Conditions: Clostridium Difficile
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 6
Healthy volunteers: no

Brief summary:
A total of 440 patients meeting enrollment criteria with a primary episode of C. Difficile Infection (CDI) will be enrolled across 3 sites. The total study time period for study procedures followed by clinical monitoring is anticipated to be about 24 months (biomarker assays and other analyses may be completed after the 24 month time period). All participants will receive oral antibiotics for CDI under the care of their physician. After consenting to participate in the study, participants will be randomized to receive either misoprostol (200 mcg po BID) or matching placebo for 14 days. Participants will be monitored for a total time-period of approximately 9 weeks with the goal of monitoring for recurrence of CDI during an 8-week follow-up period from the time that the course of antibiotic treatment is completed. Patients will have blood and stool samples (or rectal swabs if participants are unable to provide a stool sample) collected throughout the study to assess adherence, biomarkers, and to confirm recurrence of CDI (if necessary).

Detailed description:

Arms:
arm 1: None arm 2: None

Interventions: intervention 1: Misoprostol 100Mcg Tab intervention 2: Placebo
Intervention descriptions: intervention 1: Two 100mcg capsules twice per day intervention 2: Two capsules twice per day

Protocol document:
ICF_003.pdf: 
Informed Consent Document 
PROCLAIM – A Randomized, Double-Blind, Placebo-Controlled Trial to Assess the Efficacy and Safety of 
Misoprostol in the Prevention of Recurrence of Clostridium Difficile Infection in Adults 
NCT03617172 
June 23, 2021 


Institutional Review Board 
Informed Consent Document for Research 
 
Lead Site Principal Investigator:  David Aronoff, M.D. 
 
Version Date:  May 24, 2021 
Lead PI Institution/Hospital: Vanderbilt University Medical Center 
 
 
  
Study Title: Misoprostol in the prevention of recurrent Clostridium difficile infection 
 
 
1 
 
Part 1 of 2: MASTER CONSENT 
 
Name of participant: _____________________________________________Age:___________ 
 
You are being invited to take part in a research study. This study is a multi-site study, 
meaning it will take place at several different locations. Because this is a multi-site study 
this consent form includes two parts. Part 1 of this consent form is the Master 
Consent and includes information that applies to all study sites. Part 2 of the consent 
form is the Study Site Information and includes information specific to the study site 
where you are being asked to enroll. Both parts together are the legal consent form and 
must be provided to you. 
 
You do not have to be in this research study. You may choose not to be in this study and get 
other treatments without changing your healthcare, services, or other rights.  You can stop 
being in this study at any time.  If we learn something new that may affect the risks or benefits of 
this study, you will be told so that you can decide whether you still want to be in this study.    
 
What is the purpose of this study?  
You are being asked to take part in this research study because you were recently diagnosed 
with and treated for an infection caused by the bacteria Clostridium difficile (C. diff).  
 
C. diff is an infection that causes diarrhea and colitis (swelling of the colon). It is one of the most 
common healthcare-associated infections. C. diff infection (CDI) ranges in severity from mild 
diarrhea to severe colitis (sometimes resulting in septic shock and/or death). After treatment, 
CDI has a high chance of recurring (coming back). CDI recurs in about 25% of patient initially 
treated with standard of care therapy. The rate of recurrence increases with age. Once a first 
recurrence has occurred, the risk for future recurrences exceeds 40% with highest rates being 
in older adults. There are patients for whom the cycle of recurrences does not stop. 
 
The purpose of this research study is to learn about how safe and effective the drug misoprostol 
is in preventing the infection from coming back. Misoprostol (Cytotec) is FDA approved to 
decrease the chance of getting stomach ulcers related to taking NSAIDS (nonsteroidal anti-
inflammatory drugs, including aspirin). Misoprostol is investigational in this trial, meaning not 
FDA approved for C. diff.  
 
About 440 people across the country will take part in this research study. 
 
What will happen and how long will you be in the study? 
This study is a randomized, double-blind, placebo-controlled study which means that you will 
either receive capsules containing the study drug, misoprostol, or placebo (capsules that do not 
have any study drug) that look like the study drug. Randomized means that no one can choose 
who receives the study drug and who receives placebo, so it is up to chance (like flipping a 
Date of IRB Approval: 06/23/2021
Date of Expiration: 05/18/2022


Institutional Review Board 
Informed Consent Document for Research 
 
Lead Site Principal Investigator:  David Aronoff, M.D. 
 
Version Date:  May 24, 2021 
Lead PI Institution/Hospital: Vanderbilt University Medical Center 
 
 
  
Study Title: Misoprostol in the prevention of recurrent Clostridium difficile infection 
 
 
2 
 
coin). Double-blind means that you, the study doctor and the study team will not know which 
capsule you receive.  
 
If you agree to be in this study your involvement will last about 10 weeks. Below you will find 
information and what you can expect if you agree to take part in this study. 
 
In-person Visit 1 (2-7 days after you start standard treatment) – This visit will last about 1.5 
hours and could take place at the study site or at your home. Home visits will be conducted by a 
study team member in your home. If available, the stool sample that you provided to your doctor 
when you were diagnosed with C. diff may be saved for future testing. The following will occur at 
this visit: 
 
 
The study team will discuss the study with you and answer all your questions. 
 
The study team will discuss your medical history with you. 
 
The study team will confirm that you no longer have C. diff symptoms. 
 
If you are a woman who could become pregnant, a urine pregnancy test will be collected 
to make sure you are not pregnant. The study team will also discuss birth control options 
with you. 
 
A physical exam will be done by either a study physician or other health care 
professional approved by the study. 
 
You will have your blood drawn (about 4 teaspoons). 
 
A stool sample will be collected (if you are unable to provide a stool sample, you will be 
asked to provide a rectal swab). 
 
You will complete a symptom questionnaire and a quality-of-life questionnaire. 
 
You will be placed into either the study drug (misoprostol) or placebo group. 
 
You will receive the study drug to take along with an at home stool/rectal swab collection 
kit with instructions 
 
The study team will discuss the study diary with you and provide you with the diary to 
also take and return to us. 
 
Start of Study Drug (Day 7 of your standard treatment, or approximately 7-10 days after a 
positive lab test) - You will begin taking the study drug on Day 7 of your standard treatment if 
you are on a short course of antibiotics, otherwise you will begin taking the study drug 7-10 days 
after receiving a positive CDI lab test. If you have already completed the in-person study visit 1, 
the study team will contact you to remind you to begin the study drug and to answer any 
questions that you have. The study drug should be taken with food. You will take 2 study drug 
capsules 2 times a day for 14 days. You will record information daily in your study diary about 
when you took the study drug and the appearance of your stool which is important for the study.  
 
Follow Up Phone call 1 & 2 (3 days and 7 days after starting the study drug) – The study 
team will call you to ask how you are feeling and to complete the symptom questionnaire and 
Date of IRB Approval: 06/23/2021
Date of Expiration: 05/18/2022


Institutional Review Board 
Informed Consent Document for Research 
 
Lead Site Principal Investigator:  David Aronoff, M.D. 
 
Version Date:  May 24, 2021 
Lead PI Institution/Hospital: Vanderbilt University Medical Center 
 
 
  
Study Title: Misoprostol in the prevention of recurrent Clostridium difficile infection 
 
 
3 
 
quality of life questionnaire with you. If you prefer, the quality-of-life questionnaire can be sent to 
you as an email link to the survey as well. During the call they will also ask if you have been 
taking the study drug, completing the study diary and will answer any questions that you have. 
The study team will also remind you to return your diary back after you have taken your last 
dose of study drug. 
 
Follow-Up Phone calls 3 – 9 (starting after you have stopped taking the study drug) – The 
study team will contact you every week to ask how you are feeling and to complete the 
symptom questionnaire and quality of life questionnaire with you unless you have chosen to 
complete the quality-of-life questionnaire as an electronic survey.  
 
In-person Visit 2 (Day 62 of study) – This visit will last about 1.5 hours and could take place at 
the study site or as a home visit conducted by a study team member in your home. The 
following will occur at this visit:  
 
The study team will discuss how you are feeling. 
 
You will have your blood drawn (about 4 teaspoons) 
 
A stool sample will be collected (if you are unable to provide a stool sample, you will be 
asked to provide a rectal swab). 
 
You will complete a symptom questionnaire and a quality-of-life questionnaire. 
 
C. diff Recurrence Visit (if applicable) – If you have any diarrhea (loose/water stool) or if your 
doctor has confirmed that the C. diff infection has come back during the study, you will be asked 
to come in for a study visit or have a home visit and provide a stool sample or swab. If multiple 
recurrences occur, the study team may ask you to come back for each recurrence. The 
following will occur at this visit: 
 
The study team will discuss how you are feeling. 
 
You will have your blood drawn (about 4 teaspoons) 
 
A stool sample will be collected (if you are unable to provide a stool sample, you will be 
asked to provide a rectal swab). 
 
You will complete a symptom questionnaire and a quality-of-life questionnaire. 
 
If you are unable to complete an in-person visit, please contact the study team so that the stool 
sample/rectal swab, which will be in your take home kit, can be picked up at your home.  
 
Side effects and risks that you can expect if you take part in this study:  
Side effects associated with the use of misoprostol:  
Common side effects: 
 
Diarrhea (loose or watery stool) 
 
Stomach pain 
 
Date of IRB Approval: 06/23/2021
Date of Expiration: 05/18/2022


Institutional Review Board 
Informed Consent Document for Research 
 
Lead Site Principal Investigator:  David Aronoff, M.D. 
 
Version Date:  May 24, 2021 
Lead PI Institution/Hospital: Vanderbilt University Medical Center 
 
 
  
Study Title: Misoprostol in the prevention of recurrent Clostridium difficile infection 
 
 
4 
 
Misoprostol capsules taken as an FDA-approved treatment for other conditions, have been 
known to cause diarrhea, stomach pain, and/or nausea in some people, when given at higher 
doses than the dose being given in this study. In most cases these problems developed during 
the first few weeks of therapy and stopped after about a week. In this study, you will be only be 
taking the study medication for 14 days.  However, you can minimize possible diarrhea by 
making sure you take Misoprostol capsules with food and avoiding magnesium containing 
antacids. Because these side effects are usually mild to moderate and usually go away in a 
matter of days, most patients can continue to take Misoprostol capsules. If you have long-
lasting difficulty (more than 8 days), or if you have severe diarrhea, cramping and/or 
nausea, call your doctor or study team immediately. If symptoms continue the study doctor 
may reduce the dose to 1 capsule twice a day after speaking with the study team. 
 
Misoprostol Pregnancy Risks 
You cannot be in this study if you are pregnant or breastfeeding. If you are pregnant or become 
pregnant during the study, the study drug may cause harm to your unborn child. The study drug, 
misoprostol, can cause birth defects, pregnancy loss, and premature birth.  
 
If you are a female, you must not get pregnant while in this study or for one month after the 
study is over. The only certain way to not get pregnant is to not have sex. It is important to 
follow the approved birth control options below for pregnancy prevention if you are a woman of 
childbearing potential (able to get pregnant): 
 
Condoms with spermicide 
 
Condoms with diaphragm 
 
Diaphragm with spermicide 
 
Intrauterine device (IUD) with a documented failure rate of less than 1% per year 
 
Oral, injectable, or implanted hormonal contraceptives used in combination with an 
additional double barrier method such as condom or diaphragm with spermicide. 
 
If you become pregnant during the study, stop taking the study drug immediately and call your 
study doctor right away.  
 
Other Possible Risks Associated with Taking Part in this Study 
 
Blood draw: The risks of having your blood drawn include discomfort from the needle stick, 
bruising, bleeding, and rarely, infection. 
 
Stool swab collection: You may experience some discomfort with this collection, there is also 
a risk of getting stool on your skin or clothing during collection. 
 
Risks that are not known: 
Date of IRB Approval: 06/23/2021
Date of Expiration: 05/18/2022


Institutional Review Board 
Informed Consent Document for Research 
 
Lead Site Principal Investigator:  David Aronoff, M.D. 
 
Version Date:  May 24, 2021 
Lead PI Institution/Hospital: Vanderbilt University Medical Center 
 
 
  
Study Title: Misoprostol in the prevention of recurrent Clostridium difficile infection 
 
 
5 
 
Because this treatment is investigational, meaning non-FDA approved, there may be risks that 
we do not know about at this time.  
 
Good effects that might result from this study:  
a) The benefits to science and humankind that might result from this study: The results of this 
research may guide the future treatment of C. diff infection and science may gain further the 
understanding of why this infection sometimes comes back after treatment. 
b) The benefits you might get from being in this study: There is a chance that by taking part in 
this study you may be less likely to experience a recurrence of C. diff infection if you are 
randomized to the study drug. If you are randomized to the placebo group then it is unlikely 
you will experience any benefits. 
 
Other treatments you could get if you decide not to be in this study: 
You do not have to take part in this study to receive treatment. There are other options that may 
prevent C. diff from coming back. You can discuss these other options with your study doctor. 
 
Reasons why the study doctor may take you out of this study:   
You will be taken out of this study if the study doctor determines that it is no longer in your best 
interest to be in the study. If you are taken out of the study, you will be told the reason. 
 
What will happen if you decide to stop being in this study? 
If you decide to stop being part of the study, you should tell your study doctor.  Deciding not to 
be part of the study will not change your regular medical care in any way. 
 
Clinical Trials Registry:   
A description of this clinical trial will be available on www.clinicaltrials.gov, as required by U.S. 
Law.  This website will not include information that can identify you.  At most, the website will 
include a summary of the results.  You can search this Web site at any time. 
 
Confidentiality:   
All efforts, within reason, will be made to keep your personal information in your research record 
confidential but total confidentiality cannot be guaranteed. The study results will be kept in your 
research record for at least six years after the study is over or as long as we need the information for 
the study. All the information on paper will be locked in a secure location. Any information kept on a 
computer will be through REDCap (an electronic data management system), which has many 
safeguards. Only members of the study team will be able to see any of the information that would 
identify you. Any research data entered into your medical record will be kept as long as it is needed.  
 
This study receives support from the National Institutes of Health (NIH). Therefore, your study 
information is protected by a Certificate of Confidentiality.  This Certificate allows us, in some 
cases, to refuse to give out your information even if requested using legal means.  
  
Date of IRB Approval: 06/23/2021
Date of Expiration: 05/18/2022


Institutional Review Board 
Informed Consent Document for Research 
 
Lead Site Principal Investigator:  David Aronoff, M.D. 
 
Version Date:  May 24, 2021 
Lead PI Institution/Hospital: Vanderbilt University Medical Center 
 
 
  
Study Title: Misoprostol in the prevention of recurrent Clostridium difficile infection 
 
 
6 
 
It does not protect information that we have to report by law, such as child abuse or some 
infectious diseases.  The Certificate does not prevent us from disclosing your information if we 
learn of possible harm to yourself or others, or if you need medical help.  
  
Disclosures that you consent to in this document are not protected. This includes putting 
research data in the medical record or sharing research data for this study or future 
research. Disclosures that you make yourself are also not protected.  
Date of IRB Approval: 06/23/2021
Date of Expiration: 05/18/2022

Prot_000.pdf: 
Protocol 
 
PROCLAIM – A Randomized, Double-Blind, Placebo-Controlled Trial to Assess the Efficacy and Safety of 
Misoprostol in the Prevention of Recurrence of Clostridium Difficile Infection in Adults 
 
 
NCT 03617172 
 
 
May 24, 2021 


 
                                                                                            
 
 
 
 
 
Protocol 
Version 8.0: May 24, 2021 
 
 
A RANDOMIZED, DOUBLE-BLIND, PLACEBO-CONTROLLED TRIAL TO ASSESS THE EFFICACY AND 
SAFETY OF MISOPROSTOL IN THE PREVENTION OF RECURRENCE OF CLOSTRIDIUM DIFFICILE 
INFECTION IN ADULTS 
 
Short Title: PROCLAIM – Prevent Recurrence of Clostridium difficile1 Infection with 
Misoprostol 
 
 
 
 
 
 
 
 
 
David Aronoff, M.D., FIDSA 
Director, Division of Infectious Diseases 
Department of Medicine 
Vanderbilt University Medical Center 
 
 
 
 
1 Clostridium difficile was renamed to Clostridioides difficile, and all uses of each term in this document are 
equivalent   


CONFIDENTIAL 
 
Version 8.0: 24 May 2020 
2 
TABLE OF CONTENTS 
 
1. Study Description  
2. Study Design 
3. Drug Products 
4. Enrollment and Informed Consent 
5. Study Procedures 
6. Risks 
7. Reporting of Adverse Events or Unanticipated Problems 
8. Study Withdrawal/Discontinuation 
9. Statistical Considerations 
10. Privacy/Confidentiality Issues 
11. Follow-up and Record Retention 
12. References 
13. Appendices 
a. PheWAS Study Results 
b. Mouse Model of CDI Data 
c. IND Safe to Proceed Letter from FDA 
d. Misoprostol Package Insert 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
 


CONFIDENTIAL 
 
Version 8.0: 24 May 2020 
3 
 
PROTOCOL NUMBER:  
MISO-001 
INVESTIGATIONAL  
MEDICINAL PRODUCT:  
Misoprostol Oral Tablet 
 
PATIENT POPULATIONS: Patients 18 years of age or older with C. difficile Infection  
 
SPONSOR: Vanderbilt University Medical Center 
 
PARTICIPATING SITES: Vanderbilt University Medical Center (Nashville, TN), Washington 
University in St. Louis (St. Louis, MO), and The University of North Carolina at Chapel Hill 
(Chapel Hill, NC)  
 
1. OBJECTIVES, INTRODUCTION AND BACKGROUND  
 
Study Objectives 
The objectives of this study are to:  
1. Evaluate the efficacy of misoprostol for prevention of recurrent Clostridium difficile 
Infection (rCDI), by determining if misoprostol can modify the rate and severity of rCDI 
in adults ≥18 years of age during the first 8 weeks after standard of care is completed. 
2. Evaluate the safety and tolerability of misoprostol dosed orally for up to 14 days in 
adults (≥18 years of age) with Clostridium difficile Infection (CDI), measured by 
treatment emergent adverse events and standard laboratory assessments. 
3. Determine the effect of misoprostol on the gut microbiome and diarrhea 
occurrence/severity, in order to inform the design of future studies.  
Primary Hypothesis 
The treatment of patients with CDI with misoprostol, in addition to standard care, will result in a 
relative reduction in the rate of recurrence by 40% when compared to patients treated only with 
standard care. 
 
Introduction 
Clostridium difficile is a Gram-positive, spore-forming, toxin-producing bacillus that causes 
diarrhea and colitis often following exposure to antibiotics. It is emerging as one of the most 
common healthcare-associated infections. C. difficile infection (CDI) ranges in severity from mild 
diarrhea to severe colitis (sometimes resulting in septic shock and/or death). In addition, CDI has 
a high tendency to recur following treatment. First clinical recurrence of infection occurs in about 
25% of patients initially treated with antibiotic therapy with rate of recurrence increasing with 
age. Once a first recurrence has occurred, the risk for future recurrences exceeds 40% with 
highest rates being in older adults. The problem of recurrent CDI (rCDI) is a significant one, 
negatively impacting quality of life. There are patients for whom the cycle of recurrences does 
not remit. Experimental treatments such as fecal transplant are being investigated for patients 
with rCDI. Misoprostol, a prostaglandin analogue, has been shown to enhance recovery from CDI 


CONFIDENTIAL 
 
Version 8.0: 24 May 2020 
4 
in animal models, and decreased prostaglandin pathway signaling is associated generally with GI 
inflammatory conditions in a phenome wide association study (PheWAS) analysis. Misoprostol is 
FDA-approved for chronic use in the prevention of gastric ulcers given its established mucosal 
protective properties and inhibitory effects on gastric acid. This trial will evaluate the rate and 
severity of rCDI in patients treated with oral misoprostol vs placebo subsequent to the initiation 
of standard treatment(s) in adults ≥18 years of age following a primary episode of CDI. 
 
Background 
Clostridium difficile 
CDI is a serious problem worldwide, and in the United States, it comprises approximately 17.1% 
of hospital-associated infections affecting 500,000 individuals annually. It leads to worse clinical 
outcomes, more frequent readmissions, longer hospital stays, and costs billions of dollars each 
year in health care expenses.1 In this disease there are focal areas of epithelial loss and an 
exudate consisting of polymorphonuclear cells, fibrin, and cellular debris. Focal inflammation 
might arise after exposure to high local concentrations of toxins released from C. difficile 
adherent to specific small regions of the epithelium.2 There are two main toxins that cause 
damage, Toxins A and B, and these cause the symptoms of CDI.  
 
The most well-known risk factor for the infection is antibiotic use, but other risk factors include 
advanced age, hospitalization within preceding 2 months, proton-pump inhibitor use, and 
severity of underlying illness. Inflammatory bowel disease (IBD), which includes Crohn's disease 
(CD), ulcerative colitis (UC), and patients after ileal pouch anal anastomosis (IPAA), is known to 
be an independent risk factor for CDI.1 A history of CDI has also been associated with failure of 
IPAA reconstruction, which is a frequent treatment for UC.3 
 
Humoral immunity is an increasing area of interest in understanding the varied clinical 
presentation of CDI in both IBD and non-IBD patients, and also in investigating why an IBD patient 
may be more susceptible to clinical disease. Hughes et al. (2016) conducted a study to determine 
if IBD patients have any alteration in humoral response to C. difficile toxins A or B when compared 
with a group of control patients enrolled from outpatient clinics at the same medical center. They 
found that patients with UC have lower IgA levels to C. difficile toxins compared to those with 
Crohn’s disease and those after IPAA. Patients with IBD with prior CDI failed to demonstrate any 
increase in antitoxin IgG. These findings suggest that IBD patients may benefit from immunization 
strategies targeting C. difficile toxins.1 However, If prostaglandins maintain mucosal integrity and 
enhance almost all mucosal defensive mechanisms, perhaps they can protect against other 
exogenous insults, lower in the GI system.4  
 
A Prostaglandin E2 receptor 4 (EP4) receptor agonist, KAG-308, is in Phase II study for ulcerative 
colitis. Oral administration of KAG-308 suppressed onset of dextran sulfate sodium-induced 
colitis and promoted mucosal healing in a mouse model; it also prevented colorectal 
carcinogenesis by inhibiting colitis development in another mouse model (conversely, an EP4 
antagonist increased mortality).5 
 
PheWAS in drug discovery and drug repurposing 
One source of data guiding the design of this study is BioVU, a large repository of de-identified 
DNA samples that Vanderbilt University Medical Center (VUMC) has catalogued for >10 years 
from what otherwise would have been excess, discarded patient blood samples collected during 


CONFIDENTIAL 
 
Version 8.0: 24 May 2020 
5 
routine clinical testing. This biobank is a centralized resource for investigating genotype-
phenotype associations. Biospecimens within BioVU are linked to corresponding longitudinal 
clinical and demographic data derived from the Synthetic Derivative, VUMC’s de-identified 
database of EMRs.6–8 More recently, PheWAS has been introduced as a systematic and efficient 
approach to discover novel disease-variant associations and pleiotropy using BioVU.9 It is the 
comprehensive and diverse nature of the diagnostic information within EMRs that enables 
PheWAS.  PheWAS not only replicates known genetic-phenotypic associations, but also reveals 
new phenotypic associations with genetic variants, enhancing analyses of the genomic basis of 
human diseases and providing genetic support for drug discovery and drug repurposing efforts.10–
13  
 
Prostaglandin receptor function and misoprostol pharmacology 
Prostaglandin E2 (PGE2) plays a pivotal role in maintaining local homeostasis in a variety of 
pathophysiological settings, including the colon. PGE2 receptors (EPs) mediate the effects of this 
molecule and include four subtypes: EP1–4. PGE2 participates decisively in the defense of the 
colonic mucosa.  For example, misoprostol, a synthetic PGE1 analog, potently protects human 
colonic mucosa against mucosal insults. In addition, in mice, PGE2 and EP4-selective agonists 
significantly improved colitis induced by dextran sodium sulfate (DSS) treatment. Nakatsuji et al. 
(2015) investigated the effect of the recently identified EP4-association protein (EPRAP), which 
is essential for anti-inflammatory function of EP4 signaling in macrophages, on colitis and colitis-
associated tumorigenesis in mice. They observed that EPRAP deficiency exacerbated DSS-induced 
colitis and found support for the idea that EPRAP in macrophages functions crucially in 
suppressing colonic inflammation.14 
 
Misoprostol is utilized clinically as an anti-ulcer agent and signals through the protective PGE2 
EP2, EP3, and EP4 receptors.  It increases the cytoprotective levels of PGE2 necessary for 
maintaining integrity of the gastric mucosa.  The risk for nonsteroidal anti-inflammatory drug 
(NSAID)-induced gastric or duodenal ulcer is decreased with concomitant use of misoprostol.15 
Misoprostol can affect gastric, esophageal and duodenal mucosal integrity from insults related 
to acids and exodenous factors like NSAIDS. NSAIDs inhibit prostaglandin synthesis, thereby 
reducing mucus production, bicarbonate secretion, and mucosal blood flow.  Continuous blood 
flow through the microvessels is very crucial for the function and maintenance of structural 
integrity of the gastrointestinal tract.  Microcirculation delivers oxygen and nutrients to all tissues 
and cells and removes ad hoc generated toxic metabolites.  See Figure 1 below for the signaling 
pathway of these events.  
 
Ahluwalia et al. (2014) attempted to better understand the roles of PGEs and their receptors, 
signaling pathways including cAMP and CREB, and their relation to VEGF and angiogenesis in the 
tissue injury healing process (i.e. gastroduodenal and dermal ulcers).  Using an esophageal ulcer 
model in rats, they demonstrated that esophageal mucosa expresses predominantly EP2 
receptors and that esophageal ulceration triggers an increase in expression of the EP2 receptor, 
activation of CREB (the downstream target of the cAMP signaling), and enhanced VEGF gene 
expression.  Treatment of rats with misoprostol, a PGE1 analog capable of activating EP receptors, 
enhanced phosphorylation of CREB, stimulated VEGF expression and angiogenesis, and 
accelerated esophageal ulcer healing. In cultured human esophageal epithelial (HET-1A) cells, 
misoprostol increased intracellular cAMP levels (by 163-fold), induced phosphorylation of CREB, 
and stimulated VEGF expression. A cAMP analog (Sp-cAMP) mimicked, whereas an inhibitor of 


CONFIDENTIAL 
 
Version 8.0: 24 May 2020 
6 
cAMP-dependent protein kinase A (Rp-cAMP) blocked, these effects of misoprostol. These results 
indicate that the EP2/cAMP/protein kinase A pathway mediates the stimulatory effect of PGEs 
on angiogenesis essential for tissue injury healing via the induction of CREB activity and VEGF 
expression.16  
 
Figure 1. Protective Factors of the Gastric and Duodenal Mucosa 4 
 
PheWAS data supporting the use of misoprostol in CDI 
The PheWAS data summarized in Appendix A support, and are consistent with, a role for 
prostaglandin biology in various GI inflammatory diseases.  More specifically, these PheWAS data 
are consistent with evidence from a variety of sources which suggest a potential role for 
misoprostol in preventing recurrent CDI. 
 
Preclinical data supporting the use of misoprostol in CDI 
Data from mouse model (of CDI) experiments conducted in David Aronoff’s laboratory further 
suggest that misoprostol may be effective in helping to prevent recurrence of CDI infection and 
improve outcomes of patients with CDI (Data shown in Appendix B).  Mice first treated with 
cefoperazone and then challenged by inoculation with spores of C. difficile fared significantly 


CONFIDENTIAL 
 
Version 8.0: 24 May 2020 
7 
better when treated with misoprostol compared to placebo on multiple outcome variables 
including survival, diarrhea severity and intestinal permeability.    
 
2. STUDY DESIGN 
 
Experimental Design 
A total of 440 patients meeting enrollment criteria (listed below) will be consented and 
randomized across 3 sites.  All patients will receive standard care intended to treat CDI under the 
care of their physician.  Potential participants will be contacted and consent will be obtained if 
the individual is interested in participating in the study. Those who consent and enroll will be 
randomized to receive either misoprostol (200 mcg po BID) or matching placebo for 14 days. 
Participants will be monitored for a total time-period of approximately 70 days with the goal of 
monitoring for recurrence of CDI during an 8-week follow-up period from the time that standard 
care (often a short course of antibiotics) is completed.  Patients will have blood and stool samples, 
or rectal swabs if participants are unable to provide a stool sample, collected throughout the 
study (as described below) to assess biomarkers and to confirm recurrence of CDI (if necessary). 
The total study time period for study procedures followed by clinical monitoring is anticipated to 
be about 36 months (biomarker assays and other analyses may be completed after the 36-month 
time period).   
 
Screening 
Patients with a documented positive C. difficile lab test (lab tests outlined in inclusion criteria) 
will be screened with the possibility of enrollment. Outpatients meeting the inclusion criteria will 
be followed up with via phone call after a positive laboratory test.  Inpatients may be followed 
up with in person during the admission to discuss enrollment in the study.  Additionally, patients 
at other outside/community clinics who hear about the study may contact a study site to 
determine eligibility as well. 
 
Inclusion Criteria 
1. Episode of CDI, defined as both a and b below: 
a. A documented positive C. difficile toxin assay (enzyme immunoassay [EIA] or cell 
cytotoxicity assay) or NAAT for toxigenic C. difficile from a stool sample collected 
while the subject was symptomatic; and 
b. No other plausible explanation for diarrhea (e.g. laxatives). 
2. Be ≥18 years of age. 
3. Be able to provide signed and dated informed consent. 
4. Must be able to read and understand English. 
 
Exclusion Criteria 
1. Pregnant, nursing, or planning to become pregnant. 
2. If female: 
a. be pre-menopausal (cessation of menses less than or equal to 1 year) and not 
surgically/medically sterile or not following acceptable non-hormonal method of 
birth control such as abstinence, intrauterine device, or barrier control for at 
least 1 complete menstrual cycle before the enrollment visit, or 
b. not using estrogen/progestin containing products for at least 2 months before 
the enrollment visit through completion of the study. 


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3. Current or planned treatment with prostanoid therapy. 
4. Known hypersensitivity to misoprostol. 
5. Have any contraindication to oral/enteral therapy (e.g., severe nausea/vomiting or 
ileus). 
6. Have any clinically significant medical or surgical condition that in the investigator’s 
opinion could interfere with the administration of study drug, interpretation of study 
results, or compromise the safety or well-being of the subject. 
7. Unreliable access to telephone service to allow for contact with study personnel. 
 
3. DRUG PRODUCTS 
 
Misoprostol 
The study drug, misoprostol, will be purchased from Novel Laboratories, Inc. Misoprostol will be 
over-encapsulated using #00 locking capsules by each site’s Investigational Drug 
Service/Pharmacy for the participants at the respective sites. The capsules containing either 
misoprostol or placebo will be prepared in an identical fashion. To use misoprostol in this study, 
an Investigational New Drug (IND) application was submitted to the FDA and the safe to proceed 
letter from the agency (see Appendix C). 
 
The matching placebo compounded for this study is identical in appearance and will also be 
compounded at each site’s Investigational Drug Service/Pharmacy.  The Investigational Drug 
Service at the sponsor’s institution will manage all shipment of study drug products to the other 
investigational sites following federal and local shipping regulations. 
 
Dose and Mode of Administration 
Patients randomized to receive misoprostol will take two 100 mcg capsules, by mouth, twice per 
day (200 mcg po BID) for 14 days.  Per the drug label, patients will be instructed to take the 
capsules with food. Patients randomized to placebo will take two capsules, by mouth, twice per 
day for 14 days. 
 
4. ENROLLMENT AND INFORMED CONSENT 
 
Enrollment 
The study will be discussed with patients with a documented positive C. difficile lab test (lab tests 
outlined in inclusion criteria).  Outpatients will be scheduled to come into the study site for Visit 
1 of the study to be enrolled.  Alternatively, it may be arranged for a study team member, to go 
to the patient’s home for the enrollment visit.  Inpatients will be followed up with in person. 
 
Informed Consent 
Those participants meeting eligibility criteria will be informed by study staff that their 
participation is voluntary, and they may withdraw from the study at any time, for any reason 
prior to study completion. Participants will be given ample time to ask questions and will have 
their questions answered. Those that are interested will be consented. Study staff will give a copy 
of the informed consent document to the potential participant. No research procedures will be 
performed until after consent has been provided.  Consent or refusal to participate in this study 
will not affect medical care. An alteration of consent will be used in this study to allow for storage 
of the standard of care stool sample collected prior to consent if the sample is available. No 


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9 
samples will be obtained by study staff until participants have consented to participate in the 
study. 
 
Due to the timing of the clinical lab testing (which, per SOC, occurs before patients will be eligible 
to be enrolled), when possible, all available positive samples will be held by the clinical lab until 
patients consent, decline to consent and/or are determined not to be eligible to participate in 
the study.  Samples for patients who consent to participate will be frozen and kept for future 
study related testing, and all other samples will be discarded. 
 
 
5. STUDY PROCEDURES 
 
This is a randomized, double-blind, placebo-controlled study in patients age 18 years of age or 
older who have been recently diagnosed with an episode of CDI.  The study will be discussed with 
patients presenting with an episode of CDI as defined in the inclusion criteria. Patients who meet 
inclusion/exclusion criteria, will be consented and a pregnancy test will be completed for women 
of childbearing potential. If participants are pregnant, they will be determined to be a screen 
failure and will not proceed with any further study procedures. Participants will then be 
randomized to receive misoprostol or matching placebo. For patients taking antibiotics, 
consenting patients will be contacted on the last regular business day before the seventh day of 
oral antibiotic treatment, to remind all patients to begin the treatment that they were 
randomized to on day 7 of their oral antibiotic treatment; patients not taking oral antibiotics will 
start taking misoprostol 7-10 days after a positive CDI lab test. All randomized patients who were 
taking antibiotics will continue to take oral antibiotics for an additional 4-7 days after starting 
misoprostol treatment (such that those participants have a 4-7 day overlap of antibiotic and 
misoprostol treatment), and begin taking an investigational 200 mcg oral misoprostol or placebo 
two times a day (200 mcg po BID) for 14 days.  Participants will be randomized 1:1 between the 
misoprostol and placebo arms of the study. All randomized participants will be monitored for 
recurrence via phone call once per week for an additional 49 days (the total duration of study 
participation will be 62 days).  
 
In the event that a participant thinks he/she is having a recurrence of CDI, the participant will 
collect a stool sample and the sample will be tested for the presence of C. difficile.  If the 
participant has a confirmed recurrence (positive toxin test) participant will continue to 
participate in the study procedures as described in Table 1 below. The participant’s care 
subsequent to a recurrence will be the SOC at the discretion of participant’s physician (participant 
will stop taking misoprostol/placebo if recurrence occurs during misoprostol/placebo treatment).   
 
Throughout the study, participants will be followed through their inpatient or outpatient care, 
depending on their individual clinical conditions and routine care context. Safety will be 
monitored through the recording of adverse events (all AEs, including SAEs, will be collected 
while participants are on study drug, and only SAEs will be collected after participants have 
finished taking the study drug through the end of the study), and clinical safety laboratory testing. 
A diagnostic stool sample (or rectal swab samples if stool sample cannot be produced) will be 
collected (self-collection) to confirm CDI infection and at periodic study visits, including (1) the 
study visit at day -5 to 0, (2) in the case of a possible recurrence (for toxin testing), and (3) at the 
end of study period at day 62 (+4 day window for scheduling). Blood draws, when possible, will 


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also occur at periodic study visits, including (1) the study visit at day -5 to 0 (to obtain baseline 
data before treatment with study drug or placebo), (2) in the case of a possible recurrence (to 
test for antibodies and other biomarkers), and (3) at the end of study period (to test for 
antibodies and other biomarkers) at day 62 (+4 day window for scheduling) if possible at an in-
person visit. Participants who are unable to return for the day 62 visit will have the option to mail 
in a self-collected stool sample but would not contribute a blood sample. 
 
An overview of an example study timeline is presented in Figure 2 below, followed by more 
detail.  
 
 
Figure 2. Study Timeline 
 
Pre-enrollment period (Days -7 to Enrollment Study Visit): Patients with a documented positive 
C. difficile lab test (lab tests outlined in inclusion criteria) will be screened for the proposed study 
and the possibility of enrollment.  Once a positive C. difficile laboratory test (Inclusion Criteria 1b) 
is obtained, outpatients will either be asked to schedule a time to come to the study site for an 
enrollment visit or study staff may travel to the patient for the enrollment visit (if distance is 
reasonable etc.).  When possible, stool samples will be held such that stool samples from patients 
with confirmed CDI infection (defined in inclusion criteria) who give informed consent and enroll 
in the study will be available for future analyses. 
 
Enrollment, Study Visit and Randomization (Day -5 to Day 0): Participants meeting 
inclusion/exclusion criteria will be consented and upon completion of a negative pregnancy test 
result, if needed, the patient will be randomized to misoprostol or placebo.  The project 
statistician will implement a permuted block allocation scheme, likely based on block sizes of 2 
and 4, stratified by site, sex, and severity of illness, as defined by IDSA criteria for severity (if 
bloodwork is not available, the patient will be assumed to be “non-severe”): 
• Non-severe (or bloodwork unavailable): Leukocytosis with a WBC count of 
≤15,000 cells/mL and serum creatinine <1.5 mg/dL 


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• Severe: Leukocytosis with a WBS count >15,000 cells/mL or serum creatinine >1.5 
mg/dL 
• Fulminant: Hypotension or shock, ileus, megacolon 
Randomization will be accessed at each site through REDCap. The randomization module in 
REDCap allows the statistician to load a randomization table that will allow the sites to click a 
'randomize' button.  Study participants will be dispensed study drug at this visit and will be 
instructed on the use of the medication. If study participants are on a course of antibiotics, they 
will begin taking investigational therapy on Day 0 (Day 7 of antibiotics) in addition to oral 
antibiotics.  If study participants are not on a course of antibiotics, they start taking the study 
drug 7-10 days after their positive CDI lab test.  Participants will be given the study medication 
and stool diary and instructed on how to complete the diary. Blood samples (if possible) and stool 
samples (or rectal swabs) will be collected at this visit. A stool sample collection kit to collect a 
stool sample at home for future visits will be provided. 
 
Study Drug Administration Period (Days 0-13): Participants will be called (or visited in person if 
inpatient or present at clinic) on Day 0 (or on the last regular business day before day 0 occurs) 
to be reminded to begin study drug. Participants will continue taking oral antibiotics, if applicable, 
such that there is a 4-7 day overlap during at which time the participant will be taking both oral 
antibiotics and investigational therapy.  They will be instructed to record date and time of each 
study drug administration (approx. 12 hours apart), all adverse events, date and time of any 
unformed (loose or watery) stools, and any other medications taken.  
 
Follow-up phone calls 1 & 2 (Day 3 +/- 1 day, Day 7 +/- 1 day): All participants will be contacted 
on Day 3 and Day 7 of taking the study medication to review symptoms, discuss adherence to 
study drug, review stool and drug diary, and complete the CDI QOL Patient Questionnaire and 
the symptom questionnaire. 
 
Follow-up phone calls 3-9 (Days 13-55 +/- 2 days): Study personnel will contact participants at 
least once weekly through Day 62 (via telephone contacts) to inquire about any occurrence of 
diarrhea and/or loose/watery stools, completion of a symptoms questionnaire, and the CDI QOL 
Patient Questionnaire.   
 
Follow-up visit (Day 62-66): If possible, participants will return on day 62 for a study visit to 
collect blood and stool samples; a four day window is permitted in case of scheduling difficulties.  
Participants will complete the CDI QOL Patient Questionnaire, the symptom questionnaire, blood 
samples will be collected (if possible) and patients will be asked to bring in a stool sample (if a 
stool sample cannot be provided, participants will be asked to provide a rectal swab). If patients 
cannot attend this visit on site, a home visit can be arranged for the study team to come to their 
home or they will be given the option to provide a self-collected stool sample that will be picked 
up from their home. A blood sample will not be obtained if an in-person visit is not completed. 
 
Recurrence visit (Day 7 - Day 62): If possible, participants exhibiting symptoms of a recurrence 
of their CDI after completion of antibiotic treatment will come to the study site for a follow-up 
visit. At this study visit participants will complete the CDI QOL Patient Questionnaire, the 
symptom questionnaire, blood samples will be collected (if possible) and patients will be asked 
to bring in a stool sample to this visit. A home visit can also be arranged if the participant would 
prefer a site study team member to come to their home. If an in-person visit is not able to be 


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scheduled within 24 hours of the recurrence of symptoms, the participant will be asked to follow 
the instructions provided so that a stool sample/rectal swab can be picked up at their home. 
 
 
 
 
 
 
 
A schedule of the study events is detailed below in Table 1. 
 
Procedures 
Clinical Care 
Day -7 to -3 
Pre-Enrollment Period 
Day -7 to enrollment 
Enrollment Visit  
Day -5 to Day 0 
Start Study Drug 
Day 0 
Follow-up Phone Calls 
1&2  Day 3 & Day 7 
( +/- 1 day) 
Follow-up Phone Call 3 
Day 13 (+/-2 days) 
Follow-up Phone Call 4 
Day 20 (+/-2 days) 
Follow-up Phone Call 5 
Day  27 (+/-2 days) 
Follow-up Phone Call 6 
Day 37 (+/-2 days) 
Follow-up Phone Call 7 
Day 41 (+/-2 days) 
Follow-up Phone Call 8 
Day 48 (+/-2 days) 
Follow-up Phone Call 9 
Day 55 (+/-2 days) 
Recurrence Visit1 
Day 14 - Day 62 
Final Study Visit  
Day 62 (+4 days) 
Start Standard of 
Care Treatment 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Contact CDI+ 
potential 
participants 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Informed consent 
 
 
X 
X⁸ 
 
 
 
 
 
 
 
 
 
 
Demographics 
 
 
X 
X⁸ 
 
 
 
 
 
 
 
 
 
 
Medical history 
 
 
X 
X⁸ 
 
 
 
 
 
 
 
 
 
 
Pregnancy test 
 
 
X 
X⁸ 
 
 
 
 
 
 
 
 
 
 
Concomitant 
medication 
review 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Physical Exam 
 
 
X 
X⁸ 
 
 
 
 
 
 
 
 
 
 
Vital signs 
 
 
X 
 
 
 
 
 
 
 
 
 
X1 
X 
Height 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
Weight 
 
 
X 
 
 
 
 
 
 
 
 
 
X1 
X 
CDI toxin test2 
X2 
 
 
 
 
 
 
 
 
 
 
 
X1, 2 
 
Participants begin 
taking study drug 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
Confirm CDI 
symptom 
resolution 
 
 
X 
X 
 
 
 
 
 
 
 
 
 
 
Symptom 
questionnaire 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X1 
X 
CDI Patient 
Quality of Life 
Questionnaire 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X1 
X 
Participant 
Medication & 
Stool Diary 
 
 
 
X 
X 
X 
 
 
 
 
 
 
 
 
Blood draw 
 
 
X 
 
 
 
 
 
 
 
 
 
X1 
X 
CBC w/ diff2  
 
 
X2 
 
 
 
 
 
 
 
 
 
X1, 2 
X2 
Complete 
Metabolic Panel2 
 
 
X2 
 
 
 
 
 
 
 
 
 
X1, 2 
X2 
Stool Sample or 
Rectal Swab 
Collection  
X4 
 
X 
 
 
 
 
 
 
 
 
 
X1 
X 
Serum anti-toxin 
IgG to TcdA & 
TcdB6 
 
 
X6 
 
 
 
 
 
 
 
 
 
X1, 6 
X6 
Phone call to 
participants 
 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
Randomization 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 


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13 
Procedures 
Clinical Care 
Day -7 to -3 
Pre-Enrollment Period 
Day -7 to enrollment 
Enrollment Visit  
Day -5 to Day 0 
Start Study Drug 
Day 0 
Follow-up Phone Calls 
1&2  Day 3 & Day 7 
( +/- 1 day) 
Follow-up Phone Call 3 
Day 13 (+/-2 days) 
Follow-up Phone Call 4 
Day 20 (+/-2 days) 
Follow-up Phone Call 5 
Day  27 (+/-2 days) 
Follow-up Phone Call 6 
Day 37 (+/-2 days) 
Follow-up Phone Call 7 
Day 41 (+/-2 days) 
Follow-up Phone Call 8 
Day 48 (+/-2 days) 
Follow-up Phone Call 9 
Day 55 (+/-2 days) 
Recurrence Visit1 
Day 14 - Day 62 
Final Study Visit  
Day 62 (+4 days) 
Dispense study 
drug 
 
 
X 
X⁸ 
 
 
 
 
 
 
 
 
 
 
Complete Case 
Report Forms 
(CRFs) 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X1 
X 
 Collection of 
adverse events 
 
 
 
X 
X 
 
 
 
 
 
 
 
 
 
Collection of 
serious adverse 
events 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Table 1: Schedule of events 
 
Procedures 
Clinical Care 
Day -7 to -3 
Pre-Enrollment Period 
Day -7 to enrollment 
Enrollment Visit  
Day -5 to Day 0 
Start Study Drug 
Day 0 
Follow-up Phone Calls 
1&2  Day 3 & Day 7 
( +/- 1 day) 
Follow-up Phone Call 3 
Day 13 (+/-2 days) 
Follow-up Phone Call 4 
Day 20 (+/-2 days) 
Follow-up Phone Call 5 
Day  27 (+/-2 days) 
Follow-up Phone Call 6 
Day 34 (+/-2 days) 
Follow-up Phone Call 7 
Day 41 (+/-2 days) 
Follow-up Phone Call 8 
Day 48 (+/-2 days) 
Follow-up Phone Call 9 
Day 55 (+/-2 days) 
Recurrence Visit1 
Day 14 - Day 62 
Final Study Visit  
Day 62 (+4 days) 
Metabolomic 
profiling of stool 
sample3, 5 
X3,5 
 
X3,5 
 
 
 
 
 
 
 
 
 
X1,3,5 
X3,5 
Ribotyping of 
stool sample5 
X3,5 
 
X3,5 
 
 
 
 
 
 
 
 
 
X1, 3, 5 
X3,5 
Nutritional 
Immunity/Fecal 
metals analysis6 
X3,6 
 
X3,6 
 
 
 
 
 
 
 
 
 
X1,3, 6 
X3,6 
Microbiome 
sequencing of 
stool samples7 
X3,7 
 
X3,7 
 
 
 
 
 
 
 
 
 
X1, 3, 7 
X3,7 
1 – This visit will occur if participant experiences symptoms of CDI recurrence. 
2 – Completed at individual sites’ local Clinical Laboratory. 
3 – To be completed at completion of study. 
4 – Positive samples will be retained by local Clinical Laboratory until consent is obtained, all samples that are retained and consent is not obtained 
will be discarded by local Clinical Laboratory.  Note, it may not always be possible to obtain this sample. 
5 – To be completed by Microbiology lab at Washington University in St. Louis. 
6 – To be completed by sponsor investigator’s lab at Vanderbilt University Medical Center. 
7 – To be completed by Vanderbilt University Medical Center’s Genomics Core Facility. 
8 – To be completed if not obtained prior to Day 0. 
Table 2: List of the research laboratory tests that will be completed on the stool samples collected throughout the 
study.  Note, it may not be possible to collect all samples from all participants.  The exploratory analyses will be 
completed on the samples it is possible to collect. 
 
A schedule of the laboratory tests that will be completed on the stool samples collected at the 
following study visits appears above in Table 2. 
 
Misoprostol may have gastrointestinal (GI) side effects (e.g., diarrhea, abdominal cramping) 
during the first few days of use, which is not to be confused with recurrence of disease. Since 
patients do not generally experience a recurrence until after they have completed the full course 
of antibiotic therapy, any GI symptoms occurring during the first 4-7 days of investigational 
therapy (which overlaps with antibiotic treatment) will be presumed to be a side effect of 
misoprostol (captured as an adverse event) and not a recurrence of CDI. Depending on the 


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severity of symptoms (specifically GI symptoms such as diarrhea and abdominal cramping), 
misoprostol dose may be reduced to minimize discomfort (100 mcg po BID minimum dose) after 
contacting the lead center for guidance. 
 
An independent unmasked Data Safety Monitoring Board (DSMB, more detail in section 7 below) 
will review available data at predefined time points during the study.  
 
Both the participant and everyone interacting with the participant on the study team (PI, 
coordinator, nurses, etc.) will be blinded throughout the duration of the study.  There is no plan 
to ever make the treatment assignments known to the participants during or after the 
completion of the study.  In the event of a serious adverse event requiring knowledge of the 
treatment allocation to protect the safety and welfare of the participant, the blind may be broken 
following study standard operating procedures. Study personnel will remain blinded.     
 
Concomitant Medications 
All medications that a patient is taking at Day -7 through completion of final study follow-up visit 
will be collected. At each study visit, all concomitant medications will be reviewed and changes 
will be recorded. Medications will also be reviewed over the phone at each follow up phone call 
and changes will be recorded. 
 
Medication information collected will include: 
• Drug 
• Dose  
• Unit 
• Route of administration 
• Frequency 
 
Screen Failures and Study Withdrawals 
Participants not meeting inclusion/exclusion criteria will not be allowed to participate in any 
further study procedures. Participants who are no longer allowed to participate in the study or 
voluntarily remove themselves from the study after consent and prior to randomization will be 
considered a Screen Failure.  
 
Participants who are removed from the study, either by discretion of the PI or participant 
withdraws consent after randomization, will be considered a Study Withdrawal. 
 
6. RISKS 
 
Drug Related Risks 
See package insert. 
 
Misoprostol has been used clinically for several decades and potential risks are well documented 
in the medical literature.  Specific to this study, Misoprostol may have gastrointestinal (GI) side 
effects (e.g., diarrhea, abdominal cramping) during the first few days of use.  Depending on the 
severity of symptoms, misoprostol dose may be reduced to minimize discomfort (100 mcg po BID 
minimum dose). 
 


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Any clinical event of diarrhea or loose/watery stools will be recorded on a case report form. 
Participants who have ≥3 unformed (loose or watery) stools within a 24-hour period (or those 
with any diarrhea of concern to the subject or investigator) will contact study personnel as soon 
as possible for clinical assessment. If the clinical event of diarrhea occurs after completion of final 
antibiotic dose (if applicable), arrangements will be made for collection of a stool sample to 
determine whether the event is a recurrence of CDI. Whenever possible, the collection of the 
stool sample should occur at the study site or collected by a study team member at an in-home 
visit. However, because it is important to collect a stool sample while the subject is symptomatic, 
participants who are outpatients will be provided with a stool collection kit and instructions for 
stool sample collection at home; participants can bring these samples with them to the study 
site, have them available for an at-home visit, or have them picked up and delivered to the study 
site under direction of study personnel. Stool samples collected in association with an adverse 
event of diarrhea or loose/watery stools will be tested for C. difficile toxin to confirm if recurrence 
has occurred.  All other aspects of management of participants with diarrhea or loose/watery 
stools (including additional laboratory testing and any treatment for diarrhea/CDI) will be at the 
discretion of the treating physician based on clinical presentation.  
  
Other Risks 
1. Blood sampling: There are minor risks and discomforts associated with blood sampling 
and venipuncture.  This may cause a brief period of pain and possibly a small bruise at the 
site. Occasionally, a person feels faint when their blood is drawn. There is a small risk of 
bleeding after removal of the needle and possibly a bruise at the site, which can be 
prevented by tight compression on the site. Rarely, an infection develops which can be 
treated. Proper aseptic techniques will be used to minimize these side effects. 
2. Pregnancy Risks: If a subject is a female who could be pregnant, a urine pregnancy test 
will be done to make sure she is not pregnant. If she is pregnant, she will be excluded 
from the study. 
3. Other Risks: There may be risks that are not known at this time. If any new information 
becomes known, participants will be notified. 
 
7. REPORTING OF ADVERSE EVENTS OR UNANTICIPATED PROBLEMS INVOLVING RISK TO 
PARTICIPANTS OR OTHERS 
 
Adverse Event Reporting 
Serious adverse events (SAEs) may occur in older adults in the study population, but are not 
generally expected otherwise.  Note, serious adverse events related to the use of the study drug 
(misoprostol) are not expected to occur based on extensive previous human experience.  Since 
misoprostol is a marketed drug with a known safety profile, non-serious adverse events will only 
be collected while participants are receiving study drug, after completion of the study drug only 
SAEs will be collected. Individual sites must report SAEs to the sponsor within one business day 
of the study team’s awareness of the event.  Additionally, the site will report all SAEs to their IRB 
per local policy.  
 
All study personnel will be responsible for the accurate documentation, investigation and follow-
up of all serious adverse events and unanticipated problems involving risks to participants and 


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others that are possibly related to study participation.  When a serious adverse event is identified, 
the necessary medical personnel will immediately be notified, as well as the DSMB Chair.  
 
Adverse Event 
An AE is any unexpected medical occurrence in any study participant administered an 
investigational product and may or may not have a causal relationship with the treatment. An 
adverse event can therefore be any unfavorable and unintended sign (including an abnormal 
laboratory finding), symptom, or disease temporally associated with the use of a medicinal 
(investigational) product, whether or not considered related to the medicinal (investigational) 
product. 
AEs include any of the following: 
• Worsening (change in nature, severity or frequency) of conditions present at the onset of 
the trial 
• Subject deterioration due to the primary illness 
• Intercurrent illnesses 
• Drug interactions 
• Events related or possibly related to concomitant medications 
• Abnormal laboratory values or changes of vital signs, as well as significant shifts from 
baseline within the range of normal, which the Investigator considers to be clinically 
significant 
 
Serious Adverse Event 
During clinical investigations, serious AEs may occur.  If the event is suspected to be drug-related, 
the event may be significant enough to lead to important changes in the way the medicinal 
product is developed (e.g., change in dose, population, needed monitoring, consent forms). This 
is particularly true for reactions, which, in their most severe forms, threaten life or function.  A 
serious AE (SAE) is any untoward medical occurrence that: 
• Results in death 
• Is life-threatening. ‘Life-threatening” refers to an event in which the subject is at risk of 
death at the time of the event; it does not refer to an event which hypothetically might 
have caused death if it were more severe (ICH E6). 
• Requires inpatient hospitalization or prolongation of existing hospitalization 
• Results in persistent or significant disability/ incapacity (as per PI’s opinion) 
• Is a congenital anomaly/birth defect 
• Is another medically important condition.  Important medical conditions that may not 
result in death, be life-threatening or require hospitalization may be considered as SAEs 
when, based upon appropriate medical judgment, they may jeopardize the subject or may 
require intervention to prevent one of the outcomes listed in the definition above. 
Examples of such events are intensive treatment in an emergency room or at home for 
allergic bronchospasm; blood dyscrasias or convulsions that do not result in 
hospitalization; or development of drug dependency or drug abuse [Code of Federal 
Regulations Title 21, Volume 5, 21CFR312.32, revised April 1, 2006]. 
 
Please note: Serious is not synonymous with severe. An event may be severe (e.g., severe 
headache) but still be of minor medical significance. Serious refers to an event that poses a threat 
to the subject’s life or functioning. 


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Assigning Severity to an Adverse Event 
Severity of SAEs will be categorized based on Common Terminology Criteria for Adverse Events 
(CTCAE) Version 5.0.  
 
Assigning Relationship of Adverse Event to Study Drug (Causality) 
The PI will determine the relationship of each SAE to study drug (i.e., causality) by using the 
classification criteria ‘not related’, ‘possibly related’, or ‘probably related’. Descriptions of the 
three classification categories are as follows: 
 
Not Related 
Exposure to study drug has not occurred; administration of study drug and the adverse event are 
not reasonably related in time; or the AE/SAE is considered by the Investigator to be due to a 
pre-existing condition, a known manifestation of the target disease, a recurrent condition, or is 
likely explained by environmental or diagnostic therapeutic factors or was pre-existing and did 
not deteriorate. 
 
Possibly Related 
The AE/SAE occurred during or within a reasonable period of time after administration of the 
study drug, or a pre-existing event worsened within an appropriate period of time after 
administration of study drug, but the AE/SAE could be explained equally well by factors or causes 
other than exposure to the study drug.  This category will also be used if there is a lack of 
information, or insufficient or conflicting evidence exists for classifying the causality of the 
AE/SAE. 
 
Probably Related 
The AE/SAE occurred during or within a reasonable period of time after administration of the 
study drug or a pre-existing event worsened within an appropriate period of time after 
administration of study drug, and at least one of the following criteria is applicable: 
• the event could not be explained by the clinical condition or history of the subject, 
environmental or toxic factors, or other diagnostic or therapeutic measures; 
• the event was an expected ADR associated with study treatment or a class-labeled drug 
effect; 
• the AE/SAE subsided or disappeared after withdrawal or dose reduction of study 
treatment; or 
• the AE/SAE recurred after re-exposure to study treatment. 
 
Adverse and Serious Event Recording 
During study drug administration all AEs and SAEs will be recorded.  Each event occurring to a 
subject, either spontaneously revealed by the subject or observed by the Investigator, whether 
believed by the sponsor to be related or unrelated to the study drug, must be recorded on the 
AE/SAE Case Report Form within the EDC. 
 
After a participant has completed taking the study drug only SAEs (not all AEs) will be recorded.   
 
Site investigators will record all appropriate information into the CRF including time of onset, 
duration, and the precautions carried out.  


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Laboratory results will be recorded on the case report form and the investigator will indicate 
whether abnormal results (high or low) are clinically or are not clinically significant. Clinically 
significant laboratory abnormalities will be entered on the ‘AE/SAE Event Form’ of the CRF.  
Clinically significant changes in vital signs (e.g., tachycardia) or other clinically significant changes 
observed by the physician will be entered in the appropriate CRF.  
 
Serious Adverse Event Reporting 
All SAEs must be reported to the sponsor within 24 hours of knowledge of the event to the lead 
site/sponsor and will be reported to the IRB, DSMB Chair and FDA in accordance with applicable 
regulations and policies.  
 
Data Safety Monitoring Board (DSMB) 
An independent, unmasked Data Safety Monitoring Board (DSMB) will review available data at 
predefined time points during the study according to a monitoring plan.  Briefly, all SAEs will be 
reviewed by one member of the DSMB (DSMB Chair) and this individual will make a 
determination if it is clear that the SAE was not (or was) related to the use of study drug.  If it is 
unclear whether or not the SAE was not (or was) related to the use of study drug, then the case 
will be discussed by the entire DSMB.  All SAEs will be reported promptly to the sponsor and will 
be reported to the IRB and FDA in accordance with applicable regulations and policies.  
 
8. STUDY WITHDRAWAL/DISCONTINUATION 
 
Participants will be advised that they have the right to withdraw from the study at any point in 
time for any reason.  If during the course of the study they no longer want to participate, they 
will be withdrawn from the study.  Subjects who wish to withdraw from the study will be asked 
permission to allow the study team to review their medical records through the Day 62 visit 
window to determine if recurrence occurred.  
 
Participants who begin to exhibit symptoms of recurrence between the enrollment visit and  
randomization will be considered a screen fail for the study. 
 
9. STATISTICAL CONSIDERATIONS 
 
The text below is a summary of the statistical analysis plan (SAP), for full details see the SAP. 
 
Primary Hypothesis 
The treatment of patients with CDI with misoprostol, in addition to standard care, will result in a 
relative reduction in the rate of recurrence by 40% when compared to patients treated only with 
standard care. 
 
Planned Interim Analyses 
 
We will perform an interim safety analysis after 44 patients (10% of accrual) have completed 
study treatment.  It is expected that of the first 44 patients, although there might be dose 
adjustments, there will be no greater than 5 withdrawals due to diarrheal events reasonably 
attributed to misoprostol use. If more than 5 diarrheal events reasonably attributed to 


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19 
misoprostol use occur among the first 44 patients which require study withdrawal, enrollment 
will be interrupted for safety concerns. All currently enrolled participants will be followed to 
completion and the data will be analyzed as planned to estimate effect sizes; no statistical testing 
will be performed. The data will be presented to the DSMB to determine whether the study 
should proceed or not.  If no more than 5 diarrheal events (which can be attributed to treatment 
with misoprostol) occur among the first 44 patients which require study withdrawal occur, the 
study will continue without interruption of enrollment. 
 
A planned interim futility analysis at 176 participants will be conducted. This will allow the study 
to be terminated early in the event there is a less than acceptable chance of detecting the desired 
effect, therefore not wasting funds or exposing patients to unnecessary risk with no 
commensurate benefit.  We propose the interim analysis for efficacy will be completed using the 
Lan-DeMets procedure with an O’Brien-Fleming stopping boundary to account for the 2 interim 
analyses and the final analysis. The significance boundary for final analysis will be P ≤ .048. The 
interim analysis for futility will be based on the stochastic curtailment method. The interim 
analyses for futility will be performed by calculation of conditional power, an estimate of the 
probability that the study shows a statistically significant effect on the primary endpoint, i.e. a 
reduction in the rate of recurrence, given the results to date and assumptions regarding outcome 
through the end of the study. A recommendation to stop the trial for futility will require a 
conditional power below 30%, under the observed efficacy trend at the time of interim analysis, 
with two-sided type I error less than 5%. The proposed sample size of 196 per arm is adjusted for 
the interim analyses, i.e., types I and II errors. 
 
Endpoints 
To evaluate the exploratory objectives of the study assessing oral misoprostol compared to 
placebo in patients aged 18 years or older with an episode of CDI, the following endpoints will be 
measured: 
 
• Primary endpoint: Rate of clinical recurrence of CDI in patients aged 18 years or older 
during the first 42 days after standard of care therapy is stopped.  Rate of recurrence 
among misoprostol treated participants will be compared with that of placebo treated 
participants to determine if misoprostol has efficacy in preventing rCDI.  rCDI will be 
defined as people who meet criteria for CDI in the 42 day follow-up period. 
• Safety endpoint: Safety and tolerability of misoprostol in patients aged 18 years or older 
with rCDI compared to placebo as measured by treatment emergent adverse events and 
standard laboratory assessments. 
• Secondary endpoint: Number of recurrences during the follow-up period (for those who 
have a first recurrence during the follow-up time period) 
• Secondary endpoint: Time to resolution of diarrhea (TTROD; for those with recurrence). 
• Exploratory endpoint: Serum biomarkers/toxin A and B antibodies (from blood draws).  
• Exploratory endpoint: Recovery of bowel microbiota (from microbiome collection).  
• Exploratory endpoint: Titer of toxins A and B in stool (for those with recurrence). 
• Exploratory endpoint: Severity of disease (for those with recurrence), determined using a 
combination of severity criteria used in Hospital specific guidelines, SHEA/IDSA guidelines, 
Zar criteria, or other sources: 
• 
WBC count  


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20 
• 
Serum creatinine  
• 
Temperature  
• 
Albumin level  
• 
ICU or step down unit admission 
• 
Endoscopically or histologically confirmed pseudomembranous colitis 
• 
Toxic megacolon, perforation, colectomy, or septic shock requiring ICU 
admission and pressors 
• 
Hospital readmission 
• 
Validated quality of life instrument 
 
Assessments by interview, recording of adverse events, clinical laboratory tests, and other 
evaluations.  Patients will be asked to record self-administration of study drugs and symptoms. 
 
Safety analyses will include displays of adverse events and laboratory values and will include 
descriptive statistics for the study population where applicable.  Adverse events will be 
summarized by treatment, intensity, and relationship to study drug.  
 
Statistical methods 
For data summary, frequency tables will be generated for categorical variables, and continuous 
variables will be expressed as means ± SD or medians and interquartile ranges as appropriate.  
We will then compare the outcomes of interest between the patient groups who receive 
treatment vs placebo.  The primary outcome of this study is the clinical recurrence rate of CDI in 
patients aged 18 years or older during the first 8 weeks after SOC therapy is completed.  
Secondary outcomes will include treatment emergent adverse events (AE), standard laboratory 
assessment for AE, recovery of bowel microflora, time to recurrence, time to resolution of 
diarrhea, severity of diarrhea, titer of toxin in stool.  Serum biomarkers/toxin antibodies from 
blood draws will be recorded for each patient.  Comparisons between control and treatment 
patients will use t-test or Kruskal-Wallis test for continuous variables and Fisher exact tests for 
categorical variables. Secondarily, we will model outcomes using generalized linear mixed-
models with linear or logit link functions as appropriate. A two-tailed p < 0.05 will be considered 
significant. Data analyses will be performed using standard statistical software, such as the 
current version of R (http://www.r-project.org/). 
 
The sample size calculation is based on relative reduction in the proportion of participants with 
recurrent events between the placebo and misoprostol treatment group. We assume the patient 
population may be heterogeneous and has variable baseline recurrences rate due to their 
baseline variables before applying misoprostol, such as disease severity (as determined by IDSA 
guidelines), sex differences, or study site differences (stratification variables).  However, 
recurrence rates are, in general, well established, with age being a key variable influencing 
recurrence rates. For patients experiencing their first ever episode of CDI, a conservative 
estimate of recurrence rate is 20% and a recurrence rate more consistent with previous data for 
older patients is closer to 30-40%. There are demographics of patients with even higher 
recurrence rates.  We performed power calculations using a range of combinations of control 
group recurrence rate (p0) and % reduction in recurrence rate by the treatment (effect size). 


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Version 8.0: 24 May 2020 
21 
 
Figure 2. Power Curves for control recurrence rates from 20% to 60% 
As illustrated in Figure 2, for a set of control infection recurrence rates (p0) varying from 0.2 – 
0.6 (corresponding subpanels), the power curves show the sample size required (assumed same 
for the control and the treatment groups) as a function of % reduction in recurrence rate (x-
coordinate), in order to achieve the anticipated power in the 70% - 90% range (red, green, and 
blue lines).  The vertical dashed line indicates the anticipated 40% reduction in recurrence rate.  
For example, to gain 80% power for a 50% reduction in recurrence rate, we would require 119 or 
80 patients in each group when control recurrence rate is at 0.3 or 0.4 respectively.  To gain 80% 
power for a 40% reduction in recurrence rate, we would require 196 or 132, patients in each 
group when control recurrence rate is at 0.3 or 0.4 respectively.  We aim to collect data on 196 
participants in each group (392 total participants) as this will afford us 80% power to detect a 
40% reduction in recurrence when the control recurrence rate is 30%.  We aim to enroll 440 total 
participants which would allow for a 10% overall attrition rate. 
 
10. PRIVACY/CONFIDENTIALITY ISSUES 
 
Data will be maintained in paper and computer files that will be locked and password-protected. 
Only the study team will have access to identifying information.  Information will be maintained 
indefinitely by the PI. 
 
Each site will be required to have a local coordinator(s) who will be responsible for entering the 
study information into the web-based database and uploading the identifiable source 
documents/medical records associated with the study information. For Vanderbilt participants, 
access to the electronic health record (EHR) will be provided to study data monitors. A web-based 
database housing the electronic case report forms (eCRFs) has been designed for the study, which 
will improve efficiency, lower cost of the study, and speed up publication of the results. Use of 
drop-down selection lists, radio buttons, checkboxes, and validation checks will be incorporated 


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22 
to aid the speed, accuracy and consistency of data entry. The database will be backed up 
regularly. 
 
This project will utilize the Research Electronic Data Capture (REDCap) platform for data 
collection and management.  Project team members listed as Key Study Personnel with existing 
electronic health record (EHR) system access rights may also be granted use of REDCap Clinical 
Data Pull (CDP) tools.  These tools are designed to enable transfer of relevant study-related data 
directly from the EHR into REDCap. The latest version of each eCRF and any applicable source 
document worksheets will be available as a PDF file on the REDCap website for use by study 
personnel.  
 
The web-based Randomization Module will be used by authorized site personnel for the purpose 
of randomizing eligible patients. The Study Coordinator (or other appropriate study team 
member) will log onto the REDCap system via the PROCLaIM Portal (Proclaimstudy.org) using a 
unique username and confidential password. When a subject is deemed eligible, a unique subject 
ID and record will be created. Once the Study Coordinator has entered the required subject 
information and clicked “Randomize”, the computer program will display a message that the 
treatment assignment has been sent to the pharmacy for the subject.  The subject is considered 
randomized at the time the REDCap system generates the treatment assignment.  
 
VUMC employs several layers of data protection to ensure data security. The first part of security 
is physical protection of the hardware systems, access to which is limited to authorized 
personnel. By limiting access, ensuring only authorized personnel have access, and tracking all 
entry, we can ensure this risk is minimal. Additionally, REDCap has a built-in audit trail that tracks 
all user activity and changes made to the data entry fields with a date-time stamp. 
 
11. FOLLOW-UP AND RECORD RETENTION 
 
The research material obtained in this study will be recorded data available through the EMR and 
volunteered by participants, and recorded during study procedures, obtained specifically for 
research purposes and kept indefinitely. Study teams (except VUMC) will be expected to upload 
non-redacted source into the EDC for source verification. For more detail regarding data 
monitoring, please refer to the trial’s Data Safety Monitoring Plan. Blood and stool samples will 
be obtained for screening purposes and for analysis of the various parameters under study.  
Initial, available diagnostic stool specimens for patients who test positive for CDI, but do not 
enroll in the study, will follow local lab/hospital policy for discarding sample.  A separate secure 
database of identified patient information will be maintained in order to store and analyze 
collected study data.  Blood and stool samples will be stored at VUMC indefinitely.  Samples sent 
to Washington University in St. Louis will also be stored indefinitely.  All study data will be 
recorded in REDCap through the Portal will be stored indefinitely on secure servers. For more 
detail regarding sample collection and storage, please refer to the trial’s Lab Manual. Any 
research records that are kept on paper or in other electronic formats will be stored according 
to applicable laws and regulations. All research records will be accessible for inspection by 
authorized representatives of the IRB, federal regulatory agency representatives, and the 
department or agency supporting the research. 
 


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Wang X, Pacheco JA, Kho AN, Hayes MG, Weston N, Matsumoto M, Kopp PA, Newton KM, 
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DR, Roden DM, de Andrade M. Variants near FOXE1 are associated with hypothyroidism 


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and other thyroid conditions: using electronic medical records for genome- and phenome-
wide studies. Am J Hum Genet. 2011 Oct 7;89(4):529–542. PMCID: PMC3188836 
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14.  Nakatsuji M, Minami M, Seno H, Yasui M, Komekado H, Higuchi S, Fujikawa R, Nakanishi Y, 
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Associated Protein in Macrophages Ameliorates Colitis and Colitis-Associated 
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15.  Fashner J, Gitu AC. Diagnosis and Treatment of Peptic Ulcer Disease and H. pylori Infection. 
Am Fam Physician. 2015 Feb 15;91(4):236–242. PMID: 25955624 
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17.  Flicek P, Amode MR, Barrell D, Beal K, Brent S, Chen Y, Clapham P, Coates G, Fairley S, 
Fitzgerald S, Gordon L, Hendrix M, Hourlier T, Johnson N, Kähäri A, Keefe D, Keenan S, 
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Trevanion S, Vandrovcova J, Vilella AJ, White S, Wilder SP, Zadissa A, Zamora J, Aken BL, 
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Jan;39(Database issue):D800-806. PMCID: PMC3013672 
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20.  Christodoulou K, Wiskin AE, Gibson J, Tapper W, Willis C, Afzal NA, Upstill-Goddard R, 
Holloway JW, Simpson MA, Beattie RM, Collins A, Ennis S. Next generation exome 
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Implicates Other Immune Related Genes. McCarthy MI, editor. PLOS Genet. 2015 Feb 
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22.  Resler AJ, Makar KW, Heath L, Whitton J, Potter JD, Poole EM, Habermann N, Scherer D, 
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from: https://www.google.com/patents/CA2756193A1?cl=en 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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26 
APPENDIX A PHEWAS DATA TABLES 
 
The tables below show information about the variants for the relevant prostaglandin receptors 
covered in our genotyping platform, the Illumina Infinium Exomchip.  Table 1 shows basic 
information on the variants covered on our platform.  
 
Table 1. Relevant SNPs for PTGER2-4 
SNP 
rsID 
Mutation 
SIFT 
PP2 
Exome 
MAF* 
Populations with highest MAF17 
Gln379Ar
g 
rs141688168 
(PTGER3) 
Missense 
0.63 
0.0 
0.2% 
Punjabi in Pakistan 1% 
Bengali in Bangladesh 0.6% 
Tyr30Cys; 
Tyr285Cys 
rs139552094 
(PTGER2) 
Missense 
0.0 
0.566 
0.3% 
Toscani in Italy 0.9% 
Mexican ancestry in LA 0.8% 
Val294Ile 
rs111866313 
(PTGER4) 
Missense 
0.4 
0.002 
2.7% 
Finnish in Finland 3.5% 
Utah residents with 
Northern/Western European 
ancestry 3% 
Puerto Ricans in Puerto Rico 2.4% 
Cys83Gly 
rs111965614 
(PTGER2) 
Missense 
0.0 
0.999 
0.6% 
Colombians in Colombia 3.2% 
Iberians in Spain 2.8% 
Toscani in Italy 2.8% 
- 
rs11209710 
(PTGER3) 
Intronic 
- 
- 
32% 
East Asians 62% 
South Asians 57% 
* Minor allele frequency reflects the MAF in the Exomechip European ancestry population 
genotyped for this analysis.   
 
Sorting Intolerant from Tolerant (SIFT) scores at or below 0.05 are considered to be deleterious; 
those above 0.05 are considered to be tolerated.18 Polyphen2 (PP2) scores below 0.447 are 
considered benign; those higher than 0.908 are considered probably damaging; and those in 
between possibly damaging.19 The scores above are color coded to indicate benign or tolerated 
(green), possibly damaging (orange) and probably damaging (red).  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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27 
 
 
Table 2 below summarizes existing literature on the functional effects of SNPs with PheWAS 
associations that will be displayed in subsequent tables below.   
 
Table 2. Literature Describing SNP Functional Effects 
SNP 
Additional 
SNP details 
Predicted effects 
Studies of SNP effects 
rs141688168 
(PTGER3) 
 
Gln379Arg 
(missense) 
Predicted to be benign/tolerated by 
SIFT and PolyPhen2 algorithms 
None identified to date 
rs139552094 
(PTGER2) 
 
Tyr30Cys; 
Tyr285Cys 
(missense) 
Predicted to be deleterious/possibly 
damaging by SIFT and PolyPhen2 
algorithms, respectively 
None identified to date 
rs111866313 
(PTGER4) 
 
Val294Ile 
(missense) 
Predicted to be benign/tolerated by 
SIFT and PolyPhen2 algorithms 
Known IBD gene 20;  
Protective in IBD 21 
rs111965614 
(PTGER2) 
 
Cys83Gly 
(missense) 
Predicted to be deleterious/probably 
damaging by SIFT and Poly-Phen2 
algorithms, respectively 
None identified to date 
rs11209710 
(PTGER3) 
Intronic 
(no prediction data for intronic SNPs) 
No significant effect on 
cancer risk 22; no significant 
effect on ACE-inhibitor 
associated cough; patent 
application, Biomarkers for 
assessing peripheral 
neuropathy response to 
treatment with a proteasome 
inhibitor23 
 
Based on directionality of the ORs displayed in Table 3, the below SNPs would all function like 
receptor antagonists (i.e. opposite of misoprostol’s effects). If this is true, any other diseases 
associated with those SNPs with a >1 OR (risk) would be potential new therapeutic indications 
for misoprostol.  
 
Table 3. PheWAS Results Validating Current Use 
Condition 
PheWAS 
Code 
rsID 
(gene) 
p Value 
Odds 
Ratio 
Case 
Carriers 
Total 
Cases 
Gastritis and 
duodenitis 
535 
rs111965614 
(PTGER2) 
0.00476 
1.88 
22 
968 
Ulcer of esophagus 
530.12 
rs139552094 
(PTGER2) 
0.01113 
6.44 
2 
72 
 
 
 
 


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28 
 
Table 4 below shows known and potential new GI inflammatory conditions that could be 
plausible to treat with misoprostol. 
Table 4. Gastrointestinal PheWAS Results 
Condition 
PheWAS 
Code 
rsID 
(gene) 
p Value 
Odds 
Ratio 
Case 
Carriers 
Total Cases 
Ulcerative 
colitis 
555.2  
rs139552094 
(PTGER2) 
0.02431 
3.19 
4 
238 
Ulceration of 
intestine 
556.1 
rs141688168 
(PTGER3) 
0.02549 
5.01 
2 
76 
Other specified 
gastritis 
535.8 
rs11209710 
(PTGER3) 
0.01728 
1.32 
106 
161 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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APPENDIX B MOUSE MODEL OF CDI DATA 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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30 
 
 
Appendices C & D are separate documents 
 
Appendix C 
IND safe to proceed letter from FDA 
 
Appendix D 
Misoprostol Package Insert 
 
 

SAP_001.pdf: 
Statistical Analysis Plan 
 
PROCLAIM – A Randomized, Double-Blind, Placebo-Controlled Trial to Assess the Efficacy and Safety of 
Misoprostol in the Prevention of Recurrence of Clostridium Difficile Infection in Adults 
 
 
NCT 03617172 
 
 
August 26, 2021 




PROCLaIM v 1.1 
 
26 August 2021 
 
Statistical analysis plan for Misoprostol in the prevention of recurrence in CDI 
Version 1.1 
 
Page 2
 
 
MISOPROSTOL IN THE PREVENTION OF RECURRENT CDI 
 
A Randomized, Double-Blind, Placebo-Controlled Trial To Assess The Efficacy And Safety Of Misoprostol In The 
Prevention Of First Recurrence Of Clostridium Difficile Infection In Adults Aged 50 And Over 
 
Statistical Analysis Plan 
Version 1.1 
 
Yaomin Xu, Sharon Phillips, and Christopher Lindsell 
 
Introduction 
This document describes the statistical design and analysis plan for a double-blinded, randomized 1:1 controlled 
trial to test the efficacy and safety of Misoprostol for the prevention of recurring clostridium difficile infection 
(rCDI). A total of 440 adults aged 18 years and older with a recent diagnosis of CDI will be recruited from 3 sites. 
Consenting patients will be randomized 1:1 to receive either misoprostol or matching placebo. For patients taking 
antibiotics, consenting patients will be contacted on the last regular business day before the seventh day of oral 
antibiotic treatment, to remind all patients to begin the treatment that they were randomized to on day 7 of 
their oral antibiotic treatment; patients not taking oral antibiotics will start taking misoprostol 7-10 days after a 
positive CDI lab test. All randomized patients who were taking antibiotics will continue to take oral antibiotics 
for an additional 4-7 days after starting misoprostol treatment (such that those participants have a 4-7 day 
overlap of antibiotic and misoprostol treatment), and begin taking an investigational 200 mcg oral misoprostol 
or placebo two times a day (200 mcg po BID) for 14 days. All randomized patients will be monitored for 
recurrence via phone call once per week for an additional 49 days (the total duration of study participation will 
be 62 days).  
 
 
Treatment arms 
Participants will be randomized to receive either: 
a) Treatment: 200 µg oral misoprostol two times a day for 14 days 
b) Control: Placebo two times a day for 14 days 
 
Endpoints 
Clinical recurrence of clostridium difficile infection (rCDI): the primary endpoint for this trial is recurrence of a 
clostridium difficile infection within 8 weeks after vancomycin is stopped (i.e. from four days after study 
treatment has begun to 60 days after study treatment has begun). Recurrence will be defined to have occurred 
when a participant concurrently meets all criteria for a clostridium difficile infection: 
a) ≥3 unformed (loose or watery) stools within a 24-hour period; 
b) A documented positive C. difficile toxin assay (enzyme immunoassay [EIA] or cellular cytotoxicity assay) 
or DNA PCR assay for toxigenic C. difficile from a stool sample collected while the subject was 
symptomatic; and 
c) No other explanation for diarrhea (e.g. laxatives). 
 
A patient will be considered to not have experienced rCDI if all of these criteria are not met within the 8 week 
follow up period.  


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Safety and tolerability: The two secondary endpoints for this trial are safety and tolerability. A participant will 
be defined to have experienced a safety outcome if there is a treatment-related adverse event, clinically 
relevant changes in physical examinations, or clinical safety laboratory testing. A participant will be defined as 
having experienced a tolerability outcome if the participant, the investigators, or a treating physician reduces 
the dosage or stops study treatment citing side effects or adverse events as the reason. 
 
Exploratory endpoints: A number of exploratory endpoints are expected to be impacted by treatment and so 
are included in this study. These include: 
• 
Serum biomarkers/toxin A and B antibodies from blood draws 
• 
Recovery of bowel microbiota assessed from microbiome collection. 
• 
For patients with recurrence, additional endpoints are: 
o Number of recurrences during the follow-up period. 
o Time to resolution of diarrhea. 
o Serum biomarkers/toxin A and B antibodies (from blood draws). 
o Severity of disease (for those with recurrence), determined using a combination of severity 
criteria used in Hospital specific guidelines, SHEA/IDSA guidelines, Zar criteria, or other sources: 
 
 
 WBC  
 
Serum creatinine  
 
Temperature  
 
Albumin level  
 
ICU or step down unit admission 
 
Endoscopically or histologically confirmed pseudomembranous colitis 
 
Toxic megacolon, perforation, colectomy, or septic shock requiring ICU admission and 
pressors 
 
Hospital readmission 
 
Validated quality of life instrument 
 
Final analysis 
The final analysis for this trial will occur after all enrolled subjects have been followed for 8 weeks all queries 
have been resolved, and the database has been locked.  
 
Descriptive analysis 
Initially, the study sample will be described by demographics, clinical characteristics and primary, secondary 
and exploratory endpoints. The study sample will be described both overall and stratified by treatment arm. 
Categorical variables will be described using frequencies and percentages. Continuous variables will be 
described using means and standard deviations and/or medians and interquartile ranges. Data visualization 
and summary statistics will be used to evaluate distributional assumptions and to guide selection of data 
transformations or use of non-parametric alternatives where appropriate. 
 
Analysis of primary endpoints 
The main analysis will be a Fisher’s Exact test to compare the proportion (rate) of recurrence between 
treatment arms. The null hypothesis to be tested is that the proportions do not differ between treatment arms.  
 


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A difference in the primary endpoint between treatment groups will be declared if the two-tailed p-value is less 
than 0.048. Because any efficacy claim will be based on a single comparison of the primary endpoint between 
treatment arms, no adjustment for multiple testing will be made for this analysis. 
 
Analysis of secondary endpoints 
The safety and tolerability endpoints will be compared between study arms using a Fisher’s Exact test with a 
significance level of 0.025 (i.e. 0.05/2).  This controls the overall type 1 error rate for a claim on safety and 
tolerability at 5%. 
 
Intent-to-treat analysis 
The final analysis for this trial will use the intent-to-treat principle with no modifications. With this approach, 
participants will be grouped according to the arm to which they were assigned regardless of whether they 
received the control or the treatment. 
 
 
Adjusted analysis 
There are a number of key factors that might affect endpoints independently of any treatment effect. We will 
use logistic regression to separately model the odds of rCDI, safety and tolerability as a function of treatment 
arm adjusted for demographics, baseline disease severity, comorbidities, and site (as a random variable). 
Statistical models will be constructed following best practices. Briefly, the primary endpoint is rCDI is a 
dichotomous outcome of yes/no. This will be modeled as logistic regression controlling for treatment, baseline 
severity, age, sex, pertinent labs. One of the secondary endpoints is time to resolution of diarrhea which will be 
modeled using time-to-event analysis controlling for baseline severity, age, sex, labs.  
 
Subgroup analysis 
There are no expected subgroups based on mechanism of action. Subgroup analyses will be pursued if there is 
an interaction between the subgrouping variable and the treatment variable. For example, we will test the 
interaction between sex and treatment. If the interaction is significant, we will separately estimate the 
treatment effect for males and females. Recognizing that the power for finding interactions is low and the 
importance of understanding heterogeneity of treatment effects is high, we will use a significance level of 10%. 
The following subgrouping variables will be considered: 
• 
Age (<65 v ≥65 yrs) 
• 
Race (Black, white, other) 
• 
Ethnicity (Hispanic or latino v not Hispanic or latino) 
• 
Sex (male v female) 
 
 
Subgroup analyses will follow the same approach as the primary analyses whereby an unadjusted analysis will 
be used to test the primary and key secondary endpoints followed by generation of a statistical model. We will 
not make any adjustments for multiplicity in these analyses. 
 
Exploratory analyses 


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Exploratory endpoints will be compared between treatment arms. Continuous variables will be compared using 
student’s t-test or the non-parametric equivalent (Mann-Whitney U test) if distributional assumptions are not 
met. Ordinal variables will be compared using the Mann-Whitney U-test. Categorical variables will be 
compared using Fisher’s Exact test. Adjusted analyses may be utilized.  
 
Randomization 
Participants will be randomized to receive either treatment or control upon confirmation that the patient has 
responded to oral vancomycin treatment (day 7 of vancomycin treatment). Randomization will use a randomly 
permuted small blocks approach with stratification by site, sex and severity of illness, as defined by IDSA 
criteria for severity (if bloodwork is not available, the patient will be assumed to be “non-severe”): 
• 
Non-severe: Leukocytosis with a WBC count of ≤15,000 cells/mL and serum creatinine <1.5 mg/dL  
• 
Severe: Leukocytosis with a WBS count >15,000 cells/mL or serum creatinine >1.5 mg/dL  
• 
Fulminant: Hypotension or shock, ileus, megacolon  
 
 
Interim analysis 
We will perform an interim safety analysis after 44 patients (10% of accrual) have completed study treatment 
(14 days after randomization). It is expected that of the first 44 patients, although there might be dose 
adjustments, there will be no greater than 5 withdrawals due to diarrhea events during the period of co-
administration with vancomycin. If more than 5 diarrheal events occur among the first 44 patients which 
require study withdrawal during the co-administration period, enrollment will be interrupted for safety 
concerns. All currently enrolled participants will be followed to completion and the data will be analyzed to 
estimate conditional power; no statistical testing will be performed. The data will be presented to the DSMB to 
determine whether the study should proceed or not.  If no more than 5 diarrhea events occur among the first 
44 patients which require study withdrawal, the study will continue without interruption of enrollment. 
 
A planned interim analysis will be conducted after the 176th participant has completed the 63 days of follow up, 
with accrual continuing during this time. This will allow the study to be terminated early in the event there is a 
less than acceptable chance of detecting the desired effect, therefore not wasting funds or exposing patients to 
unnecessary risk with no commensurate benefit.  We propose the interim analysis for efficacy will be 
completed using the Lan-DeMets procedure with an O’Brien-Fleming stopping boundary to account for the 2 
interim analyses and the final analysis. The significance boundary for final analysis will be P ≤ .048. The interim 
analysis for futility will be based on the stochastic curtailment method. The interim analyses for futility will be 
performed by calculation of conditional power, an estimate of the probability that the study shows a 
statistically significant effect on the primary endpoint, i.e. a reduction in the rate of recurrence, given the 
results to date and assumptions regarding outcome through the end of the study. A recommendation to stop 
the trial for futility will require a conditional power below 30%, under the observed efficacy trend at the time 
of interim analysis, with two-sided type I error less than 5%. The proposed sample size of 196 per arm is 
adjusted for the interim analyses, i.e., types I and II errors. 
 
 
Power and sample size calculations  
Participants will be considered enrolled at the time of randomization. With 196 participants in each group (392 
total participants), there will be 80% power to detect a relative reduction in rCDI of 40% when the control rCDI 
rate is 30%. We expect a drop-out rate of 10%, therefore, we will randomize 440 participants to account for 
attrition and sill achieve the expected power.  
 
The participants who drop-out will be described and balance checked between the two groups of participants. 


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Missing data 
Participants are not evaluable on their primary outcome will be excluded from the primary analysis. Similarly, 
participants not evaluable on the key secondary outcomes will be excluded. For statistical modeling, multiple 
imputation will be used to resolve missing data on predictor variables.  
 
Sensitivity analyses will be conducted including these participants under various assumptions of treatment 
effect (e.g. treatment resulting in the worst outcome and placebo the best; treatment resulting in the best 
outcomes and placebo the worst.)