followed by the number of subjects randomized for each treatment group and lastly the number 
of subjects randomized and treated.  The proportion of randomized subjects in each treatment 
group and overall who complete the study and who discontinue the study will be presented.  
Separately, a table or listing will be created to document the subjects that are included in each 
analysis population; ITT, mITT, PP and SP.  
 
Discontinuation will be categorized by reason as a percentage of the number of subjects in each 
analysis population and overall.  Additionally, subjects who were successfully treated but 
withdraw prior to the 8-week primary endpoint evaluation will be identified along with the 
reason for withdrawal to document which analysis population(s) to which they contribute.  
 


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7.2 
Protocol Deviations 
Protocol deviations will be documented in the clinical database.  The information available will 
be summarized by treatment arm and deviation type and listed by individual site. 
 
7.3 
Baseline Assessments 
Baseline assessments are collected within 30 days before or on the day of study treatment 
administration.  Summaries for age by group (< 65 and > 65), sex, ethnicity, and race will be 
provided by analysis group and treatment arm.  Baseline height, weight and employment status 
will also be summarized by analysis group and treatment arm.  CDI history will be summarized 
by total number of episodes experienced prior to blinded treatment.  Additionally, the following 
will be summarized for each episode of CDI: duration (days), treatment administered, 
hospitalization with duration (if applicable) and available C. difficile test results.  Additionally, 
the severity of the qualifying CDI episode will be documented for each subject by ATLAS score 
and the baseline comorbidities will be scored for each subject using the Charlson Comorbidities 
Index.   
 
The following baseline assessments will be provided in summary table and listings by treatment 
arm: 
• Medical history 
• CDI history 
• Laboratory testing (hematology) 
• Vital signs (systolic/diastolic blood pressure [mmHg], pulse rate [beats/min], respiration 
rate [breaths/min], temperature [°C]) 
 
7.4 
Demographic and Other Baseline Characteristics 
All baseline summaries will include the ITT, mITT and PP population and will be provided by 
treatment arm.  Descriptive statistics (number of subjects [N], mean, standard deviation [SD], 
median, interquartile range [IQR], minimum, and maximum for continuous variables and number 


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of subjects [N] and the percentage for categorical variables) will be provided for all baseline 
measures.  
  
7.5 
Prior and Concomitant Therapy  
The current World Health Organization (WHO) Drug Dictionary will be used to classify 
medications by preferred term and WHO Anatomical Therapeutic Chemical (ATC) at the highest 
level of ATC.  Listings of all concomitant medications will be made by WHO ATC classification 
of ingredients and by preferred term tabulated by each treatment arm.  All listings of usage of 
medications will be based on the Safety population. 
 
7.6 
Analysis of Efficacy 
7.6.1 Analyses Supporting the Primary Efficacy Endpoint 
7.6.1.1 Treatment Success 
A hierarchical, closed-testing procedure will be utilized for the primary endpoint and secondary 
endpoints.  Analyses of the single primary endpoint, recurrence of CDI within 8 weeks of blinded 
treatment, will be analyzed as the proportion of treatment success of Group A Placebo (PPLA) and 
Group B RBX2660 (PRBX). This analysis will be performed on the ITT, mITT and PP 
populations, with the mITT being the analysis of import for reporting purposes.   
 
The primary efficacy analysis will be a Bayesian hierarchical model which formally incorporates 
data from the previous Phase 2B study (Protocol 2014-01) of RBX2660. Study success will be 
declared if the posterior probability of superiority exceeds 0.99943 at the first two interim 
analysis.  If the trial continues to the maximum enrollment, the success criteria will be chosen so 
that the overall alpha is a nominal 0.00125. Success at this level is considered appropriate for 
approval based on results from a single study.  If success at this level is not met at the final 
analysis, success may also be declared if the posterior probability exceeds 0.97706. Success at 
this secondary threshold still constitutes a successful study, although it may not be sufficiently 
strong evidence to request regulatory approval based on a single study without further FDA 
discussion.  Details regarding the analysis can be found in Section 10 (Bayesian Analysis Plan). 


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The secondary endpoints will be analyzed using a hierarchical testing approach if sufficient 
evidence is found to claim study success. 
 
The primary efficacy analysis will be conducted when all subjects in each group reach their 8-
week assessment following blinded treatment or early termed from the study.  Full study results 
in the form of a final analysis and report will be completed once the last subject has completed 
the 6-month telephone assessment, at which time the study will be considered complete.  This 
includes subjects receiving unblinded RBX2660 due to a documented treatment failure. 
 
7.6.1.2 Sensitivity Analysis for the Primary Efficacy Endpoint 
The primary efficacy analysis will be repeated using the ITT and PP population to assess the 
sensitivity of the primary endpoint regardless of the outcome of the mITT primary efficacy 
analysis.  The ITT analysis will serve as the conservative analysis as it considers all subjects who 
exit prior to the 8-week efficacy assessment as treatment failures regardless of treatment failure 
documentation.  
 
Three groups of sensitivity  analyses will be performed to assess the sensitivity of the results to 
the borrowing of historical data from the previous Phase 2B study (Protocol 2014-01) of 
RBX2660.  
1) A Pearson’s chi-square test will be used to test the null hypothesis that the response rate 
in the treatment group is equal to that of the control group. In addition, two-sided 95% 
confidence intervals for the difference in response rate between arms will be calculated 
using a normal distribution approximation. This sensitivity analysis will be conducted 
using the mITT, ITT, and PP populations using data from the 2017-01 study only.  
2) Sensitivity of the primary analysis (Bayesian analysis) to the analysis population (ITT, 
PP) using Bayesian analysis. 
3) Sensitivity of the primary analysis (Bayesian analysis) to the following different 
sensitivity analysis populations from the 2014-01 study using Bayesian analysis.  The 
2014-01 Sensitivity Analysis Population #1 represents the ITT population from the 
2014-01 study, and the 2014-01 Sensitivity Analysis Population #2 represents the 


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population used for the primary analysis inclusive of one subject in Group C who was 
exposed to product but did not receive the full enema. 
 
2014-01 Sensitivity Analysis Population #1 
2014-01 Sensitivity Analysis Population #2 
2014-01 
Treatment 
Group 
# Subjects 
# Responders 
Response 
Rate 
2014-01 
Treatment 
Group 
# Subjects 
# Responders 
Response 
Rate 
A 
45 
25 
0.556 
A 
41 
25 
0.610 
B 
44 
19 
0.432 
B 
44 
19 
0.432 
C 
44 
25 
0.568 
C 
43 
25 
0.581 
 
 
Efficacy analysis will be repeated without the borrowing of historical data from the Phase 2B 
study (Protocol 2014-01) using the mITT, ITT, and PP populations. 
 
An analysis of time to CDI recurrence after completion of the assigned blinded treatment will be 
analyzed and presented by the Kaplan-Meier procedure and median duration presented by days 
(and weeks) using the ITT, mITT, and PP populations. The Kaplan-Meier estimate of the median 
time (days) and the associated 95% confidence interval will be presented for each treatment 
group. A graphic presentation of the Kaplan-Meier estimates will supplement the tabular 
presentation. Between group comparisons will be performed using the stratified log-rank test 
based on the stratification factor at randomization. The corresponding hazard ratio of the 
treatment effect along with the 95% confidence interval will be calculated using a Cox 
proportional hazard model with treatment and the stratification factor at randomization as 
explanatory variables. 
 
Time to CDI recurrence is defined as the number of days from enema administration to 
first assessment indicating recurrence for those subjects who were deemed Treatment Failures 
with CDI recurrence.  All subjects who are Indeterminate or discontinued for any reason prior to 
assessment of efficacy will be censored at the last assessment date at/prior to Week 8. For the 
analysis of Time to CDI recurrence within 8 weeks, all subjects considered as a treatment 
success will be censored at the date of their 8-week assessment.  For the analysis of Time to CDI 


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recurrence through 6 months, all subjects considered to have achieved sustained clinical response 
will be censored at the date of their 6-month assessment. 
 
Finally, the sensitivity of the primary efficacy endpoint will be assessed in a multivariate logistic 
regression adjusting for possible interactions of the following covariates with treatment group: 
age (< 65 years, ≥ 65 years), sex (Female, Male), race group (White, Non-white), ethnicity 
(Hispanic-Latino, not Hispanic-Latino), site geography (outside the US, Eastern US*, Southern 
US*, Northern US*, Western US*), randomization strata (i.e., treatment received at the 
qualifying event) and number of previous episodes of CDI recurrence at baseline. Other baseline 
variables will be compared for balance using the appropriate statistical test. This analysis will be 
performed on all analysis populations where at least 20 subjects in each sub-group are available 
for analysis to fully understand the observed treatment effect. 
*States included in the identified US geographical regions are as follows: 
• Eastern US: Connecticut, Delaware, District of Columbia, Maine, Maryland, 
Massachusetts, New Hampshire, New Jersey, New York, Ohio, Pennsylvania, Rhode 
Island, Vermont, Virginia, West Virginia 
• Southern US: Alabama, Arkansas, Georgia, Florida, Kansas, Kentucky, Louisiana, 
Mississippi, North Carolina, Oklahoma, South Carolina, Tennessee, Texas 
• Northern US: Illinois, Indiana, Iowa, Michigan, Minnesota, Missouri, Nebraska, North 
Dakota, South Dakota, Wisconsin 
• Western US: Arizona, California, Colorado, Idaho, Montana, Nevada, New Mexico, 
Oregon, Utah, Washington, Wyoming 
 
7.6.2 Analysis Secondary Efficacy Endpoint 
A hierarchical, closed-testing procedure will be utilized for the secondary endpoint:  If the null 
hypothesis for the primary efficacy endpoint is rejected, the secondary endpoint will be analyzed.  
The secondary endpoint will be tested at a two-sided 0.05 significance level.      
 


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7.6.2.1 Sustained clinical response of RBX2660 vs. Placebo. 
The null hypothesis of interest is that the rates of sustained clinical response (i.e., the rate of CDI 
occurrence with treatment success of the presenting CDI recurrence and no new CDI episodes 
for greater than 8 weeks after completing a blinded study treatment) are the same between the 
RBX2660 group and the Placebo group during the 6 months of follow-up.  The alternative is that 
there is a difference in sustained clinical response rates.  A Pearson’s chi-square test will be used 
to test the null hypothesis that the response rate in the treatment group is equal to that of the 
control group. In addition, two-sided 95% confidence intervals for the difference in response rate 
between arms will be calculated using a normal distribution approximation.  The null hypothesis 
will be rejected at the two-sided 0.05 significance level.  Subjects who were deemed a Treatment 
Success for the efficacy analysis and exited prior to their 6 month follow-up will be 
conservatively counted as a sustained treatment failure for the purpose of this analysis. 
 
An analysis of time to CDI occurrence after completion of the assigned blinded treatment will be 
analyzed and presented by the Kaplan-Meier procedure and median duration presented by days 
(and weeks) using the ITT, mITT, and PP populations.  The estimates will be presented for each 
of the time points at which CDI recurrence assessments are scheduled to occur.  A graphic 
presentation of the Kaplan-Meier estimates will supplement the tabular presentation.  Between 
group comparisons will be performed using the log-rank test. 
 
Time to CDI occurrence is defined as the number of days from enema administration to 
first assessment indicating recurrence, for those subjects who were deemed Treatment Failures or 
Indeterminate.  Randomized subjects who do not complete the assigned blinded treatment will be 
censored at Day 0.  All subjects who are discontinued for any reason prior to the 6-month 
timepoint will be censored at the last assessment date at/prior to Month 6.  All subjects 
considered as a sustained treatment success will be censored at the date of their 6-month 
assessment. 
 
 


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7.6.3 Other Endpoint Analysis 
An initial assessment will be completed during the primary efficacy analysis as follow-up data 
allows with a complete analysis to be provided with the full study results for applicable other 
analyses.  Additionally, the other analyses will be conducted on other efficacy populations as 
needed.  
7.6.3.1 Comparison of baseline characteristics in subjects successful with one dose of 
RBX2660 vs. those who were unsuccessful. 
A multivariate analysis or regression analysis will be used to identify predictors (if any) for 
subject success with one RBX2660 dose vs. those who were unsuccessful.   
 
7.6.3.2 Cdiff32 scores at Screening, 1-week, 4-week, and 8-week assessment visits, and 
3 and 6 month phone assessments. 
Changes from Screening in the Cdiff32 scores will be summarized using descriptive statistics by 
treatment arm.  Last observation carried forward (LOCF) will be used to impute missing post-
baseline data.  No inferential statistics will be performed. 
 
7.6.3.3 Subject fecal microbial composition at Screening, and 4 and 8- weeks and 3 and 
6 months after blinded study treatment. 
Descriptive statistics will be used to present the changes from Screening fecal microbial 
composition to those at 4 weeks, 8 weeks, 3 months and 6 months after blinded study treatment 
for those subjects treated with RBX2660 and those receiving Placebo.   
 
7.6.3.4 Charlson Comorbidity Index at Screening, 8 week visit, and 3-, 6- month phone 
assessments. 
A comorbidity index score will be documented for all subjects at Screening and compared to 
subsequent scores at 8 weeks, 3 months and 6 months.  An initial assessment will be completed 
during the primary efficacy analysis as follow-up data allows with a complete analysis to be 
provided with the full study results.  Additional analysis may be done of Placebo subjects who 


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were documented treatment failures after blinded treatment then treatment successes following 
unblinded RBX2660 treatment.  
 
7.6.3.5 Atlas score for CDI severity of qualifying CDI. 
Quantification of CDI severity scores for subjects at the time of the CDI recurrence that qualified 
them for enrollment into the study based on the ATLAS Scoring System.  Data from the 
qualifying CDI event will be used (see table below), including the protocol required eligibility 
criteria of treatment with systemic antibiotics prior to randomization.  However, if data points are 
not available, baseline data may be substituted.  
Parameter 
0 Points 
1 Point 
2 Points 
Age (years) 
< 60 
60 – 79 
≥ 80 
Temperature (oC) 
≤ 37.5 
37.6 – 38.5 
≥ 38.6 
Leukocyte count 
(total)/mm3 
< 16,000 
16,000 – 25,000 
> 25,000 
Albumin (g/L) 
> 35 
26 – 35 
≤ 25 
Systemic concomitant 
antibiotics during CDI RX 
No 
- 
Yes 
 
 
7.6.3.6 Recurrence of CDI within 8 weeks in Placebo subjects who were documented 
study treatment failures and then received RBX2660 through open-label. 
Treatment success for documented Placebo failures after receiving RBX2660 will be completed 
using a Chi-square test comparing the treatment success of blinded placebo subjects as compared 
to the success of placebo subjects who received RBX2660.  This analysis will be completed with 
the full study results rather than the primary efficacy analysis as it requires data on the outcome 
following any unblinded RBX2660 enemas received. 
 
7.6.3.7 Recurrence of CDI within 8 weeks of unblinded (open-label) RBX2660 treatment 
in subjects who received a blinded dose of RBX2660. 
Determine the treatment success rate of those subjects randomized to RBX2660 who were 
deemed an initial treatment failure then went on to receive an unblinded RBX2660 enema (two 
RBX2660 enemas).  This success rate will be calculated at 8 weeks following the second 


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RBX2660 enema and compared to the blinded success rate of the RBX2660 arm using a Chi-
square test.  
 
7.6.3.8 Occurrence of CDI through 6 months in all subjects receiving a single dose of 
RBX2660. 
Determine the combined treatment efficacy for RBX2660 randomized subjects plus Placebo 
randomized subjects who were documented failures after blinded treatment then received a 
unblinded RBX2660 enema.  This analysis will be completed with the full study results rather 
than the primary efficacy analysis as it requires data on the outcome following any unblinded 
RBX2660 enemas received.  Both treatment success and treatment durability will be analyzed; 
• Treatment success rate for the prevention of CDI recurrence 
• Treatment success rate for the prevention of new CDI episodes (those with onset 
occurring beyond 8 weeks after study treatment) 
 
7.6.3.9 Concentration of vancomycin resistant enterococcus in stool samples for 
subjects who were carriers at baseline. 
Lab generated values for the presence of vancomycin resistant enterococcus based on submitted 
stool samples at baseline and at 1-, 4-, 8-weeks, and 3 and 6 months will be assessed for all 
subjects to identify any trends in the clearance of vancomycin resistant enterococcus following 
enema treatment.  
 
7.6.3.10 Presence of C. difficile in stool samples at Screening, and 4-weeks, 8-weeks, 3-
months and 6-months after study treatment. 
Lab generated values for the presence of C. difficile based on submitted stool samples at baseline 
and at 1-, 4-, 8-weeks, and 3 and 6 months will be assessed for all subjects to identify any trends 
in the clearance of C. difficile following enema treatment.  
 
 


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7.7 
Safety Analysis 
The Safety population will be used to summarize all adverse event data, unless otherwise 
specified. Safety data will be summarized separately, using the same approach, for both the 
blinded and unblinded enemas.  Adverse events will be summarized for each randomization 
group through the 8-week primary endpoint follow-up.  Adverse events occurring after the 8-
week follow-up visit will be grouped into the following categories: 
1. Group A, placebo randomized subjects who did not receive an unblinded RBX2660 
2. Group B, RBX2660 randomized subjects who did not receive an unblinded RBX2660 
3. Group C, placebo randomized subjects who went on to receive an unblinded RBX2660 
4. Group D, RBX2660 randomized subjects who went on to receive an unblinded RBX2660 
 
 
7.7.1 Adverse Events 
Adverse events will be summarized using counts of the number of events and frequencies 
(counts and percentages) of the number of subjects by treatment group and overall.  The 
incidence of each preferred term (PT) within the primary system organ class (SOC) as well as 
overall primary SOC incidence will be presented.  Summaries may also be presented by PT, 
irrespective of SOC.  A subject with multiple events coded to the same PT within a primary SOC 
will be counted only once for the PT within the primary SOC.  Likewise, a subject with multiple 
events coded to the same SOC will be counted only once within the SOC. 
 
An overall summary of AEs will present the incidence of (1) all TEAEs, (2) TEAEs related to IP, 
(3) TEAEs related to enema procedure, (4) TEAEs related to C. difficile disease, (5) TEAEs 
related to a preexisting condition, (6) serious TEAEs, (7) serious TEAEs related to IP, (8) serious 
TEAEs related to the enema procedure, (9) serious TEAEs related to C. difficile disease, (10) 
serious TEAEs related to a preexisting condition, and (11) TEAEs leading to death. 
 
Frequency of occurrence and number and percent of subjects experiencing an event through 6 
months follow-up will be presented by treatment for both the blinded and unblinded enemas as 
follows: 


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• All TEAEs 
• TEAEs by maximum severity 
• TEAEs by relatedness to IP 
• TEAEs by relatedness to C. difficile disease 
• TEAEs by relatedness to enema procedure 
• TEAEs by relatedness to preexisting condition 
• TEAEs resulting in subject withdrawal 
• TEAEs resulting in death 
• Serious TEAEs 
• Serious TEAEs by maximum severity 
• Serious TEAEs by relatedness to IP 
• Serious TEAEs by relatedness to C. difficile disease 
• Serious TEAEs by relatedness to enema procedure 
• Serious TEAEs by relatedness to preexisting condition 
• Serious TEAEs resulting in subject withdrawal 
• Serious TEAEs resulting in death 
 
For the summary of AEs by maximum severity, if a subject has multiple events occurring in the 
same SOC or same PT, then the event with the highest severity will be counted.  For AEs 
reported by causality, if a subject has multiple events occurring in the same SOC or same PT, the 
event with the highest association will be summarized. 
 
A summary of TEAE and serious TEAE characteristics (severity, relatedness to IP, relatedness to 
C. difficile disease, relatedness to enema procedure, and relatedness to preexisting condition by 
outcome) will also be presented by treatment and overall for both the blinded and unblinded 
enemas. 
 
Incidence of AEs and serious AEs will also be presented by onset interval after the blinded and 
unblinded enemas for the following intervals:  
• Baseline (onset date prior to date of blinded enema);  


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• IP administration (onset date on the date of the blinded or unblinded enema as applicable);  
• and the following intervals relative to the last enema date (blinded or unblinded as 
applicable):  
o 1 Week (day 8), 4 Weeks (day 29), 8 Weeks (day 57), 3 Months (Day 90) and 6 
Months (Day 180). 
 
At the time of the primary efficacy analysis, all safety endpoints related to events occurring 
within 8 weeks following blinded treatment will be assessed.  Additionally, other safety 
endpoints will be analyzed as the data allows during the primary efficacy analysis with a 
complete analysis to be provided with the full study results in the final analysis. 
 
For data presentation, SOC will be ordered alphabetically, with PT sorted by decreasing total 
frequency.  No inferential statistics will be provided for the AE data. 
 
Subject listings will be presented for all AEs including SAEs. 
 
7.7.2 Other Analyses Related to Safety 
7.7.2.1 Subject Diary including Solicited Adverse Events and Compliance 
All solicited adverse events recorded in the Subject Diary will be summarized utilizing 
frequencies (number of events), proportions (number of subjects with the event) and 95% CIs for 
categorical variables and using the descriptive statistics as listed in Section 8.1 for continuous 
variables.  
 
For each solicited event, severity scores will be summarized as both categorical and continuous 
values.  For continuous summaries, severity scores will be identified as either pre-treatment or 
post-treatment (all diary entries captured after the blinded and unblinded enemas), and the 
average for each period will be calculated across individual subjects.  Baseline for continuous 
summaries will be considered the average during the pre-treatment period.  For categorical 
summaries, if a subject reports more than one severity score on the same date, the maximum 


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severity will be used. Baseline for categorical summaries will be considered the last non-missing 
assessment prior to the blinded enema. 
 
For each categorical summary of event frequency, a two-sided 95% CI will be calculated using 
the normal approximation of the binomial. 
 
The following summaries will be produced separately for the blinded enemas and unblinded 
enemas: 
• Frequency of severity scores on day of enema administration by treatment and overall 
• Frequency of severity scores on day 1 through 1-week post-treatment by treatment and 
overall 
• Frequency of maximum post-treatment severity score by treatment and overall 
 
The following summaries will be produced for the double-blind phase only: 
• Summary of average severity score and change from baseline by treatment and overall 
• Shift from baseline to 1-week post-treatment severity score by treatment and overall 
• Shift from baseline to maximum post-treatment severity score by treatment and overall  
 
The following summary will be produced during the final analysis as related to the overall 
scores: 
• Summary of average severity scores by treatment and overall 
 
Compliance in returning the Subject Diary will be calculated by dividing the number of diaries 
turned in, whether complete or not, by the number of diaries expected to be turned in.  
Furthermore, compliance will be summarized by calculating the proportion of subjects who 
turned in “complete” diaries, where complete is defined as answering all 11 daily questions for at 
least 80% of the expected number of diaries.  Compliance will be calculated following both the 
blinded and unblinded enemas separately and summarized by treatment group. 
 


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7.7.2.2 Major Complications of CDI events 
Major complications of CDI events include death, septic shock, toxic megacolon, colonic 
perforation, emergency colectomy, or ICU admission collected through the 6-month telephone 
assessment.  Incidence of major complications of CDI events will be summarized by treatment 
arm after both the blinded and unblinded enemas. 
 
7.7.2.3 Hospitalizations for Recurrent CDI 
The number of subjects requiring hospitalization due to recurrent CDI through 8 weeks 
following the completion of the randomized study treatment will be summarized, along with the 
length of hospital stay, the number of subjects requiring ICU admission, and the length of ICU 
stay during hospitalization. 
 
A listing of all hospitalizations due to CDI throughout the study will be generated. 
7.7.2.4 Rate of onset of new chronic conditions comparing those treated with RBX2660 
vs. Placebo. 
Documentation of physician diagnosed chronic conditions for all subjects will be completed at 
baseline and compared to new physician diagnosed chronic conditions reported at 8 weeks, 3 
months, and 6 months.  Chronic condition is defined as a condition lasting >3 months.  An initial 
assessment will be completed during the primary efficacy analysis as follow-up data allows with 
a complete analysis to be provided with the full study results.  Additional analysis may be done 
of Placebo subjects who were documented treatment failures after blinded treatment then 
treatment successes following unblinded RBX2660 treatment.  
 
 
8 
STATISTICAL METHODS 
8.1 
General Methodology 
All statistical tests will be two-sided with a significance level of α=0.05, unless specified 
otherwise, and will be performed using SAS Version 9.3 or higher.  Confidence intervals (CI) 
will be constructed at the level of 95% unless specified otherwise.  For continuous variables, 


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descriptive statistics (number of subjects [N], mean, standard deviation [SD], median, inter-
quartile range [IQR], minimum, and maximum) will be generated.  For discrete/categorical 
variables, the number and percentage of non-missing subjects will be generated.  Descriptive 
statistics will be provided by treatment arm for all subjects.  Standard operating procedures 
(SOPs) will be followed in the creation, validation and quality control of all data displays and 
analyses. 
 
Subject listings of all data from the eCRFs as well as any derived variables will be presented. 
 
8.2 
Adjustments for Covariates 
Adjustments for covariates will be made in sensitivity analyses of the primary endpoint if 
applicable due to imbalance of a particular covariate. 
 
8.3 
Handling of Dropouts or Missing Data 
No imputations will be used in analyses/summaries.  However, a last observation carried forward 
analysis will be used for evaluation of the Cdiff 32 scores.  Missing safety data will not be 
imputed beyond the adjustments for adverse event data mentioned in Section 6.5.2. 
 
8.4 
Medical Monitor Adjudication 
The Medical Monitor reviews and adjudicates all serious adverse events and other adverse events 
of interest to provide an objective, qualified judgment of the events.  Adverse event review and 
adjudication includes the event description, onset date in relation to the treatment date, and the 
investigator’s determination of causal relatedness to IP, the enema procedure, and pre-existing 
condition, as well as rating the event’s severity and seriousness.  The Medical Monitor will 
remain blinded throughout the study.  However, the DSMB will be unblinded as needed to 
adjudicate specific adverse events as per the study stopping rules (see Section 8.5.1, below). 
 


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8.5 
Efficacy Analyses and Data Monitoring 
The EAC will adjudicate individual efficacy data at the time of the interim and final analyses. 
Interim analyses evaluating the primary efficacy endpoint are planned once 160 subjects 
(minimum) and 220 subjects in the mITT population have completed (i.e., completed the 8 week 
follow-up assessment and/or completion of the Treatment Outcome form).  At each interim 
analysis, the study may declare early success, in which case enrollment is stopped and the 
sponsor will file for immediate approval.  The study may also declare futility, resulting in the 
cessation of enrollment and follow-up. 
 
A primary efficacy and safety analysis will be performed when 100% of subjects reach their 8-
week assessment following blinded treatment or are documented treatment failures by the site 
investigator. Safety analysis will be completed for safety endpoints defined at or prior to 8 weeks 
following blinded treatment. Full safety results as well as unblinded RBX2660 efficacy data will be 
reported after all available subjects complete their last 6-month telephone assessment following 
their last study enema (blinded or unblinded). 
 
To decrease the chance of introducing bias, the primary efficacy analyses will use efficacy data 
as reviewed and confirmed by the EAC. The study sites, subjects, and the medical monitor will 
remain blinded to the specific results and to individual randomized treatment assignments until 
the study is completed. 
 
8.5.1 Study Stopping Rules 
Enrollment will be paused if any of the following events are identified and the DSMB determines 
that there is probable cause that the IP or enema procedure contributed to the event: 
1. There is probable cause that IP or enema procedure (e.g., due to transfer from an RBX2660 
donor) contributed to a pathogenic intestinal infection in the stool of any subject, or 
 
2. Any series of events of major significance such as death or other serious outcome for 
which a causal connection with the IP is plausible represents an excess of the important 
adverse event(s) in one of the study arms.  
 
  


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8.6 
Multi-center Studies and Pooling of Centers 
The study will be conducted at up to 80 clinical study sites in the US and Canada.  
 
This is a multicenter study and before data from all sites are pooled, the effect of site will be 
assessed for the primary efficacy endpoint.  A sufficient number of subjects (≥ 7 meeting the PP 
definition) will be required within each investigative site to determine if there are any outlying 
sites.  If an investigative site is unable to enroll a sufficient number of subjects, it may be 
necessary to combine smaller sites (where enrollment is too low to detect differences) to create 
“pseudo-sites”.  Sites with < 7 subjects will be considered eligible for pooling.  Eligible sites will 
be pooled sequentially with other sites in the same region until a sufficient number of subjects 
are reached (at least seven). 
 
In this process, sites with < 7 subjects will be ordered numerically in ascending order, based on 
the investigative site number.  Sites with < 7 subjects will then be sequentially pooled with other 
sites in the same region in ascending order by site number until the resulting pseudo-site has at 
least 7 subjects meeting the PP population definition, thus creating a new “pseudo-site”.  The 
process will begin again with the next sequential small site until all small sites have been pooled 
with other sites in the same region into a pseudo-site.  Pooling of the centers will be done prior to 
study unblinding. 
 
The proportions and 95% confidence intervals for the primary efficacy endpoint data will be 
presented by pseudo-site to determine if any outlying sites exist. 
 
8.7 
Multiple Comparisons/Multiplicity 
A hierarchical closed-testing procedure will be utilized for the primary and the secondary 
efficacy endpoint as noted in Section 7.6.1.1. 
 
8.8 
Examination of Subgroups 
Subgroup analysis of age (< 65 years, ≥ 65 years), sex (Female, Male), race group (White, Non-
white), ethnicity (Hispanic-Latino, not Hispanic-Latino), site geography (outside the US, Eastern 


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US*, Southern US*, Northern US*, Western US*) and number of previous episodes of CDI 
recurrence at baseline will be conducted on the primary efficacy analysis if a sufficient sample 
size exists for each subgroup.  The primary efficacy analysis will also be conducted based on 
vancomycin use for the qualifying CDI episode ≤14 days and > 14 days using subjects who were 
stratified based on Vancomycin Alone. These analyses will include all analysis populations 
(mITT and PP) to understand any differences.  
*States included in the identified US geographical regions are as follows: 
• Eastern US: Connecticut, Delaware, District of Columbia, Maine, Maryland, 
Massachusetts, New Hampshire, New Jersey, New York, Ohio, Pennsylvania, Rhode 
Island, Vermont, Virginia, West Virginia 
• Southern US: Alabama, Arkansas, Georgia, Florida, Kansas, Kentucky, Louisiana, 
Mississippi, North Carolina, Oklahoma, South Carolina, Tennessee, Texas 
• Northern US: Illinois, Indiana, Iowa, Michigan, Minnesota, Missouri, Nebraska, North 
Dakota, South Dakota, Wisconsin 
• Western US: Arizona, California, Colorado, Idaho, Montana, Nevada, New Mexico, 
Oregon, Utah, Washington, Wyoming 
 
Other sub-groups may be considered if there are enough subjects in each group such as; 
qualifying CDI event treated as inpatient vs. outpatient, the severity of the qualifying event based 
on the ATLAS scores, treatment length or type of antibiotics taken at the time of the CDI 
qualifying event, and others yet undetermined.  
Cochran-Mantel-Haenszel tests will be used to assess homogeneity of the odds ratios in the 
subgroups. 
 
 
9 
COMPUTER SOFTWARE 
All analyses will be performed by Medpace using Version 9.3 or later of SAS software. All 
summary tables and data listings will be prepared utilizing SAS software. 
 
 
 
 


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10 APPENDIX: BAYESIAN ANALYSIS PLAN 
 


 
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Adaptive Design Report for the Phase 3 Trial (2017-01) 
for Evaluating the Efficacy and Safety of Rebiotix 
RBX2660 (microbiota suspension) for the Treatment of 
Recurrent Clostridium Difficile Infection 
Submitted to Rebiotix 
August 7, 2019 
Introduction 
This document describes the adaptive design for a randomized clinical trial to support 
administration of Rebiotix RBX2660 (microbiota suspension) via enema for the 
prevention of recurrent Clostridium difficile infection (CDI).  The trial will enroll and 
treat up to 270 subjects with recurrent CDI and includes up to two interim analyses at a 
minimum of 160 and 220 subjects complete (i.e., completed the 8-week follow-up 
assessment and/or completion of Treatment Outcome form after receiving the blinded 
treatment).  The first interim analysis may occur after 160 subjects complete, pending 
FDA review of the proposed adaptive design.  Early trial success or failure may be 
declared at either of the interims depending on the strength (either positive or negative) of 
the observed data.  The primary analysis of the trial will be a Bayesian hierarchical 
model, which formally incorporates data from a previous randomized Phase 2b study 
(Protocol 2014-01) of RBX2660. 
Treatment Arms 
Subjects will be randomized to one of the following treatments: 
• 
Treatment: A single dosing of RBX2660 via enema 
• 
Control: A single dosing of placebo via enema 
A 2:1 randomization ratio will be used to incentivize patient enrollment in the trial. 
Primary Analysis Population 
The primary analysis will be conducted using the Modified Intent-to-Treat (mITT) 
population described in the statistical analysis plan (SAP).  Subjects in the mITT 
population who withdraw due to CDI related symptoms will be counted as treatment 
failures. Up to 270 patients will be treated in the trial.  Patients in the mITT population 
who dropout prior to completion will be treated as failures; the effect of dropout is 
described in the section Operating Characteristics Under Dropout. 


 
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Primary Endpoint 
The primary endpoint of the trial is recurrence of CDI within 8 weeks of blinded 
treatment.  Treatment success is defined as the absence of CDI diarrhea through 8 weeks 
after completing the blinded study treatment.   
Statistical Model 
Historical Data 
The primary statistical analysis will use a hierarchical model to dynamically borrow 
information about the treatment effect from the previous Phase 2b trial (Protocol 2014-
01).  The efficacy objectives of the previous study were to assess the effectiveness of two 
dosing strategies of RBX2660 in patients with CDI. In one dosing group, patients 
received two enemas of RBX2660 (administered 7± 2 days apart).  In the alternate dosing 
group, patients received a single enema of RBX2660 along with a placebo (administered 
7± 2 days apart).  Both treatments were compared to patients in a control arm who 
received 2 administrations of placebo via enema (administered 7± 2 days apart).  Data 
from this trial are shown in Table 1. 
Table 1: Data from Phase 2b trial (Protocol 2014-01). 
Group 
Dosing 
# Subjects 
# Responders 
Response Rate 
A 
2 doses of RBX2660 
41 
25 
0.610 
B 
Placebo 
44 
19 
0.432 
C 
1 dose of RBX2660 
42 
25 
0.595 
The hierarchical model will incorporate data from groups B and C, excluding group A 
due to the difference in treatment dosage.   
The patients enrolled in this study are expected to be enrolled from a population similar 
to the Phase 2b trial.  In the case that subjects from the Phase 2b trial differ substantially 
from the current study, the proposed borrowing strategy allows for heterogeneity between 
study populations and dynamically borrows less information when the data from the new 
study differs from the Phase 2b study.   
For example, one difference between the studies is that patients with a single recurrence 
of CDI may be enrolled in this trial whereas they were excluded from the Phase 2b trial.  
We expect that this change will minimally impact the baseline rate of CDI recurrence 
observed in the new study.  However, if we did observe a control response rate that 
differed substantially from the Phase 2b study, the design would borrow less information, 
reducing the role of the historical data in our inference.  This effect is described in the 
section Strength of the Borrowing. 


 
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Hierarchical Model 
The data from the Phase 3 trial and Phase 2b trial will be analyzed using a Bayesian 
hierarchical model.  The analysis will be conducted using a modified intent to treat 
population.  Let 𝑁𝑁𝑘𝑘,𝑠𝑠 be the number of subjects assigned to treatment 𝑘𝑘 (k = 𝑇𝑇 for 
patients who receive a single dose of RBX2660 and k = 𝐶𝐶 for patients who receive the 
control) in study 𝑠𝑠 (𝑠𝑠= 1 for the current Phase 3 study, 𝑠𝑠= 2 for the previous Phase 2 
study).  We model the number of responders, 𝑋𝑋𝑘𝑘,𝑠𝑠 , in each arm/study as  
𝑋𝑋𝑘𝑘,𝑠𝑠∼Binomial(𝑁𝑁𝑘𝑘,𝑠𝑠, 𝑝𝑝𝑘𝑘,𝑠𝑠) 
where 𝑝𝑝𝑘𝑘,𝑠𝑠 is the underlying event rate for arm 𝑘𝑘 in study s.  The event rates are 
transformed to the log-odds scale and modeled as:  
log ቆ
𝑝𝑝𝐶𝐶,𝑠𝑠
1 −𝑝𝑝𝐶𝐶,𝑠𝑠
ቇ= 𝛼𝛼𝑠𝑠 
for the control arms and  
log ቆ
𝑝𝑝𝑇𝑇,𝑠𝑠
1 −𝑝𝑝𝑇𝑇,𝑠𝑠
ቇ= α𝑠𝑠+ 𝜃𝜃𝑠𝑠 
for the treatment arms.  The parameter 𝜃𝜃𝑠𝑠 represents the effect of RBX2660, relative to 
placebo, on the log-odds scale for trial 𝑠𝑠.  Hierarchical models are used to borrow 
information about the treatment and control effects across studies.  The following prior is 
used for the control rates across the two trials: 
 𝛼𝛼𝑠𝑠∼𝑁𝑁(𝛼𝛼, 𝜏𝜏𝛼𝛼
2 ) for 𝑠𝑠= 1, 2 
α ∼𝑁𝑁(0, 102) 
τ𝛼𝛼
2 ∼Inverse Gamma(0.001, 0.1) 
A similar prior is used for the treatment effects across the two trials: 
𝜃𝜃𝑠𝑠∼𝑁𝑁൫𝜃𝜃, 𝜏𝜏𝜃𝜃
2 ൯ for 𝑠𝑠= 1, 2 
θ ∼𝑁𝑁(0, 102) 
τ𝜃𝜃
2 ∼Inverse Gamma(0.01, 0.01) 
The priors on α and 𝜃𝜃 are chosen to be conservative and emphasize a prior assumption of 
a control response rate near 0.5 with a treatment effect centered around 0.  The priors on 
the hierarchical variance terms τ𝛼𝛼
2 and τ𝜂𝜂
2 are parameterized by their location 𝜇𝜇 and 
weight 𝜔𝜔 (rather than the traditional 𝑠𝑠ℎ𝑎𝑎𝑎𝑎𝑎𝑎= 𝜔𝜔/2 and 𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠= 𝜇𝜇2 ⋅𝜔𝜔/2  
parameterization).   These priors were chosen to encourage dynamic borrowing between 
the studies. This distribution is defined by the density:  
𝑓𝑓(𝑥𝑥|𝜇𝜇, 𝜔𝜔) ∝𝑒𝑒−𝜇𝜇2𝜔𝜔/2𝑥𝑥
𝑥𝑥𝜔𝜔/2+1  


 
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The selection of the borrowing hyper parameters is discussed in the Strength of the 
Borrowing Section of this design report. 
Success Criteria 
Our primary interest lies in the treatment effect (TE) for the current Phase 3 study (s =
1):  
𝑇𝑇𝑇𝑇= 𝑝𝑝𝑇𝑇,1 −𝑝𝑝𝐶𝐶,1 
The primary goal of the trial is to demonstrate the efficacy of RBX2660 by testing the 
hypothesis:  
𝐻𝐻0: 𝑇𝑇𝑇𝑇≤0  vs.  𝐻𝐻𝐴𝐴: 𝑇𝑇𝑇𝑇> 0 
We test this hypothesis by calculating the posterior probability of superiority, Pr( 𝑇𝑇𝑇𝑇>  0 
| Data) , which is equivalent to Pr(𝜃𝜃1 > 0 | Data).  The thresholds for defining success at 
each analysis are summarized in Table 2. 
Table 2: Success criteria at each analysis.  The * indicates that the success criteria may 
be updated at the final analysis to ensure the correct amount of 𝛼𝛼−spend. 
Analysis 
Criteria 
Subjects complete 
Interim 1 
Pr(𝑇𝑇𝑇𝑇> 0 | Data) > 0.99943 
Minimum 160 
Interim 2 
Pr(𝑇𝑇𝑇𝑇> 0 | Data) > 0.99943 
220 
Final  
 Pr(𝑇𝑇𝑇𝑇> 0 | Data) > 0.99943∗ 
Maximum 270 
Final  
Pr(𝑇𝑇𝑇𝑇> 0 | Data) > 0.97706 
Maximum 270 
The success criteria of 0.99943 was chosen using a Pocock spending function for analysis 
performed at 160, 220, and 270 patients complete with a cumulative 𝛼𝛼 spend of 0.00125.  
Success at this level is considered appropriate for approval based on results from a single 
trial.   
Uncertainty from several factors make assessing the exact 𝛼𝛼−spend at each analysis 
difficult to quantify.  First, the definition of the mITT population allows for the final 
analysis to be performed with fewer than 270 complete subjects.  In addition, the first 
interim analysis may be performed when more than 160 subjects are enrolled and treated. 
Finally, the second interim analysis may be omitted if the first interim analysis occurs 
sufficiently close to 220 subjects enrolled.  Therefore, to account for this uncertainty, the 
success criteria at the final analysis will be updated to reflect the information spend at 
each interim analysis and ensure a cumulative 𝛼𝛼 spend of 0.00125.  This change will be 
made based on the fraction of information at each interim analysis and does not depend 
on the outcome data for the trial. 
The lower, secondary, threshold of 0.97706 at the final analysis allows for success at the 
less demanding 𝛼𝛼= 0.025 level.  The second threshold at the final analysis controls the 
overall Type I error rate for the trial at 2.5% (without borrowing), accounting for the 𝛼𝛼 
spend at the interims and higher look at the final analysis.   


 
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Futility Stopping 
The trial will stop for futility at either interim if the predictive probability (PP) of success 
at the final analysis is sufficiently small.  The predictive probabilities will be computed 
using a final threshold of 0.97706 and assuming a final enrollment of 270 subjects. 
Bayesian predictive distributions incorporate the uncertainty around the model 
parameters as well as the uncertainty in the outcomes of subjects not yet enrolled.  The 
trial will stop for futility if there is limited probability that the trial will reach the success 
criterion, even at the maximum sample size.  The criteria for declaring futility at each 
interim are shown in Table 3.  
Table 3: Futility criteria at each analysis. 
Analysis 
Criteria 
Subjects complete 
Interim 1 
PP(Success) < 0.01 
Minimum 160 
Interim 2 
PP(Success) < 0.01 
220 
Strength of the Borrowing  
The hyper-priors on τ𝛼𝛼
2 and τ𝜃𝜃
2 are chosen to encourage dynamic borrowing from the 
Phase 2b trial. Dynamic borrowing is the idea that when the observed data in the current 
trial is similar to the previous trial, the strength of our conclusions should increase. On 
the other hand, if the newly observed data is quite different from the previous data, the 
prior data should contribute less information to our inference. 
Consider two extremes.  The first is the case of full borrowing, where data from the 
previous study is added to data from the new trial and inference is performed using the 
full weight of both studies.  Alternatively, we could do no borrowing, ignoring 
information from the previous trial and conducting our analysis with only the new 
information.  The hyper-priors on τ𝛼𝛼
2 and τ𝜃𝜃
2 are specified to provide dynamic borrowing, 
behaving similarly to the full borrowing approach when the observed data resembles the 
Phase 2b data and mimicking the no borrowing approach when the results differ in the 
two trials.  The effect of the dynamic borrowing is illustrated in Figure 1.  
Figure 1: Effective sample size (ESS) borrowed on the treatment arm for different 
observed treatment and response rates at the first interim. 


 
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Figure 1 summarizes the amount of borrowing from the hierarchical model for different 
observed treatment success proportions (𝑋𝑋𝑇𝑇,1 𝑁𝑁𝑇𝑇,1
⁄
, the x-axis) and different observed 
control proportions (𝑋𝑋𝐶𝐶,1 𝑁𝑁𝐶𝐶,1
⁄
, differently colored lines).  At the first interim analysis, 
104 subjects are assumed to be enrolled to the treatment arm with 56 subjects enrolled in 
the control arm.  The y-axis shows the effective number of additional treatment patients 
borrowed by the model, given by the equation 
Additional patients = Var൫𝜃𝜃𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓൯
Var(𝜃𝜃1) (𝑁𝑁1 + 𝑁𝑁2) −𝑁𝑁 2 
The quantity Var(θ1) is the posterior variance of the treatment effect under the 
hierarchical model and Var(θfull) is the variance of the treatment effect under the full 
borrowing model.  The ratio of these terms is the relative effective sample size, which 
measures the uncertainty in the hierarchical model relative to the full model.  When this 
ratio is small, there is substantially more uncertainty in the hierarchical model compared 
to the full model.   
The horizontal dashed lines correspond to 0 (no borrowing from the Phase 2b data) and 
86 (full borrowing of the Phase 2b data of Groups B and C).  The hierarchical model 
borrows the most patients from the Phase 2b trial when the observed treatment response 
rate is close to the treatment response rate in the previous study (a rate of 0.595).  As the 
observed control rate differs from the historical rate, fewer additional patients from the 
previous trial are used and the amount of borrowing diminishes.  When the observed 
control rate differs from the historical rate, borrowing is strongest when the log-odds of 
the treatment effect are similar to the phase 2b data. The chosen hyper-priors create 
appropriate dynamic behavior, decreasing uncertainty when then observed data is in 
concordance with the previous trial and increasing uncertainty when the Phase 3 trial 
results differ from the Phase 2b trial. 


 
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Example Trials 
This section contains several example trials selected to highlight aspects of the analysis 
and design.  For each example trial result, we show the following information at each 
analysis. 
• Observed data: a bar plot showing the number of patients with complete data 
enrolled per arm (total width of the bar) as well as the number of responders 
(darker shaded region). 
• Credible intervals: plot showing the fitted response rate for each arm (the X) and 
95% credible interval for each arm. 
• Pr(Treatment > Control): the posterior probability of a positive treatment effect 
in the Phase 3 trial, used for declaring trial success according to Table 2. 
• Pr(Success at N = 270): the predictive probability of success at the final analysis.  
The trial stops for futility when this probability drops below 0.001. 
• Pr(Treatment Response): posterior mean of the treatment arm response 
probability 
• Pr(Control Response): posterior mean of the control arm response probability 
Example Trial 1 
The first interim occurs when the minimum of N=160 subjects have complete data (i.e., 
completed their 8-week follow-up assessment and/or completion of Treatment Outcome 
form after receiving the blinded treatment). Example 1.1 shows the status of the trial at 
the first interim. 
Example 1.1: Status of the trial at the 1st interim 
 
The response rate on the control arm is 44.4%, which is similar to the Phase 2b data, and 
the response rate on the treatment arm is 69.8%, which is larger than observed in the 
Phase 2 trial.  The posterior estimates of the treatment response rate are smaller than the 
observed rate due to the borrowing from the Phase 2b data.  The posterior probability of a 
positive treatment effect exceeds 0.99943 and success is declared.  


 
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Example Trial 2 
The first interim occurs when N=170 subjects have complete data. Example 2.1 shows 
the status of the trial at the first interim. 
Example 2.1: Status of the trial at the 1st interim 
 
The observed response rates in this scenario are much smaller than in the Phase 2b data, 
with an observed treatment effect of 0.066.  While the predictive probability of success is 
small (0.324), it is sufficient to continue the trial to the second interim. 
Example 2.2: Status of the trial at the 2nd interim 
 
The second interim, shown in Example 2.2, occurs when 220 patients have complete 
data.  The observed response rates have increased in both arms, though the observed 
treatment effect is smaller (0.014).  While the estimated treatment effect is positive, the 
probability of declaring success at the final analysis is 0.003 which is sufficiently small to 
stop the trial for futility. 
Example Trial 3 
In this example, the first interim analysis occurs when 180 subjects have been enrolled. 
The observed control rate is 0.5, somewhat higher than seen in the Phase 2b trial.  
Example 3.1 shows the status of the trial at the first interim 
Example 3.1: Status of the trial at the 1st interim 
The posterior estimate of the control response rate (0.473) is somewhat lower than 


 
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observed due to the borrowing and the posterior probability of a treatment effect is 
insufficiently high to declare success at the first interim. 
Example 3.2: Status of the trial at the 2nd interim 
 
At the second interim (N=220, shown in Example 3.2), the observed response rates in 
both arms have been reduced.  The trial has not shown sufficient evidence to declare 
success or futility and continues enrollment, though the predictive probability of success 
at the final analysis remains optimistic (0.797). 
Example 3.3: Status of the trial at the final analysis 
 
The final analysis is conducted when 270 patients have completed follow-up and 260 
subjects qualify for the mITT population (Example 3.3).  The single trial success criteria 
is updated to 0.99931, using the information fraction and 0.99943 criteria at the first two 
interims (0.692 and 0.846) to ensure an overall 𝛼𝛼−spend of 0.00125.  The trial is 
declared a success at the 0.025 level, though it does not meet the single trial success 
criteria. 
Performance Characteristics 
The performance characteristics of the design were determined through trial simulation.  
We assume several scenarios for the underlying response probability for the control and 
treatment arms, considering control probabilities in {0.2, 0.3, 0.4, 0.45, 0.5, 0.6, 0.7} and 
additive treatment effects of {0.0, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3}.  The scenario with a 
control probability of 0.45 and a treatment effect of 0.15 corresponds most closely to 
what was observed during the Phase 2b trial.  The scenarios with treatment effects of 0 
correspond to null scenarios.  For the purposes of simulation, we assume that patients are 
randomized in 3 patient blocks, that interim analysis were performed with exactly 160 
and 220 subjects completed, and that the 270 mITT subjects have complete data at the 
final analysis.   


 
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Operating Characteristics 
We simulated 1,000,000 virtual trials for each scenario.  The results of the simulations 
are summarized using the following operating characteristics. 
• Pr(Success): the cumulative probability of declaring success at the analysis.  For 
example, Pr(Success) under the section Interim 2 includes the probability of 
declaring success at either Interim 1 or Interim 2. For the final analysis, 
Pr(Success) (0.99943) and Pr(Success) (0.97706) include the cumulative 
probability of declaring success at the single trial and lower success levels 
respectively.  
• Pr(Futility): the cumulative probability of declaring futility at the analysis 
• # Subjects: the average number of subjects enrolled in the trial 
• Bias: the average error in estimating the true (simulated) treatment effect. 
Table 4 shows an example set of operating characteristics when the simulated control 
response probability is 0.45.  
Table 4: Operating characteristics of the design when the underlying control response 
probability is 0.45. 
 
Under the null scenario where both the control and treatment arms have a 45% true 
response rate, 45% of trials stop for futility at the first interim analysis (N = 160) and 
0.1% of trials meet the success criterion.  By the second interim, 73% of trials have 
stopped for futility.  Assuming a final threshold of 0.97706, the probability of trial 
success at or before the final analysis is 0.042.  The type I error of this trial is inflated 
beyond the nominal 2.5% level because of the borrowing from the positive phase 2 data.  
In the scenario with a treatment effect of 0.15 (which most closely reflects the observed 
data from Phase 2), the trial has 83% power.  Broken down by analysis, 14% of trials 
declare success at the first interim, another 12% of trials declare success at the second 
interim, 9% of trials reach the higher success threshold of 0.99875 at the final analysis 
and 48% of trials declare success at the lower hurdle at the final analysis.  When the true 


 
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effect is 0.10, the overall power drops to 52% with 77% of trials progressing to the final 
analysis. 
Operating characteristics for simulations with control response probabilities in {0.2, 0.3, 
0.4, 0.5, 0.6, 0.7} are included at the end of this section.  
Decision Rules 
The decision rules of the trial at each analysis can be characterized in terms of the 
observed effect sizes (# responders / # subjects enrolled) in each arm.  The effects leading 
to either futility or success, colored by result, are shown in in Figure 2. 
Figure 2: Scatterplots of the observed response rates for both arms, colored by result.  
 
The design has a clear decision boundary at the first interim, declaring futility or success 
when the observed treatment effect is sufficiently large or small.  The gap between 
success and futility narrows at the 2nd interim, as more information about the arms 
informs decision making.  The effect size required to win is smallest at the final interim. 
Operating Characteristics with No Borrowing 
For comparison we include the operating characteristics of an alternate design without 
borrowing.  This design uses the same interim schedule as the borrowing design but does 
not include stopping for futility.  Table 6 compares the cumulative probability of 
declaring success for at each interim for both designs when the simulated control 
response rate is 0.45. 
Table 6: Operating characteristics for the borrowing design (borrow) and alternate 
design (none) at each interim analysis when the simulated response rate is 0.45. 
 


 
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For a simulated treatment effect of 0.15, the borrowing design has 20% more power than 
the alternate design.  Broken down by interim, the borrowing design is 7% more likely to 
declare success at the first interim, 12% more likely to declare success by the second 
interim,  and 14% more likely to reach the higher threshold by the final analysis.  The 
borrowing design is more likely to declare success when the simulated treatment effect is 
0, with a 4.2% chance of declaring success compared to 2.2% for the alternate design. 
Operating Characteristics Under Drop Out 
Per the protocol, subjects who drop out of the mITT population prior to the final analysis 
for CDI related symptoms will be treated as treatment failures (regardless of arm). 
Therefore, simulated dropout has the practical effect of reducing the simulated control 
and treatment rates and the tables in the following second can be used to understand the 
performance of the design under a range of dropout scenarios. 
For example, suppose that the true response rate on the control arm was 0.45, the true 
treatment effect was 0.20 and the dropout rate was 10%. Then, this trial would have the 
same operating characteristics of a trial with a simulated 0.405 control rate, a 0.145 
treatment effect, and no dropout. The operating characteristics in this scenario can be 
adequately approximated using Table 8. 
Additional Operating Characteristics 
This section contains operating characteristics for control response probabilities in {0.2, 
0.3, 0.4, 0.5, 0.6, 0.7}.  A description of these tables can be found under the operating 
characteristics section. 
Table 6: Operating when the underlying control response probability is 0.2 
 
Table 7: Operating when the underlying control response probability is 0.3 
 


 
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Table 8: Operating when the underlying control response probability is 0.4 
 
Table 9: Operating when the underlying control response probability is 0.5 
 
Table 10: Operating when the underlying control response probability is 0.6 
 
Table 11: Operating when the underlying control response probability is 0.7 
 
Computational Details 
Simulations were run using R (R Core Team 2017) version 3.4.3. Bayesian computations 
were performed using the Stan (Stan Development Team 2018) version 2.17.2. 


 
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Many samples from the posterior distribution are required to determine whether the 
posterior probability is greater than 0.99943 with sufficient precision.  For the power 
calculations, for each trial we ran a single MCMC sampler for 10,000 iterations.  When 
executing the trial, if the posterior probability of a positive treatment effect is close to the 
boundary we recommend using more samples to obtain sufficient precision.  For 
example, using 1,000 independent Markov chains with 10,000 post burn-in iterations 
each.