Clinical trial NCT02765256
Conditions: Crohn's Disease
Sponsor: University of Pennsylvania
Start date: 2016-08-01; Completion date: 2018-12-01
Enrollment: 8 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
To determine the effect of a novel gut microbiota-targeted therapeutic regimen (bowel lavage and antibiotics with or without an antifungal) in the management of active Crohn's Disease (CD) that is refractory to conventional, immunosuppressive therapy.

Detailed description:

Arms: arm 1: Vancomycin 500 mg oral suspension four times daily (Day 1-14), plus neomycin 1000 mg orally three times daily (Days 1-3), plus ciprofloxacin 750 mg orally twice daily (Day 4-14), plus Polyethylene Glycol 3350 (Miralax) 238 g dissolved in 64 ounces of Gatorade or Crystal Lite on day 2, plus fluconazole 400 mg orally once daily (Day 1-14). PRN (as needed) Promethazine 12.5mg up to every four hours (Days 1-3). arm 2: Vancomycin 500 mg oral suspension four times daily (Day 1-14), plus neomycin 1000 mg orally three times daily (Days 1-3), plus ciprofloxacin 750 mg orally twice daily (Day 4-14), plus Polyethylene Glycol 3350 (Miralax) 238 g dissolved in 64 ounces of Gatorade or Crystal Lite on day 2, plus placebo for fluconazole. PRN (as needed) Promethazine 12.5mg up to every four hours (Days 1-3).
Interventions: intervention 1: Fluconazole intervention 2: Vancomycin intervention 3: Neomycin intervention 4: Ciprofloxacin intervention 5: Polyethylene Glycol 3350 intervention 6: Promethazine intervention 7: Fluconazole placebo
Intervention descriptions: intervention 1: 400mg orally once daily (Day 1-14) intervention 2: 500mg oral suspension 4 times daily (Day 1-14) intervention 3: neomycin 1000 mg orally three times daily (Days 1-3) intervention 4: ciprofloxacin 750 mg orally twice daily (Day 4-14) intervention 5: 238 g dissolved in 64 ounces of Gatorade or Crystal Lite on day 2 intervention 6: PRN (as needed) Promethazine 12.5mg up to every four hours (Days 1-3). intervention 7: Once daily

Protocol documents:
Prot_SAP_000.pdf: 
  
V10.0.20170811 
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sponsor 
 
Fundamental Modification of the Gut Microbiota in the Treatment of 
Refractory Crohn’s Disease 
 
 
Principal Investigator:  
Lindsey Albenberg, DO 
 
 
 
 
Division of Gastroenterology, Hepatology and Nutrition 
 
 
 
 
Children’s Hospital of Philadelphia 
 
 
 
 
3401 Civic Center Blvd., 7NW26 
 
 
 
 
Philadelphia, PA 19104 
 
 
 
 
(215) 590-1680 
 
 
 
 
albenbergL@email.chop.edu 
 
Sub-Investigators: 
 
Gary D. Wu, MD 
 
 
 
 
James D. Lewis, MD, MSCE 
 
Funding Sponsor: 
 
The Broad Medical Research Program at CCFA 
 
Investigational Product: 
Neomycin, vancomycin, ciprofloxacin, fluconazole, polyethylene glycol 
3350, promethazine 
 
 
IRB Number: 
 
 
823635 
 
IND/IDE Number: 
 
IND Exemption granted by Penn Office of Clinical Research 
 
 
Initial version: 2015-10-13 v1.0 
Amended: 
v2.0.20160502 
Amended: 
v3.0.20160623 
Amended: 
v4.0.20160729 
Amended: 
v5.0.20160810 
Amended: 
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Amended: 
v7.0.20170210 
Amended: 
v8.0.20170313 
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v9.0.20170512 
Amended: 
v10.0.20170811 
 
 
 
 
 
 
 
 


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Table of Contents 
 
Contents 
Contents ................................................................................................................................................... ii 
List of Abbreviations ........................................................................................................................... v 
Study Summary ..................................................................................................................................... 1 
Background and Study Rationale .................................................................................................... 3 
Introduction ...................................................................................................................................................... 3 
1.1 
Background and Relevant Literature ........................................................................................... 3 
1.1.1 
Inflammatory Bowel Disease: Current Therapies and Treatment Challenges ......................... 3 
1.1.2 
History of Gut Microbiota-Targeted Therapies for IBD .................................................................... 4 
1.1.3 
Preliminary Data ............................................................................................................................................. 5 
1.2 
Investigational Regimen and Rationale ....................................................................................... 7 
2 
Study Objectives ............................................................................................................................ 8 
2.1 
Primary Objective ................................................................................................................................ 8 
2.2 
Secondary Objectives ......................................................................................................................... 8 
3 
Investigational Plan ...................................................................................................................... 8 
3.1 
General Design ...................................................................................................................................... 8 
3.1.1 
Screening Phase ............................................................................................................................................... 8 
3.1.2 
Intervention Phase .......................................................................................................................................... 9 
3.1.3 
Optional Intervention Extension Phase (14 day repeat drug regimen) ...................................... 9 
3.1.4 
Follow Up Phase ............................................................................................................................................. 10 
3.2 
Study Endpoints ................................................................................................................................. 10 
3.2.1 
Primary Study Endpoints ............................................................................................................................ 10 
3.2.2 
Secondary Study Endpoints ....................................................................................................................... 10 
4 
Study Population and Duration of Participation ............................................................. 10 
4.1 
Inclusion Criteria ............................................................................................................................... 10 
4.2 
Exclusion Criteria .............................................................................................................................. 11 
4.3 
Participant Recruitment ................................................................................................................. 12 
4.4 
Duration of Study Participation .................................................................................................... 12 
4.5 
Total Number of Participants and Sites ..................................................................................... 12 
4.6 
Vulnerable Populations: .................................................................................................................. 12 
5 
Study Intervention ..................................................................................................................... 12 
5.1 
Intervention Description and Regimen ...................................................................................... 12 
5.2 
Randomization ................................................................................................................................... 13 
5.3 
Receipt ................................................................................................................................................... 13 
5.4 
Storage ................................................................................................................................................... 13 
5.5 
Preparation and Packaging ............................................................................................................ 13 
5.6 
Administration and Accountability ............................................................................................. 13 
5.7 
Participant Compliance Monitoring ............................................................................................ 14 
5.7.1 
Return or Destruction of Investigational Product .......................................................................... 14 
6 
Study Procedures ....................................................................................................................... 15 
Table 1: Schedule of Study Procedures .................................................................................................. 15 


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6.1 
Screening Phase (Visit 0) ................................................................................................................ 16 
6.2 
Enrollment Phase (Visit 1) ............................................................................................................. 16 
6.3 
Intervention Phase (Days 1-15 including Visits 2-4): In-person Study Visits ............... 17 
6.3.1 
Daily Surveys .................................................................................................................................................. 17 
6.3.2 
Study Visit 2 (Day 5) .................................................................................................................................... 17 
6.3.3 
Study Visit 3 (Day 8) .................................................................................................................................... 17 
6.3.4 
Study Visit 4 (Day 15) ................................................................................................................................. 18 
6.4 
Follow-Up Phase (Days 16- 6 months including Visits 5-8): Telephone and In-person 
Study Visits ...................................................................................................................................................... 18 
6.4.1 
Telephone Follow-Up: Visit 5 (Day 22) ............................................................................................... 18 
6.4.2 
Study Visit 6 (Day 29) ................................................................................................................................. 18 
6.4.3 
Telephone Follow-Ups: 3 Months & 6 Months .................................................................................................. 19 
6.5 
Rescue Therapy .................................................................................................................................. 19 
6.6 
Participant Withdrawal ................................................................................................................... 19 
6.7 
Early Termination Visits ................................................................................................................. 19 
7 
Study Evaluations and Measurements ................................................................................ 19 
7.1 
Medical Record Review .................................................................................................................... 19 
7.2 
Physical Examination ....................................................................................................................... 20 
7.3 
Vital Signs ............................................................................................................................................. 20 
7.4 
EKG ......................................................................................................................................................... 20 
7.5 
Laboratory Blood Evaluations ....................................................................................................... 20 
7.6 
Pregnancy Testing ............................................................................................................................. 21 
7.7 
Stool Collection, Shipping, and Analysis .................................................................................... 21 
7.8 
Rectal Swabs ........................................................................................................................................ 21 
7.9 
Efficacy Evaluations .......................................................................................................................... 22 
7.9.1 
HBI and short CDAI scoring ...................................................................................................................... 22 
7.9.2 
Fecal calprotectin ......................................................................................................................................... 22 
7.9.3 
High-sensitivity C-reactive protein (hs-CRP) ................................................................................... 22 
7.9.4 
Microbial DNA sequencing ....................................................................................................................... 22 
7.10 
Safety Evaluations ......................................................................................................................... 23 
8 
Statistical Plan ............................................................................................................................. 23 
8.1 
Primary Endpoint .............................................................................................................................. 23 
8.2 
Secondary Endpoints ........................................................................................................................ 23 
8.3 
Statistical Methods ............................................................................................................................ 23 
8.3.1 
Safety Analysis ............................................................................................................................................... 24 
9 
Safety and Adverse Events ...................................................................................................... 25 
9.1 
Definitions ............................................................................................................................................ 25 
9.1.1 
Adverse Event ................................................................................................................................................ 25 
9.1.2 
Serious Adverse Event ................................................................................................................................ 25 
9.2 
Recording of Adverse Events ......................................................................................................... 25 
9.3 
Relationship of AE to Study ............................................................................................................ 25 
9.4 
Reporting of Adverse Events and Unanticipated Problems ................................................ 25 
9.4.1 
Study SAE Notification to Investigator ................................................................................................ 26 
9.4.3 
Follow-up report ........................................................................................................................................... 27 
9.4.4 
Investigator Reporting: Notifying the Penn IRB ............................................................................. 27 
9.5 
Medical Monitoring ........................................................................................................................... 27 
9.5.1 
Data and Safety Monitoring Plan ........................................................................................................... 27 
10 
Study Administration, Data Handling and Record Keeping ..................................... 28 


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10.1 
Confidentiality ................................................................................................................................ 28 
10.2 
Data Collection and Management ............................................................................................. 28 
10.3 
Records Retention ......................................................................................................................... 28 
11 
Study Monitoring, Auditing, and Inspecting .................................................................. 28 
12 
Ethical Considerations .......................................................................................................... 28 
12.1 
Risks ................................................................................................................................................... 29 
12.2 
Benefits ............................................................................................................................................. 29 
12.3 
Risk Benefit Assessment .............................................................................................................. 29 
12.4 
Informed Consent Process / HIPAA Authorization ............................................................ 29 
13 
Study Finances ......................................................................................................................... 30 
13.1 
Funding Source ............................................................................................................................... 30 
13.2 
Conflict of Interest ......................................................................................................................... 30 
13.3 
Participants Stipends or Payments .......................................................................................... 30 
14 
Publication Plan ...................................................................................................................... 30 
15 
References ................................................................................................................................. 30 
16 
Appendix A: Adverse Reactions ......................................................................................... 33 
16.1 
Ciprofloxacin ................................................................................................................................... 33 
16.2 
Vancomycin ...................................................................................................................................... 34 
16.3 
Neomycin .......................................................................................................................................... 34 
16.4 
Fluconazole ...................................................................................................................................... 35 
16.5 
Miralax (PEG 3350) ....................................................................................................................... 35 
16.6 
Promethazine .................................................................................................................................. 36 
17 
Appendix B: Investigational Drug Service Documentation ...................................... 37 
18 
Appendix C: CCFA-BMRP Sponsor Guidelines ............................................................... 39 
 
 
 
 
 
 


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List of Abbreviations 
AE 
 
 
Adverse event 
CBC 
 
 
Complete blood count 
CD 
 
 
Crohn’s disease 
CDAI                              Crohn’s Disease Activity Index 
CHOP  
 
The Children’s Hospital of Philadelphia 
CMP                               Comprehensive metabolic panel 
CMV                               Cytomegalovirus 
CRF 
 
 
Case Report Form 
CRP 
 
 
C-reactive protein 
CTRC  
 
Clinical and Translational Research Center 
EKG 
 
 
Electrocardiogram 
ESR 
 
 
Erythrocyte sedimentation rate 
FCP 
 
 
Fecal calprotectin 
GI 
 
 
Gastrointestinal 
HBI 
 
 
Harvey Bradshaw Index 
hsCRP  
 
High-sensitivity CRP 
IBD                                 Inflammatory bowel disease 
IBDU 
 
 
Indeterminate colitis 
ICE 
 
 
Idiopathic chronic enterocolitis 
IDS 
 
 
Investigational Drug Service 
IRB 
 
 
Institutional Review Board 
ITS 
 
 
Internal Transcribed Spacer 
LOR 
 
 
Loss of responsiveness 
MRN 
 
 
Medical record number 
PEG 
 
 
Polyethylene glycol 
PENN  
 
University of Pennsylvania 
PCDAI  
 
Pediatric Crohn’s Disease Activity Index 
PUCAI  
 
Pediatric Ulcerative Colitis Activity Index 
SAE 
 
 
Serious adverse event 
sCDAI  
 
Short CDAI 
TUNPRC 
 
Tulane University National Primate Center 
UC 
 
 
Ulcerative colitis 
 
 
 
 
 
 
 
 


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Study Summary 
Title 
Fundamental Modification of the Gut Microbiota in the Treatment of Refractory 
Crohn’s Disease 
Short Title 
Holiday 
IRB Number 
823635 
Protocol Number 
N/A 
Phase 
Phase II 
Methodology 
Randomized, placebo-controlled, double blind 
Study Duration 
Individual participant participation will last 6-7 months; full study procedures 
are expected to last 2 years. 
Study Center(s) 
University of Pennsylvania  
Objectives 
Primary Objective: 
• 
To determine the effect of a novel gut microbiota-targeted therapeutic 
regimen (bowel lavage and antibiotics with or without an antifungal) in 
the management of active CD that is refractory to conventional, 
immunosuppressive therapy.  
 
Secondary Objective(s): 
• 
To correlate the effectiveness in reducing bacterial 16S rRNA copy 
number and fungal 18S rRNA copy number, with the use of the 
regimen in the primary objective, with improvement of disease activity 
in patients with CD refractory to immunosuppressive therapy. 
• 
To determine the relationship between the effectiveness of the 
proposed therapies with changes in gut microbiota composition. 
Number of 
Participants 
30 participants will be enrolled with an estimated yield of 20 evaluable 
participants. 


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Main Inclusion and 
Exclusion Criteria 
Inclusion Criteria: 
• 
Participants ages 18-75 
• 
Active moderate CD defined as Harvey Bradshaw Index (HBI) ≥ 7 
• 
Have had primary nonresponse or an initial response for 8 or more 
weeks, followed by loss of responsiveness (LOR) (self-reported 
worsening of symptoms for at least 7 days), to one or more of the 
following therapies*: azathioprine, 6-mercaptopurine, methotrexate, 
adalimumab, certolizumab, golimumab, infliximab, natalizumab,  
vedolizumab, or ustekinumab 
 
*Must be administered at standard, therapeutic dosages  
 
Exclusion Criteria: 
• 
Known or suspected stricturing disease producing obstructive 
symptoms 
• 
Known allergy or intolerance to any of the medications used in this 
study 
• 
Elevated creatinine 
• 
Prolonged QTc interval as seen on enrollment EKG 
 
Investigational 
Product (drug, dose, 
route, regimen) 
 
Vancomycin 500 mg suspension four times daily (Day 1-14), plus neomycin 
1000 mg orally three times daily (Days 1-3), plus ciprofloxacin 750 mg orally 
twice daily (Day 4-14), plus Polyethylene Glycol 3350 (Miralax) 238 g 
dissolved in 64 ounces of Gatorade or Crystal Lite on day 2, plus fluconazole 
400 mg orally once daily (Day 1-14) or placebo. PRN Promethazine 12.5mg up 
to every four hours (Days 1-3). 
Duration of 
administration 
14 days 
Reference therapy 
N/A 
Statistical 
Methodology 
Our estimates of efficacy will use a disease activity score (HBI) for clinical 
response, FCP for mucosal inflammation, and hsCRP for systemic 
inflammation. The two treatment groups will be compared using standard 
descriptive statistics. Categorical variables will be compared using Fisher’s 
exact test and continuous variables will be compared using the unpaired t-test 
or Wilcoxon rank sum test if the data are not normally distributed.  
Safety Evaluations  
Primary measurements that will be used to assess safety include vital signs, 
laboratory results, EKG for QTc interval, disease activity scores, and screening 
for medication side effects and adverse events. 
Data and Safety 
Monitoring Plan  
Monitoring is risk based. 


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BACKGROUND AND STUDY RATIONALE 
This study will be conducted in full accordance with all applicable University of Pennsylvania Research 
Policies and Procedures and all applicable Federal and state laws and regulations including 45 CFR 46, 
21 CFR Parts 50, 54, 56, and Good Clinical Practice: Consolidated Guidelines approved by the 
International Conference on Harmonization (ICH). All episodes of noncompliance will be documented. 
 
This document is a clinical research protocol and the investigators will perform the study in accordance 
with this protocol, will obtain consent, and will report unanticipated problems involving risks to participants 
or others in compliance with the University of Pennsylvania Research Policies and Procedures and all 
federal requirements.  Collection, recording, and reporting of data will be accurate and will ensure the 
privacy, health, and welfare of research participants during and after the study.  
Introduction 
Recent evidence suggesting that the commensal gut microbiota, or the “gut microbiome,” is responsible 
for stimulation of the intestinal immune system in inflammatory bowel disease (IBD) has caused many 
researchers and providers in the field to question current therapeutic approaches.  Though most IBD 
providers would agree that immunosuppressive medications are currently the most reliable therapies, 
such research indicates that if gut flora plays a role in the pathogenesis of IBD, then perhaps targeting 
commensal microbes rather than or in addition to the immune system would be more efficacious.  
 
This protocol seeks to test the hypothesis that a therapeutic approach that deeply alters the composition 
and bacterial/fungal load of the gut microbiota will be efficacious in patients with refractory Crohn’s 
Disease (CD) who have primary nonresponse or secondary loss of response to conventional, immune 
suppressive therapies. This study will evaluate the efficacy of a novel treatment regimen employing non-
immunosuppressive medications in the management of refractory CD.  Patients will be treated with a 
combination of gut microbiota-targeted therapies to deeply modify the dysbiotic gut microbiota that has 
been consistently reported in patients with CD[1].  We believe this strategy will both treat the gut 
inflammation associated with IBD, as well as rescue response to biologic or immunomodulator therapies.    
1.1 
Background and Relevant Literature  
1.1.1 
Inflammatory Bowel Disease: Current Therapies and Treatment Challenges 
Inflammatory bowel disease (IBD), including CD and UC, affects approximately 1.5 million Americans and 
the incidence is increasing worldwide [2].  Current evidence indicates that the pathogenesis of IBD 
involves an inappropriate and persistent inflammatory response to the gut microbiota in genetically 
susceptible individuals [3].  In support of this notion, several studies utilizing animal models have shown 
that the development of intestinal inflammation requires microbial colonization of the gut [4, 5].  Clinical 
observations of IBD further implicate the role of the commensal gut microbiota, as IBD usually affects 
intestinal regions with the highest bacterial load, and both fecal diversion and antibiotic treatment can be 
effective in the management of CD [6, 7]. 
 
However, standard treatments for IBD do not focus on restoring immune tolerance to commensal 
microbes but rather depend on immunosuppression.  Although effective in inducing and maintaining 
remission for many patients, there is substantial risk of side effects associated with the use of 
immunosuppressive 
medications, 
namely 
steroids, 
immunomodulators 
(e.g., 
thiopurines 
and 
methotrexate) and biologics (e.g., infliximab and adalimumab) [8].  In addition, a significant proportion of 
patients will experience primary nonresponse or a loss of response (LOR) to immunosuppression over 
time in a way that cannot be explained by the pharmacokinetics of the drug [9]. 
 
When nonresponse or LOR occurs, providers must escalate dosage or change the treatment 
medication(s).  Because of the limited number of medications and concern for the exhaustion of non-


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surgical options, maintaining patients with IBD on an effective medication for as long as possible 
becomes a priority of clinical care.   
 
1.1.2 
History of Gut Microbiota-Targeted Therapies for IBD 
 
Antibiotics 
There is clear evidence for the effectiveness of antibiotics in the treatment of inflammation in animal 
models of IBD [10-12].  For example, IL-10 knockout mice develop a phenotype comparable to human 
IBD and at least two studies have shown that antibiotics such as neomycin, ciprofloxacin, vancomycin, 
and metronidazole may both prevent and treat intestinal inflammation [10, 12].  However, the evidence for 
the effectiveness of antibiotics in the treatment of humans with IBD has historically been less robust.  
Within the past several years, two meta-analyses of randomized controlled trials have documented a 
small but statistically significant benefit of antibiotics to induce remission in both CD and UC [13, 14].  In 
addition, several studies have now shown that antibiotic combination therapy significantly improves rates 
of remission and also steroid withdrawal in UC [15-18].   
 
Most notably, Turner and colleagues have recently published their experience using a two- to three-week 
course of combination antibiotic therapy in pediatric UC and indeterminate colitis refractory to standard 
immunosuppressive medications [18]. Patients were treated with combination oral amoxicillin, 
metronidazole, and doxycycline, except in children 2-7 years old where doxycycline was substituted with 
ciprofloxacin, and in infants under 2 years old where doxycycline was substituted with gentamicin.  In 
addition, in cases of allergy the allergenic drug was substituted with gentamicin, and in hospitalized 
children vancomycin was added to the regimen. The antibiotic regimen was definitively effective in 7/15 
(47%) of patients, inducing complete clinical remission as defined by the Pediatric Ulcerative Colitis 
Activity Index (PUCAI).  For the patients defined as primary responders (n=9), i.e., clinical remission at 
three weeks after initiating antibiotic therapy, reduction in CRP and PUCAI were statistically significant.  
Finally, all patients repeatedly tested negative for fecal C. difficile and bacterial cultures indicative of 
developed resistance. Only one patient tested positive for CMV and was subsequently treated with 
ganciclovir.  These findings demonstrate the potential efficacy of an antibiotic therapeutic strategy in the 
treatment of refractory IBD.   
 
Antifungals 
In addition to the bacterial component, there appears to be a relationship between IBD and the fungal gut 
microbiota. The authors of a recent study demonstrated that mice lacking Dectin-1, a C-type leptin 
receptor that recognizes β-glucans in the fungal cell wall, had increased susceptibility to chemically 
induced colitis due to their altered immunological responses to indigenous fungi [19]. Significantly, a 
polymorphism in the gene encoding Dectin-1 (CLEC7A) was found to be associated with a severe form of 
ulcerative colitis in humans [19]. A connection between high dietary concentrations of yeast and 
increased disease activity in patients with CD has also been suggested [20].   
 
In terms of fungal-targeted therapies for IBD, preliminary evidence suggests that fluconazole treatment 
may reduce intestinal inflammation in animal models of colitis and in patients with IBD [21]. We recently 
demonstrated a significant difference in the composition of the fungal microbiota in pediatric patients with 
IBD as compared to healthy controls [2], further suggesting a relationship between fungi and the 
pathogenesis of IBD.  
 
Bowel Lavage 
Given the potential role of the gut microbiota in the pathogenesis of IBD, therapies that deplete 
commensal flora in the gastrointestinal (GI) tract have been suggested. For example, in the 1980’s, 
intestinal lavage with normal saline was studied in patients hospitalized with acute exacerbations of 
severe CD. A small, controlled study demonstrated reduction in disease severity and also duration of 
hospitalization [23, 24]. Failure to further develop bowel lavage as an independent therapeutic option is 


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Figure 2: Time course of 16S rRNA gene copy number during oral antibiotic treatment (14 days of 
vancomycin plus neomycin) and upon discontinuing antibiotics on day 15 compared to control. 
likely secondary to advancements in pharmacological therapies. Recent evidence has indeed shown that 
bacterial diversity in the gut is significantly decreased following colonoscopy preparations [25].   
1.1.3 
Preliminary Data 
 
Effectiveness of Antibiotics in Mouse Models versus Humans, What’s the Difference? 
The observation that antibiotics as currently used have only modest efficacy in the treatment of CD and 
UC represents a challenge to the notion that antimicrobials could be used to deplete the microbiota in 
patients with IBD. One possible explanation is that the use of antibiotics in animal models is more 
effective in reducing intestinal bacterial load in mice than in humans. Indeed, in preliminary data [3], we 
show that two specific oral antibiotics dramatically reduce bacterial load by greater than 4 logs in mice 
within 72 hours, as quantified by 16S gene copy number.   
 
 
We believe that an antibiotic therapeutic strategy capable of significantly reducing bacterial load in 
patients with CD will show greater levels of efficacy in the treatment of active disease than in previously 
reported studies [26-28]. Additionally, based on data suggesting the potential importance of gut fungi in 
IBD, we hypothesize that to focus exclusively on gut bacterial load may represent a missed opportunity. 
 
Efficacy of the Holiday Regimen for Treatment of Chronic Enterocolitis in Rhesus Macaques 
(Macaca mulatta) 
Idiopathic chronic enterocolitis (ICE) is one of the most significant causes of morbidity and mortality in 
captive nonhuman primates and remains a diagnostic and therapeutic challenge for veterinarians working 
with these species.  Current evidence suggests that ICE is a multifactorial disease involving perturbations 
in gastrointestinal bacterial populations, as well as the response of the host immune system to these 
changes [29].  Our group is currently collaborating with Tulane University National Primate Center 
(TUNPRC) on a study where rhesus macaques with ICE and a negative stool infectious work-up (n=6 at 
present) were treated with an anti-microbial regimen similar to the one proposed here.  The animals 
receive the following regimen: 125 mg total vancomycin hydrochloride four times daily, 50 mg/kg 
neomycin twice daily, and fluconazole 2 mg/kg twice daily.  Therapy was administered for a total of 14 
days.  Thus far, four of the six animals have experienced no signs of diarrhea since completion of the 
treatment protocol.  Soft stool was observed for one day in the remaining two monkeys since completion 
of treatment, with normal stool observed on other days for both animals.  Based upon daily observations 
of mentation/behavior, activity level, and appetite as well as pre-treatment and intra-treatment CBC and 
serum chemistries, this treatment regimen was well-tolerated by all animals with no adverse effects noted. 
 
 
 


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Efficacy of Combination Antibiotic Therapy for Refractory IBD at CHOP 
Since the paper by Turner and colleagues [18] was presented at a national meeting in 2014, physicians 
within the CHOP Center for Inflammatory Bowel Disease have been utilizing a combination antibiotic 
approach clinically for patients with IBD refractory to standard therapy.  As a means of preliminary 
efficacy evaluation, we performed a retrospective study (CHOP IRB #15-011806) to review our 
experience treating refractory IBD with combination antibiotic therapy.  Information collected included 
patient demographics, disease characteristics, immunotherapy history, indication for antibiotic therapy, 
and type, dosage, and duration of antibiotics prescribed.  Eligible patients were ages 3-21 and prescribed 
treatment with three or more oral antibiotics concomitantly for the treatment of IBD.  Clinical outcomes 
were evaluated based on changes in disease activity, as measured by the Pediatric Crohn’s Disease 
Activity Index (PCDAI) in participants with CD, or the Pediatric Ulcerative Colitis Activity Index (PUCAI) in 
patients with UC or indeterminate colitis, at the time of initiation of combination antibiotic therapy and at 
multiple subsequent time points.  Disease morbidity outcomes were additionally measured, including 
referral to and/or scheduling of surgery, as well as escalation of therapy to an experimental medication.  
The incidence of adverse reactions during and following combination antibiotic therapy was also 
assessed. 
  
Of the enrolled patients (n=14), four participants had CD (29%) and the remainder had UC or 
indeterminate colitis (IBDU) (Table 1).  In 13 participants, the indication for combination antibiotic therapy 
was disease refractory to standard immunosuppressive therapies and in the remaining participant the 
indication for antibiotic therapy was induction and maintenance of remission.  Participants were 
prescribed combination antibiotic therapy for an average of 29.5 days (range 9-71).  
 
TABLE 1: PEDIATRIC IBD PATIENTS TREATED AT CHOP WITH COMBINATION ANTIBIOTICS 
Case 
Number 
Agea 
IBD 
Type 
LOR to 
Medicationsb 
Antibiotic 
Regimenc 
Duration 
in Days 
Surgery Referral 
Experimental 
Therapyd 
Rescue of 
Response 
1 
13.2 
UC 
IFX+MTX 
MAC 
14 
Cancelled 
No escalation 
IFX+MTX 
2 
12.3 
UC 
IFX 
MADV 
71 
Delayed (96 days) 
n/a 
IFX 
3 
16.4 
CD 
IFX+MTX, ADA+MTX 
MCV 
23 
n/a 
No escalation 
ADA+MTX 
4 
10.8 
CD 
IFX+MTX 
MAC 
9 
Proceeded 
n/a 
n/a 
5 
13.1 
IBDU 
IFX, ADA+MTX 
MAC 
24 
Cancelled 
n/a 
ADA+MTX 
6 
15.7 
CD 
IFX+MTX 
MACV 
28 
n/a 
Escalation 
n/a 
7 
13.6 
UC 
IFX+MTX 
MAC 
21 
n/a 
n/a 
n/a 
8 
18.5 
UC 
IFX+MTX 
MAC 
59 
Cancelled 
No escalation 
n/a 
9 
11.5 
UC 
IFX 
RCV 
35 
n/a 
n/a 
IFX+MTX 
10 
10.7 
IBDU 
IFX+MTX, ADA 
MCV 
19 
Delayed (22 days) 
No escalation 
n/a 
11 
6.5 
CD 
IFX+MTX, ADA+MTX 
MCV 
36 
n/a 
n/a 
ADA+MTX 
12 
16.1 
CD 
IFX 
MCDV 
31 
n/a 
No escalation 
IFX+MTX 
13 
12.3 
UC 
n/a 
MCV 
21 
n/a 
n/a 
n/a 
14 
10.4 
UC 
IFX 
MCDV 
22 
Cancelled 
n/a 
n/a 
 
 
 
 
 
 
 
 
 
aAge at initiation of antibiotic therapy 
 
bAll demonstrated LOR; IFX=infliximab monotherapy, IFX+MTX=IFX plus methotrexate dual therapy, ADA+MTX=adalimumab plus 
methotrexate dual therapy 
cA=amoxicillin, C=ciprofloxacin, D=doxycycline, M=metronidazole, R=rifaximin, V=vancomycin 
 
dReferral or discussion of eligibility screening for vedolizumab (Entyvio) or ustekinumab (Stelara)  
 
 
Because of the limitations inherent to the review of existing medical records, disease activity scores at all 
study time points were not available for each participant.  However, for those participants with available 


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data, PUCAI and PCDAI scores showed decreases in disease activity both two weeks post-initiation and 
post-completion of their prescribed antibiotic combination regimen. 
 
There were four documented adverse reactions that occurred during the course of therapy or shortly 
following therapy termination.  All were mild and only one was probably related to the medication regimen 
(black, hairy tongue); all resolved without sequelae.  There were two severe and one life-threatening 
adverse event; however, all occurred two weeks after termination of therapy and based on clinical 
expertise were unlikely related to the antibiotic therapy.  Only one additional event was possibly related to 
the prescribed antibiotic therapy – a vaginal yeast infection that occurred four weeks post-termination of 
therapy and which also resolved without sequelae. 
 
This proposal will test the hypothesis that the gut microbiota is fundamentally involved in the perpetuation 
of CD that is refractory to conventional strategies and that a strategy that dramatically alters the 
composition and/or biomass of the gut microbiota (both bacteria and fungi) will lead to clinical 
improvement and reduction of inflammation.   
 
1.2 
Investigational Regimen and Rationale  
In this protocol, we will allow the gastrointestinal tract a “Holiday” from gut microbes that may be 
perpetuating the inflammatory response.  We will attempt to reduce the microbial load in the gut through 
an intestinal lavage followed by short-term (14 days) treatment with broad-spectrum antibiotics with or 
without an antifungal.  The intestinal lavage protocol was chosen based on the standard bowel cleanse 
protocol utilized at The University of Pennsylvania hospitals prior to colonoscopy and are also widely 
accepted in the literature [30].  In our animal study above (Figure 1), we were able to significantly reduce 
16S rRNA gene copy number with a combination of two oral antibiotics – neomycin and vancomycin.  In 
this protocol, we have adapted the regimen for human use because of the risk of ototoxicity, which is 
associated with long-term neomycin therapy. Thus, we will use neomycin only for the first three days and 
then it will be replaced with ciprofloxacin. Ciprofloxacin will be an appropriate replacement as it is well 
known to be deeply disruptive to the gut microbiota [4]. For the antifungal, we have chosen to use 
fluconazole, which is used to treat a variety of fungal infections, because it is considered to be a safer 
option than other systemic antifungals such as amphotericin.   
 
We know from our animal studies that the 16S rRNA gene copy number decreases after three days of 
therapy and returns to baseline five days after antibiotics are discontinued. Fourteen (14) days of therapy 
was chosen for this study so that there would be continued suppression of bacterial load to allow 
sufficient time for healing of the gastrointestinal tract. We chose standard adult dosing for the treatment of 
systemic or gastrointestinal infections (see Section 5.1 for dosages). 
 
Participants in this study will not be required to stop any prior medications (including 
immunosuppressive medications), with the exception of probiotics and antibiotics. If potential 
participants are on prescribed antibiotics related to their IBD, they are required to stop those 
antibiotics in order to participant in this study. If potential participants are on antibiotics for a non-
IBD related reason, they will be required to complete their current course before being enrolled in 
this study. Furthermore, participants already on steroids as part of their IBD regimen will be 
required to maintain their dosage and regimen during the 14 days (Days 1-14) of the study drug 
intervention. If dosage is increased during the 14 day study drug regimen, the participant will be 
considered to have started rescue therapy and study drugs will be stopped. 
 


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2 
Study Objectives 
2.1 
Primary Objective 
The primary objective of this study is to determine the efficacy of a novel gut microbiota-targeted 
therapeutic regimen, comprised of a bowel lavage and oral antibiotics with or without an antifungal, in the 
management of active CD that is refractory to conventional, immunosuppressive therapy. 
2.2 
Secondary Objectives 
The secondary objectives will be to: 
 
• 
Correlate effectiveness in reducing bacterial 16S and fungal 18S rRNA gene copy number, by the 
use of the regimen in the primary objective, with improvement in disease activity for patients with 
CD refractory to immunosuppression. 
 
• 
Determine the relationship between the effectiveness of the proposed therapies with changes in 
gut microbiota composition 
 
3 
Investigational Plan  
3.1 
General Design 
“Holiday” will be a randomized, placebo controlled, double blind Phase 2a trial, with an enrollment goal of 
30 adult patients to yield an estimated 20 evaluable participants.  Once 10 adult participants have 
completed the intervention phase, if there are an acceptable number and severity of adverse events, we 
will submit an amendment and joint protocol application to The Children’s Hospital of Philadelphia 
(CHOP) for an ultimate enrollment goal of 30 adults and 30 children to yield 20 evaluable participants in 
each group. Recruitment will target CD patients who have demonstrated nonresponse or LOR to 
immunosuppressive therapy, and may be facing escalation of therapy or even surgery.  Participants will 
be treated with bowel lavage and oral antibiotics with or without an antifungal over a period of 14 days. 
Clinical efficacy will be assessed at defined time points through scoring on the Harvey-Bradshaw Index 
(HBI) and the short Crohn’s Disease Activity Index (sCDAI), both validated measures of CD disease 
activity, the fecal calprotectin (FCP), a clinical marker of intestinal inflammation, and the high-sensitivity 
C-reactive protein (hs-CRP), a marker of systemic inflammation.  At the stool collection time points, the 
composition of the gut microbiota will also be determined using 16S gene qPCR, 18S gene qPCR, 16S 
gene sequencing, and internal transcribed spacer (ITS) gene sequencing to determine effects on 
bacterial and fungal load and microbiota composition. 16S gene qPCR determines bacterial load. 18S 
gene qPCR determines fungal load. 16S gene sequencing determines bacterial microbiota composition. 
ITS gene sequencing determines fungal microbiota composition. 
3.1.1 
Screening Phase 
Potential participants will be identified through outpatient clinic schedules and iConnect, as well as 
physician referrals.  For participants recruited through the outpatient clinic, written informed consent will 
be obtained at that outpatient visit, a REDCap Screening Questionnaire will be completed together, and 
consequently, an enrollment Clinical and Translational Research Center (CTRC) visit will be scheduled.  
Participants consented at an outpatient clinic visit will be given a stool collection kit, so that they may 
bring a stool sample to the CTRC visit.  For participants identified as meeting inclusion criteria for the 
study and referred to the study team by their physician, participant contact will be made via telephone 
using an IRB approved pre-screening script to obtain verbal consent. Once verbal consent is obtained, 
potential participants will be emailed a REDCap Screening Questionnaire. Participants who seem to meet 
all inclusion criteria (see Section 4.1 and 4.2) will be scheduled for a CTRC visit and will be shipped a 
stool collection kit via mail. Written informed consent for participants identified through physician referrals 
will be obtained as the first step of the enrollment CTRC visit. This same sequence of events will take 
place for iConnect recruits. For all participants, the enrollment CTRC visit will include a physical 


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examination by a study investigator or CTRC nurse practitioner to determine eligibility based on clinical 
parameters.  For eligible participants, blood and stool samples will also be obtained at the enrollment visit 
for baseline values.  If necessary, (no documentation of a negative test within the previous 4 weeks), the 
stool sample will be tested to rule out Clostridium difficile infection.  However, it will not be necessary to 
wait for the result of this test to begin the study regimen as vancomycin, one of the study medications, is 
standard treatment for C. difficile infection. If a participant tests positive for C. difficile infection at 
enrollment, they will be withdrawn from the study and their primary GI physician will be notified 
so that antibiotic therapy can be continued on a clinical basis.  The enrollment visit blood sample will 
also be assessed for serum creatinine to ensure no participants with impaired kidney function are actively 
enrolled. Female participants will have a urine pregnancy test. All participants will undergo 
electrocardiogram (EKG) to assess QTc interval at baseline. Participants will also be asked to complete 7 
days of an online “Daily Survey” through REDCap to record their baseline symptoms before starting any 
study drug. 
3.1.2 
Intervention Phase  
For participants who are fully eligible, medications will be shipped to the participant’s home address. 
There will be no more than 17 days between the enrollment visit and the start of the intervention phase. 
The antimicrobial regimen will include 3 days of vancomycin and neomycin followed by 11 days of 
vancomycin and ciprofloxacin. Participants will also be randomized in a double-blinded fashion to 
fluconazole or placebo for 14 days. See Section 5.1 for details about dose, frequency and duration of all 
study drugs. Promethazine can be taken on an as needed basis on Days 1-3 for nausea secondary to 
neomycin. 
 
On Day 2, participants will also undergo a bowel preparation with polyethylene glycol (PEG) 3350 (see 
Table 5.1). On this day, the diet will be limited to clear liquids. There will be no other dietary modifications 
during the course of the study. 
 
On Days 1-14, participants will record their symptoms daily through a secure, web-based portal 
(REDCap).  This information will be used to calculate the sCDAI score.  In– person study visits will take 
place on days 5, 8, and 15. The indicated clinical and laboratory parameters will be assessed at the time 
of these visits. See table in Section 6 for details. 
 
Serum creatinine levels of participants will also be intermittently monitored on Days 8, 15, and 29. At any 
time during the course of the intervention if a participant develops an elevated serum creatinine of greater 
than 1.3 in women and 1.5 in men or an increase of 0.25, study medications will be immediately stopped 
and participants will be brought in for an unscheduled visit to re-test their serum creatinine level. If their 
re-test results are within the normal range and/or less than 0.20 above their baseline, participants will 
resume study medications for the duration of the scheduled regimen, but if their levels are still elevated, 
they will be referred to their primary care physician (PCP) for clinical care. Events as described above will 
be considered an Adverse Event (AE) and there will be appropriate follow-up as detailed in Section 9 of 
this protocol to ensure that serum creatinine levels have normalized. 
 
There is a +/- 1 day window within which the Day 15 visit must occur. 
3.1.3 
Optional Intervention Extension Phase (14 day repeat drug regimen) 
Participants who achieve clinical response (reduction of the HBI by 3 or more points) or clinical remission 
(HBI < 5) to the study intervention at Day 15, but have relapsed (HBI ≥ 7 or sCDAI ≥ 220) between days 
30 and 64 (2 months), will be offered the option to repeat the 14 day study regimen. Once the study 
coordinator is aware of a participant’s disease relapse, the participant’s symptoms will be observed for 2 
weeks through the daily survey (but on a weekly basis) before starting their extension phase to ensure 
relapse. Participants who repeat the regimen will not require rescreening or a repeat enrollment visit as 
the repeated regimen will exclude fluconazole/ placebo, eliminating the risk of a prolonged QTc interval. 
The PEG lavage will also be excluded. An extra telephone visit (visit 6.1) will be added to the total 
number of the study visits. This visit will take place at the end of the extra 14 day regimen and so will vary 


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depending on the relapse date of each participant. All participants will have a 3 month visit (visit 7) and 
the last study visit for all participants will be at 6 months. 
3.1.4 
Follow Up Phase 
The follow-up phase will include Day 16 until 6 months following the end of the intervention phase.  The 
purpose of the follow-up phase is to determine the durability of the effect of the intervention over time.  A 
telephone follow-up will occur on Day 22.  An in-person study visit will take place on Day 29.  The 
indicated clinical and laboratory parameters will be assessed at the time of these visits (See Table 1 in 
Section 6). There is a +/- 3 day window within which these two visits (Day 22 and 29) must occur. Finally, 
telephone follow-ups will occur at 3 months and 6 months following the intervention phase.  There will be 
a 14-day (2 weeks) window in which the 3 month and 6 month follow-up telephone visits must occur for 
the data to be included in the analysis. 
3.2 
Study Endpoints  
3.2.1 
Primary Study Endpoints 
The primary endpoint will be the change in disease activity, as measured by HBI score and FCP 
concentration, between the enrollment visit and Day 15. All participants who withdraw for any reason prior 
to day 15 will be considered treatment failures. 
 
• 
Participants with an HBI < 5 at Day 15, and a FCP concentration ≤ 200 mcg/g at Day 15, will be 
defined as having achieved full clinical remission. 
• 
Participants with an HBI < 5 at Day 15, but with elevated FCP (i.e. > 200mcg/g), will be 
considered responders. 
• 
Participants with a decrease in HBI score of 3 or more points but HBI remains > 5 at Day 15, 
regardless of FCP concentration level, will be considered partial responders.  
3.2.2 
Secondary Study Endpoints 
Secondary endpoints will include: 
• 
The change in CRP (or hsCRP) between the enrollment visit and day 15 
• 
Correlation of effectiveness in reducing bacterial 16S and fungal 18S rRNA copy number, by the 
use of oral antimicrobials combined with bowel lavage, with improvement of disease activity in 
patients with CD refractory to immunosuppression. 
• 
The relationship between the effectiveness of the proposed therapies with changes in gut 
microbiota composition 
 
• 
Safety and tolerability of the treatment regimen based on medication side effects and/or adverse 
events (AEs). 
4 
Study Population and Duration of Participation  
4.1 
Inclusion Criteria 
• 
Participant is capable of giving informed consent 
• 
Males or females 18-75 years of age 
• 
Normal kidney function (defined by normal serum creatinine [male: <1.27mg/dL; female: 
<1.03mg/dL]) 


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• 
Normal AST (<41U/L), ALT (<63U/L), and alkaline phosphatase (<126U/L) 
• 
Active CD defined as HBI ≥ 7* 
*Patients who have a self-report HBI of at least 6 (excluding the abdominal mass component of the 
index, which requires a physical examination by a clinician) will be considered eligible for scheduling 
of an enrollment visit and will only be actively enrolled with a definite HBI score of 7 or above, 
inclusive of the physical examination and abdominal mass component.  
• 
hs-CRP >10mg/L (or 1mg/dL) or fecal calprotectin (FCP) > 350mcg/g (within one month of 
enrollment) 
• 
Have been treated with one of the following therapies** for at least 8 weeks with primary 
nonresponse or an initial response, followed by LOR (self-reported worsening of symptoms for ≥ 
7 days): azathioprine, 6-mercaptopurine, methotrexate, adalimumab, certolizumab, golimumab, 
infliximab, natalizumab, vedolizumab, or ustekinumab 
• 
If taking steroids, dose must be stable for at least 2 weeks prior to screening 
**These medications must have been administered at standard, therapeutic dosages. 
4.2 
Exclusion Criteria 
• 
Unwillingness to provide informed consent  
• 
Allergy or intolerance to the medications used in this study 
• 
Pregnant or lactating females 
• 
Known or suspected stricturing disease producing obstructive symptoms 
• 
Active Clostridium difficile infection 
• 
History of cirrhosis 
• 
Baseline QTc interval on EKG > 430ms in males or > 450ms in females 
• 
Current use of antibiotics 
• 
Starting  or increasing the dose of a IBD related medication within 4 weeks of screening including: 
o 
Azathioprine or 6-mercaptopurine (6MP) 
o 
Infliximab, adalimumab, certolizumab, or golimumab 
o 
Natalizumab or vedolizumab 
o 
Methotrexate 
o 
Any 5-ASA compound (e.g. Lialda, Asacol, etc) 
o 
Prednisone, budesonide or other steroids delivered orally or rectally 
• 
Participants who, in the opinion of the investigator, may be non-compliant with study schedules or 
procedures 


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4.3 
Participant Recruitment  
Potential participants will be identified through outpatient clinic schedules and physician referrals from the 
Hospital of the University of Pennsylvania (HUP) or Penn Presbyterian Hospital (PPCM). The study will 
also be posted on iConnect and participants may be reminded to ask their primary GI provider or contact 
a study team member directly about this study through blasts in the MyPennMedicine portal. The team 
will procure weekly patient lists from the Penn Data Analytics Center (DAC) and target this population by 
sending out an IRB approved template email via MyPennMedicine. This has been approved by the OCR 
PennChart Research workgroup. Lastly, the study will also be posted on the following website: 
http://i3studypenn.weebly.com/associated-studies.html. An IRB approved flyer may also be posted on this 
website. Lastly, other web-based advertising avenues may be utilized in recruitment efforts including 
targeted advertising through local disease/health foundations such as the Crohn’s and Colitis Foundation 
of America (CCFA). Recruitment will also be conducted using social media platforms such as Facebook 
and Twitter using IRB approved language and hashtags. If there are questions regarding initial eligibility, 
necessary information from the participant’s medical record will be obtained by the treating physician 
and/or a member of the study team. Whenever possible, for participants recruited through the outpatient 
clinic, written consent will be obtained at that outpatient visit, a REDCap Screening Questionnaire will be 
completed, and participants will be scheduled for an enrollment CTRC visit. For participants recruited 
through physician referrals, patients will be contacted and screened via telephone using an IRB approved 
pre-screening script to obtain verbal consent. Once verbal consent is obtained, potential participants will 
be emailed a REDCap Screening Questionnaire. Participants who seem to meet all inclusion criteria (see 
Section 4.1 and 4.2) will be scheduled for a CTRC enrollment visit. Crohn's disease history will be 
obtained. A stool collection kit will be given to or mailed to all participants so that a sample can be brought 
to the enrollment CTRC visit. No samples will be received or analyzed before obtaining written consent.  
 
All participants must provide written consent before undergoing any other study procedures. The study 
procedures will be explained to each participant at the time of pre-screening and written consent. If the 
participant agrees to further participate, the written informed consent form will be signed in-person at 
either the outpatient clinic visit or the enrollment CTRC visit.   
4.4 
Duration of Study Participation 
The study duration per participant will be approximately 6-8 months, with up to 30 days for screening 
(from telephone verbal consent to the enrollment visit), 2 weeks for the intervention phase, and up to 6 
months for the follow-up phase.   
 
Participants who meet criteria and opt to undergo the extension phase of the antimicrobial regimen will 
prolong their participation in the study by approximately 2 weeks.  
4.5 
Total Number of Participants and Sites  
The study will be conducted at the clinical centers at the University of Pennsylvania (HUP and PPMC).  
Recruitment will stop when approximately 20 participants are fully eligible.  It is expected that 
approximately 30 participants will be enrolled (screened) to produce 20 evaluable participants.  
4.6 
Vulnerable Populations:  
Children, pregnant women, fetuses, neonates, or prisoners are not included in this research study. 
 
5 
Study Intervention 
5.1 
Intervention Description and Regimen 
Drug 
Dosage Form 
Dosage 
Regimen 
Vancomycin 
500 mg oral suspension 
500 mg (1 syringe) every 6 hours 
Days 1-14 


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Neomycin 
500 mg tablet 
1 g (2 tablets) PO every 8 hours 
Day 1-3 
Ciprofloxacin 
750 mg capsule 
750 mg (1 capsule) PO every 12 hours 
Days 4-14 
Encapsulated fluconazole 
or placebo capsule 
100 mg tablet 
encapsulated 
400 mg (4 capsules) PO daily 
Days 1-14 
Miralax (PEG 3350) 
Powder 
238 g dissolved in 64 oz liquid with 
electrolytes 
Day 2 
Promethazine 
Tablet 
12.5mg PO as needed every  4 hours 
Days 1-3 
5.2 
Randomization 
Stratified randomization to fluconazole versus placebo will be performed using a 1:1 ratio of treatment 
arms. The randomization order will be created by University of Pennsylvania’s Investigational Drug 
Service (IDS) 
5.3 
Receipt  
After confirmation of eligibility, participants will be sent the antimicrobial and lavage regimen listed above 
directly from IDS to their home mailing address.  All study medications will be shipped in one parcel via 
UPS.  Participants will be provided with a checklist of all study medications and will be instructed to report 
any discrepancies to a member of the study team. 
5.4 
Storage 
Study medications will be prepared, stored, and dispensed by IDS under controlled conditions.  
5.5 
Preparation and Packaging 
To promote participant compliance and minimize potential confusion, study medications will be packaged 
into 2 blister packs: one for Days 1-7 and the other Days 8-14. The rows of the blister packs will be 
labeled as “Day 1” “Day 2” etc., through “Day 14”, with each dose separated into blisters by meal times. 
The vancomycin suspensions will be packaged in separate Ziploc bags per day.  Each bag will contain 
four syringes containing 500 mg/20 mL suspension vancomycin. The Day 2 bag will additionally contain a 
bottle with 238 g of PEG 3350.  At the enrollment visit, participants will be provided with a 64 ounce water 
pitcher plus either 2 32 ounce bottles of Gatorade or packets of Crystal Lite. This will depend on 
participant preference. However, for participants who have diabetes, Crystal Lite will be required. 
 
With the exception of vancomycin and fluconazole, all study medications will be distributed in their original 
formulation as per the manufacturer. For vancomycin, IDS will compound 1,120 mL of oral suspension 
(500 mg/20 mL concentration) drawn up into 56 syringes per participant (see Appendix B). For 
fluconazole, IDS will encapsulate the original formulation tablets so that they will look identical to placebo 
capsules. 
5.6 
Administration and Accountability    
Participants will be provided with all study medications by tracked courier (UPS) to their home address 
following the enrollment visit and consequent confirmation of their eligibility.  Participants will take all 
medications orally. At the final in-person study visit, all unused study medications and empty containers 
will be collected from each participant.   


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5.7 
Participant Compliance Monitoring 
At all encounters following the enrollment visit, compliance will be monitored through study staff 
observation of empty blister packs at each in person visit (see Section 5.7). At each in-person visit, 
participants will be asked to bring in their used and unused blister packs, syringes, and PEG bottle for 
review by study staff.  Compliance will be reviewed with the participants at each in-person visit. 
5.7.1 
Return or Destruction of Investigational Product 
Records of study drug receipt and disposition will be maintained by the IDS. Investigational drug orders 
and dispensing records will be examined quarterly by a study coordinator. The study medication will be 
prescribed by Study Investigators and may not be used for any purpose other than that described in this 
protocol. At study completion, all drug supplies including partially used and empty containers must be 
returned to Investigator or a study coordinator. 
 
 
 


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5.8 
Unblinding procedure 
 
At the end of the enrollment visit, eligible participants will be randomly assigned to one of the two 
treatment groups (fluconazole or placebo) according to the randomization schedule generated by Penn 
IDS. Neither the participant, the study team, nor the clinical site personnel will know the treatment group 
to which any participant is randomized.   
 
If there is a serious adverse event, which is thought by the study team to be possibly or probably related 
to the coded medication, the principal investigator, when necessary for the safety of the participant, will 
unblind treatment group assignment.  The following procedures will be taken when the sponsor or 
principal investigator deems that unblinding is necessary: The IDS will be notified by the study team that 
unblinding is necessary.  IDS will unblind the treatment group assignment and provide it to the principal 
investigator and the study coordinator. 
 
Unblinding of treatment assignment is anticipated to be an uncommon occurrence and is highly 
discouraged.  Unblinding should only be performed if deemed necessary for the safety of the participant.   
 
6 
Study Procedures 
Table 1: Schedule of Study Procedures  
 
Screening/ 
Recruitment 
Enrollment/ 
Day 0 
Day 5 
Day 8 
Day 15 
Day 22 
Day 29 
 
Day 36-64 
(Optional 
visit) 
3 mos 
6 mos 
Study Visit 
Visit 0 
Visit 1 
Visit 2 
Visit 3 
Visit 4 
Visit 5 
Visit 6 
Visit 6.1 
Visit 7 
Visit 8 
Phase of Study 
 
 
Intervention Phase 
 
 
 
Follow-up Phase 
Telephone visit 
X 
 
 
 
 
X 
 
X 
X 
X 
Web-based 
screening survey 
X 
 
 
 
 
 
 
 
 
 
Daily Survey 
 
 
X 
X 
X 
X 
X 
X (weekly) 
X 
(weekly) 
X 
(weekly) 
Medical record 
review 
X 
X 
 
 
 
 
 
 
X 
X 
Physical exam 
including vital signs 
 
X 
X 
X 
X 
 
X 
 
 
 
Urine pregnancy test 
for female 
participants 
 
X 
 
 
 
 
 
 
 
 
Stool collection for 
microbiome analysis 
 
X 
X 
X 
X 
X 
X 
 
 
 
Stool C. difficile 
toxin 
X (within 1 
month) 
X (if not done 
within 1 
month) 
 
 
 
 
 
 
 
 
Rectal swabs for 
mucosal analysis 
 
X 
 
X 
X 
 
X 
 
 
 
FCP 
X 
X 
 
X 
X 
X 
X 
 
 
 
CBC (no diff) 
 
X 
Blood 
draw to 
store 
for 
future 
X 
X 
 
X 
 
 
 
CMP 
 
X 
X 
X 
 
X 
 
 
 
ESR 
 
X 
X 
X 
 
X 
 
 
 


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hsCRP 
 
X 
use 
X 
X 
 
X 
 
 
 
Infliximab or 
adalimumab level** 
 
X 
 
 
 
 
 
 
 
HBI 
X (within 1 
month) 
X 
X 
X 
X 
 
X 
 
 
 
EKG 
 
X 
 
X 
 
 
 
 
 
 
 
**For participants on infliximab or adalimumab 
6.1 
Screening Phase (Visit 0) 
Potential participants will be identified through outpatient clinic schedules as well as physician referrals. 
For participants recruited through the outpatient clinic, written consent will be obtained at that outpatient 
visit and participants will be scheduled for an enrollment CTRC visit. Screening for these participants will 
proceed as follows: 
• 
Written informed consent at outpatient clinic visit 
• 
Email or complete with coordinator screening questionnaire through REDCap. If seems eligible, 
schedule enrollment CTRC visit  
• 
Distribute stool kit in-person at outpatient clinic visit or via courier service (UPS) 
 
Or for participants identified through physician referrals or iConnect, potential participants will be 
contacted and screened after obtaining verbal consent via telephone using a pre-screening script, 
emailed a screening questionnaire and scheduled for an enrollment CTRC visit. Screening for these 
participants will proceed as follows: 
• 
Receive physician referral or iConnect alert 
• 
Contact potential participant via telephone, obtain verbal consent, review study procedures and 
eligibility criteria 
• 
For applicable and interested participants, email screening questionnaire through REDCap. If 
seems eligible, schedule an enrollment CTRC visit 
• 
Distribute stool kit via courier service (UPS) 
6.2 
Enrollment Phase (Visit 1) 
Eligible participants will be seen by a study team investigator and study coordinator at the CTRC for the 
enrollment visit.  The written informed consent form may be signed at this visit if not already completed.  
Enrollment will proceed as follows: 
• 
Written informed consent (if applicable) 
• 
Review and documentation of eligibility criteria  
• 
Obtain collected stool sample (kit provided during screening phase) for analysis of C. difficile 
toxin, fecal calprotectin, and microbiome 
• 
Vital signs 
• 
Anthropometric measurements 
• 
Physical exam, including CD disease symptom assessment for HBI 
• 
Rectal swabs (2) for mucosally-associated microbiome analysis 
• 
Blood sample (CBC, CMP, hsCRP, ESR, biologic levels if applicable with extra stored for future 
use) 
• 
Urine pregnancy test for female participants 
• 
EKG 
• 
Participant counseled to continue all current medications except probiotics 
• 
Medication education 
• 
Distribution of Gatorade or Crystal Lite (and a pitcher) 
 
On the same day as enrollment, one (1) stool collection kit will be given to each participant so that a 
sample can be brought to us on Day 8. 


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6.3 
Intervention Phase (Days 1-15 including Visits 2-4): In-person Study Visits 
Day 1 is defined as the day that a participant starts the study drug regimen. Regardless of the day that 
participants receive the study drugs in the mail from IDS, they will be instructed call the coordinator, who 
will instruct them to start taking study drugs on the upcoming Monday, Thursday, or Friday, in order to 
enhance compliance and ensure that in-person study visits do not fall on weekends. This was also allow 
coordinators to track medication packages.  
6.3.1 
Daily Surveys 
After verbal and/or written consent is obtained from participants, they will be asked to complete 7 days of 
online daily surveys via REDCap before Day 1 to obtain baseline data. From Day 1 through Day 29 of the 
study (intervention phase and first 15 days of follow-up phase), participants will complete the same daily 
survey through REDCap so that the study team can monitor disease activity by the sCDAI.  Participants 
will be prompted to identify any concerns including medication side effects and/or worsening of 
symptoms.  From Day 30 through 6 months, the survey including disease activity questions only will be 
completed once weekly. On in-person study visit days, the daily survey may be completed with the study 
coordinator during the visit. 
 
Survey responses will be monitored by study staff through REDCap on a regular basis. If more than two 
(2) daily surveys are not completed in a week, the coordinator will contact the participant via phone and/or 
email to ensure compliance. If a weekly survey is not completed, participants will also be contacted by the 
study coordinator. 
 
If a participant is enrolled in the study that does not have daily access to the internet and/or email, a daily 
survey paper diary will be provided to them at the start of their study participation. This will include all the 
REDCap daily (and weekly) surveys. In order to ensure compliance for this group, participants will be 
required to ship the completed paper surveys to the study team on a weekly basis. UPS or FedEx 
shipping labels will be provided in advance to enable this practice. 
6.3.2 
Study Visit 2 (Day 5) 
Study Visit 2 will take place on Day 5.  Participants will be seen at the Penn CTRC by a study coordinator 
and/or CTRC nurse.  The following will be required: 
• 
Vital signs 
• 
Anthropometric measurements 
• 
Physical exam, including CD disease symptom history for HBI 
• 
Blood sample for future use (metabolomics and neomycin levels) 
• 
Collect stool sample (kit provided at previous visit) 
• 
Adverse event assessment (see Section 9) 
• 
Study drug compliance assessment 
• 
Update current medications log 
• 
Daily Survey (optional) 
6.3.3 
Study Visit 3 (Day 8) 
Study Visit 3 will take place on Day 8.  Participants will be seen at the Penn CTRC by a study coordinator 
and/or CTRC nurse.  The following will be required: 
• 
Vital signs 
• 
Anthropometric measurements 
• 
Physical exam, including CD disease symptom assessment for HBI 
• 
EKG 
• 
Rectal swab for mucosally-associated microbiome analysis 
• 
Blood sample (CBC, CMP, hsCRP, ESR with extra stored for future use) 
• 
Collect stool sample (kit provided at previous visit) 
• 
Adverse event assessment (see Section 9) 


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• 
Study drug compliance assessment 
• 
Update current medications log 
• 
Daily Survey (optional) 
On Day 8, one (1) stool collection kit will be given to each participant so that a sample can be brought to 
us on Day 15.  
6.3.4 
Study Visit 4 (Day 15) 
Study Visit 4 will take place on Day 15.  Participants will be seen at the Penn CTRC by a study 
coordinator and/or CTRC nurse.  The following will be required: 
• 
Vital signs 
• 
Anthropometric measurements 
• 
Physical exam, including CD disease symptom assessment for HBI 
• 
Rectal swab for mucosally-associated microbiome analysis 
• 
Blood sample (CBC, CMP, hsCRP, ESR with extra stored for future use) 
• 
Collect stool sample (kit provided at previous visit) 
• 
Adverse event assessment (see Section 9) 
• 
Study drug compliance assessment and collect any unused study drug 
• 
Update current medications log 
• 
Daily Survey (optional) 
 
On Day 15, two (2) stool collection kits will be given to each participant so that 1) a sample can be 
collected and mailed to us by Day 22 and 2) a sample can be brought to us on Day 29 (Study Visit 6). 
6.4 
Follow-Up Phase (Days 16- 6 months including Visits 5-8): Telephone and In-person Study 
Visits 
The follow-up phase will include Day 16 through 6 months following the end of the intervention phase.  
Telephone follow-up will occur on Day 22 (see Section 6.4.1). An in-person study visit will take place on 
Day 29.  Day 30 onwards, until 6 months, participants will continue to record symptoms in REDCap, once 
weekly. Telephone follow-up will occur at 3 months and 6 months following the beginning of the 
intervention. 
6.4.1 
Telephone Follow-Up: Visit 5 (Day 22) 
A telephone follow-up will be conducted on Day 22 by the study coordinator. During the telephone 
session, questions will be asked to assess the general state of their Crohn’s Disease and their current 
medication regimen. The coordinator will also look at their electronic medical record to record any recent 
surgeries, hospitalizations, and C diff. test results since their last study visit. Participants will be reminded 
to ship a stool sample collected within 24 hours of this call 
6.4.2 
Study Visit 6 (Day 29) 
Visit 6 will take place on Day 29.  Participants will be seen at the Penn CTRC by a study coordinator and 
investigator or CTRC nurse.  The following will be required: 
• 
Vital signs 
• 
Anthropometric measurements 
• 
Physical exam, including CD disease symptom assessment for HBI 
• 
Rectal swab for mucosally-associated microbiome analysis 
• 
Blood sample (CBC, CMP, hsCRP, ESR with extra stored for future use) 
• 
Collect stool sample (kit provided at previous in-person visit) 
• 
Adverse event assessment (see Section 9) 
• 
Study drug compliance assessment 
• 
Update current medications log 


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• 
Daily Survey (optional) 
6.4.3 
Telephone Follow-Ups: 3 Months & 6 Months 
Telephone follow-up will also occur at 3 months and 6 months after the intervention phase.  For these 
follow-up sessions, we will allow a two-week window. During the telephone session, questions will be 
asked to assess the general state of their Crohn’s Disease and their current medication regimen. The 
coordinator will also look at their electronic medical record to record any recent surgeries, 
hospitalizations, and C diff. test results since their last study visit.  
6.5 
Rescue Therapy 
If a participant’s condition worsens during the course of the study, the PI in conjunction with the 
participant’s primary provider will make a decision, based on clinical expertise, to withdraw the participant 
from the study.  If the participant receives a rescue medication after the study intervention regimen is 
completed (Day 15 or later) the participant has the option to continue to participate in the study, at his or 
her discretion.  For participants who need to receive rescue medication(s) during Days 1-14 of the study 
from their primary GI physician, study drug will be stopped and they will be considered to have not 
responded to therapy. With their permission, these participants will be followed to the end of the 6 month 
time point of the study schedule. 
6.6 
Participant Withdrawal  
Participants may withdraw from the study at any time without prejudice to their clinical care.  They may 
also be discontinued from the study at the discretion of the Investigator for lack of adherence to study 
treatment or visit schedules, AEs, lack of response, reasons of safety, administrative reasons and/or need 
for rescue medication during the enrollment or intervention phase.  Participants who require rescue 
medication or surgery after completion of the intervention regimen (Day 15 or later) will continue to be 
followed by the study with their permission.  It will be documented whether or not each participant 
completes the clinical study. If the Investigator becomes aware of any serious, related adverse events 
after the participant completes or withdraws from the study, they will be recorded in the adverse event log 
of the study. 
6.7 
Early Termination Visits   
If a participant would like to withdraw their participation, research study staff will document when and why 
the participant withdrew from the study. In this case, no further action will be needed. Participants will be 
made aware in the consent process that data collected prior to their withdrawal may still be used for study 
analysis. Participants who withdraw consent to participate in the study or who are withdrawn by the 
principal investigator during the intervention phase will be seen for one final in-person visit to collect the 
investigational product.  
7 
Study Evaluations and Measurements  
7.1 
Medical Record Review 
The following is a list of elements that may be abstracted from the participant’s medical chart (either 
paper or electronic): 
• 
Date of birth 
• 
MRN 
• 
Mailing address 
• 
Email address 
• 
Telephone number 
• 
Sex 
• 
Race 
• 
Most recent height 


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• 
Most recent weight 
• 
Crohn’s disease history (including date of diagnosis, and disease location) 
• 
Prior use of medications 
• 
Current medications 
• 
Medication allergies 
• 
Surgical history (including all surgeries regardless of relation to IBD) 
• 
Co-morbid medical conditions 
• 
History of stricturing or fistulizing disease (from review of records for history of bowel obstruction 
and imaging studies) 
• 
Patient report of bowel frequency 
• 
Patient report of abdominal pain 
• 
Patient report of general well-being 
• 
Most recent physical examination 
• 
Laboratory tests (including ESR, CRP, albumin, hematocrit, hemoglobin, fecal calprotectin, stool 
C. difficile toxin, drug levels for biologics) 
7.2 
Physical Examination 
A physical examination will be performed in order to calculate the HBI at all in-person study visits. A study 
investigator or CTRC nurse practitioner will document whether there is an abdominal mass (0=none, 
1=dubious, 2=definite, 3=definite and tender). A study investigator or CTRC nurse practitioner will also 
document any extraintestinal manifestations of disease, including arthralgia, uveitis, erythema nodosum, 
aphthous ulcers, Pyoderma gangrenosum, anal fissure, new fistula, and/or abscess. A brief overall 
physical exam may also be completed with documentation being completed in EPIC by creating an “out of 
office” encounter. A copy of this documentation should be printed out from EPIC and added to the 
participant’s study file. 
7.3 
Vital Signs 
Oral temperature will be obtained using a digital thermometer.  Heart rate and blood pressure will be 
obtained using an automated device while the patient is seated.  Height and weight will also be obtained. 
Height will only be measured once at the enrollment visit. All anthropometric measurements will be 
conducted with the participants wearing light clothing without shoes. Weight (0.1 kg) will be measured on 
a digital electronic scale (Seca, Munich, Germany), and stature (0.1 cm) on a stadiometer (Holtain, 
Crymych, UK). Clinical anthropometric measurements taken may be used for the study as well.  
7.4 
EKG 
A 12-lead EKG will be performed by trained CTRC staff on Day 0 (the enrollment visit) and on Day 8 (Visit 
3). EKG is indicated because both ciprofloxacin and fluconazole carry some risk of QTc interval 
prolongation. The QTc interval will be assessed by a study investigator using manual determination with a 
caliper. A prolonged QTc interval is defined as >430msec in males and >450msec in females. 
Participants with a prolonged QTc interval at baseline will be excluded. Participants with a prolonged QTc 
interval on Day 8 will discontinue the fluconazole/ placebo aspect of the study medication regimen. The 
study coordinator will remove the fluconazole/ placebo from the blister pack to avoid accidental doses. 
7.5 
Laboratory Blood Evaluations 
Blood will be collected by trained CTRC staff and processed either by the Hospital of the University of 
Pennsylvania clinical laboratory (Pepper Lab) or by ARUP laboratories.  The following testing will be 
performed: 
• 
ESR  
• 
hsCRP 
• 
CMP 
• 
CBC without differential 
• 
*Drug levels for participants on biologics stored for future testing 


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• 
**Extra blood for storage for future testing 
* At the enrollment visit, blood will be obtained for drug level and antibody testing for patients who are 
being treated with infliximab or adalimumab (ARUP laboratories). Serum will be stored frozen in ARUP 
transport tubes. Serum will be stored for future use so that it will be possible to correlate response to this 
regimen with biologic drug levels and presence or absence of biologic medication antibodies. 
**An extra 8mL of blood will be drawn and will be stored for future metabolomics studies at the enrollment 
visit, day 5, day 8, day 15, and day 29. 
7.6 
Pregnancy Testing 
A urine pregnancy test will be performed at the enrollment visit in the CTRC for all female participants.  
7.7 
Stool Collection, Shipping, and Analysis 
Each participant will be provided with five (5) stool collection kits to take home during the course of the 
study. Each kit will contain a collection hat, ice packs, shipping bag, a box to hold the collection container, 
a specimen bag, and a prepaid shipping label (for the one specimen that will be shipped). Each kit will 
also include a small spoon top vial with pre-measured ethanol to preserve the freshest stool sample. 
Samples being shipped with an ethanol aliquot will be appropriately packaged with an “Excepted 
Quantities” label. This aliquoting will be completed by the participants before shipping the sample. Stool 
collection and aliquoting instructions will be provided and verbally explained to all participants in advance. 
The participants will also be asked to complete a form identifying their sample from the standard Bristol 
Stool Chart. 
 
Participants will be instructed to collect a bowel movement at 5 time points – enrollment, Day 8, Day 15, 
Day 22, and Day 29. Stool samples must be collected no more than 24 hours before an in-person visit 
delivery time or being picked up by UPS for shipping. The Day 22 sample will be shipped to the study 
team. All other samples will be delivered to the study team on the day of the in-person study visits. For 
Day 22, participants will be counseled that stool samples should not be collected or shipped Friday-
Sunday. Alternatively, the participant may also choose to deliver the Day 22 sample to a study team 
member in-person. 
 
All specimens remaining after the study is complete will be retained for possible future use unless the 
participant requests that the specimen be destroyed (opt-out) or it is deemed by the investigators that the 
specimens are no longer needed. 
An aliquot of stool from the enrollment sample will be sent to Penn microbiology laboratory for C. difficile 
testing if necessary (not already obtained clinically as previously described). An aliquot of stool from each 
of the 5 stool samples will be sent to a study lab for FCP testing. The remaining stool will be aliquoted 
and stored in the laboratory of Dr. Gary Wu (sub-investigator). The tube containing ETOH will be stored at 
-80°C. The excess stool will be aliquoted as follows and stored at -80°C: 4 spoon-top tubes (Sarstedt) – 3 
dry and 1 containing RNALater®, 1 coring tube (Globe Scientific) to obtain frozen stool cores for use with 
CryoXtract 350. All analyses will occur in Dr. Wu’s laboratory or at the PennCHOP Microbiome Center. 
7.8 
Rectal Swabs 
At the enrollment visit, Study Visit 3, Study Visit 4, and Study Visit 6, participants will undergo a rectal 
swab for assessment of the mucosally-associated gut microbiota.  Based on results from the Stool/Swab 
Pilot study (CHOP IRB #11-008506), differences were identified in the composition of the gut microbiota 
in rectal swabs as compared to traditional stool samples. Thus, we will study microbial differences 
between collected rectal swabs and stool samples [5]. 
 
Rectal swabs will be obtained during the physical examination by the CTRC nurse practitioner or study 
investigator. Two (2) sterile swabs will be inserted 3 cm into the rectum, turned 360 degrees, removed, 


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placed into a sterile tube, and frozen at -80 degrees celcius until analysis. One (1) swab will be waved in 
the air and collected as a control. 
 
For the first 5 subjects, one of the swabs at each time point will be delivered immediately to Dr. Wu’s 
laboratory for anaerobic culture. This is in order to validate microbiome PCR results with anaerobic 
culture. 
 
All frozen swabs will be analyzed by the PennCHOP Microbiome Center. 
 
7.9 
Efficacy Evaluations 
7.9.1 
HBI and short CDAI scoring 
The Harvey-Bradshaw Index (HBI) is a validated, non-invasive scale that is widely used in clinical trials to 
assess Crohn’s disease activity among adults [31, 32].  Devised as a “simple” but accurate alternative to 
the full Crohn’s Disease Activity Index (CDAI), which requires patients to recall and average daily 
symptoms over the course of a week, the HBI scale incorporates self-reported symptoms and physical 
examination findings for an overall score of 0 to >16 [31].  In accordance with clinical trial standards, the 
current study will define active disease as an HBI score ≥ 7. To be defined as having reached full clinical 
remission, patients must achieve an HBI score < 5, and a FCP concentration ≤ 200 mcg/g.  Participants 
with HBI < 5 but with elevated FCP (i.e. > 200 mcg/g) will be considered partial responders.  Participants 
who achieve clinical response with a reduction in HBI score of three or more, but without remission (HBI < 
5), will also be considered partial responders.  The short CDAI (sCDAI), a modified, validated score of 
disease activity that includes only patient self-report elements [33], will be administered through the daily 
REDCap surveys on Day 1-29 and then weekly thereafter (see Section 6.3.1).  
7.9.2 
Fecal calprotectin 
Evidence suggests that fecal calprotectin (FCP) represents an excellent surrogate marker of intestinal 
inflammation and disease activity. Calprotectin is 36 kDa calcium- and zinc-binding protein that 
represents 60% of cytosolic proteins in granulocytes [34].  Furthermore, calprotectin is highly stable in 
feces when stored at room temperature for up to 1 week [6].  Clinically, the concentration of calprotectin 
in feces (FCP) is used as a non-invasive measure of neutrophilic infiltrate in the bowel mucosa, and thus 
intestinal inflammation. The correlation of decreased FCP concentration to mucosal healing has been 
demonstrated by endoscopy in ulcerative colitis and Crohn’s disease [7]. This has been confirmed in both 
adult and pediatric populations with Crohn’s disease [8, 9]. A recent meta-analysis identified a cut point of 
250 mcg/g as optimal to distinguish the presence or absence of endoscopically detectable mucosal 
inflammation [40]. However, reduction in FCP is also used to assess improvement. For example, following 
treatment with anti-TNF therapy, FCP has been demonstrated to dramatically decline [41].   
In this study, FCP concentration will be assessed using ELISA methods ( study labs). 
7.9.3 
High-sensitivity C-reactive protein (hs-CRP) 
C-reactive protein is produced mainly in hepatocytes in response to acute phase stimuli such as 
inflammation.  Its production is driven by circulating cytokines. C-reactive protein is commonly used to 
screen the activity of chronic inflammatory diseases including IBD. In general, patients with CD have a 
high CRP when the disease is active and a normal CRP when the disease is quiescent.  High-sensitivity 
CRP (hs-CRP) assays measure CRP levels that were previously thought to be under detection limits.  Hs-
CRP levels are thought to correlate with disease activity in IBD. Hs-CRP will be tested through Pepper 
Labs at the University of Pennsylvania. 
7.9.4 
Microbial DNA sequencing 
DNA will be prepared in the PennCHOP Microbiome Center. Samples will be sent to the center de-
identified and coded with a study number. We will perform 16S rRNA and ITS gene sequencing to 
evaluate the bacterial and fungal microbiota, respectively, in stool and rectal swab samples. Isolated DNA 
will be quantified using the Picogreen system and 50 ng of DNA will be amplified.  Pyrosequencing will be 


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carried out using barcoded primers as previously described [10].  For pyrosequencing of bacteria, primers 
annealing to the V1V2 region of the 16S bacterial gene will be used.  The development of the ITS1 fungal 
primers is described in [10].  For pyrosequencing, we will use the Roche/454 Genome Sequencer Junior.  
Sequence data will be processed using QIIME [11]. If warranted by our preliminary data, we may analyze 
samples further using a metagenomic approach, in which DNA samples are nebulized, ligated to linkers, 
and subjected to pyrosequencing (IlluminaHiSeq).  This allows enumeration of the types of genes present 
in a sample. To determine bacterial and fungal load, respectively, we will determine 16S and 18S gene 
copy number from the stool and rectal swab samples. The qPCR methods including the details of the 
primers as well as PCR cycling conditions have been previously described [12]. 
 
7.10 Safety Evaluations 
Participant safety will be monitored by monitoring adverse events, medication side effects, vital signs, 
physical examinations, and laboratory data. The Principal Investigator and study team will specifically 
monitor participants for fever, nausea/vomiting, increased abdominal pain, and increased diarrhea from 
baseline (see Section 9).  Adverse events will be compared to all known side effects of the antimicrobials 
prescribed through the current study (see Appendix A). All adverse events will be tracked and assessed 
by the PI. Adverse events will be reported according to Penn Research Policy and Procedure – please 
see Section 9.5 for additional safety monitoring details for this study. 
8 
Statistical Plan 
8.1 
Primary Endpoint 
The primary endpoint will be the change in disease activity, as measured by HBI and FCP concentration, 
between the enrollment visit and Day 15.All participants who withdraw for any reason prior to day 15 will 
be considered treatment failures.  
 
• 
Participants with an HBI < 5 at Day 15, and a FCP concentration ≤ 200 mcg/g at Day 15, will be 
defined as having achieved full clinical remission. 
• 
Participants with an HBI < 5 at Day 15, but with elevated FCP (i.e. > 200mcg/g), will be 
considered responders. 
• 
Participants with a decrease in HBI score of 3 or more points but HBI remains > 5 at Day 15, 
regardless of FCP concentration level, will be considered partial responders.  
8.2 
Secondary Endpoints  
Secondary endpoints will include the following: 
• 
The change in CRP (or hsCRP) between the enrollment visit and Day 15 
• 
Correlation of effectiveness in reducing bacterial 16S and fungal 18S rRNA copy number, by the 
use of oral antimicrobials combined with bowel lavage, with improvement of disease activity in 
patients with CD refractory to immunosupression. 
• 
The relationship between the effectiveness of the proposed regimen with changes to the 
composition of the gut microbiota 
• 
Safety and tolerability of the treatment regimen based on medication side effects and/or adverse 
events (AEs). 
8.3 
Statistical Methods 
The two treatment groups will be compared using standard descriptive statistics. Categorical variables will 
be compared using Fisher’s exact test and continuous variables will be compared using the unpaired t-
test or Wilcoxon rank sum test if the data are not normally distributed.   
 


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Our estimates of efficacy will use four outcome measures: clinical response, clinical remission, reduction 
in FCP concentration, and reduction in hsCRP. For clinical response (reduction of score on HBI by 3 or 
more points) we will report the proportion and binomial 95% confidence intervals. Similar methods will be 
used for remission (HBI < 5). Although 250 mcg/g has been recently recommended, there is no standard 
definition of a clinically meaningful reduction in FCP and many definitions have been used. Therefore, we 
will use a paired t-test (after applying a log transformation if necessary) to establish whether the FCP 
concentration is lower following therapy than at baseline and we will also report the proportion of patients 
with reduction in FCP to less than 250 mcg/g among the subset with baseline FCP > 350 mcg/g.  
Similarly, there is no standard definition of a clinically meaningful reduction in hsCRP.  Therefore, we will 
use a paired t-test (after applying a log transformation if necessary) to establish whether the hsCRP is 
lower following therapy than at baseline. 
 
As a Phase 2a study to determine the effectiveness of the two treatment arms to induce a clinical 
response or remission, with 20 participants, it is possible to generate the required data [43].  
 
To detect reduction in bacterial and fungal load by 16S and 18S gene copy number we will use a paired t-
test after applying a log transformation. Based on our preliminary data, we assumed that the standard 
deviation of change will be approximately 0.4 log and that a minimum of a 2 log drop in copy number 
would be clinically significant. To have 90% power, this requires only 3 participants. Even if the standard 
deviation is greater, say 1 or 2 log, the required sample size is 5 or 13, respectively.  We will measure the 
Pearson’s correlation to determine the relationship between copy numbers and the outcome measures 
listed above (HBI, FCP, and hsCRP). If all patients do not have >2 log drop in 16S and 18S copy number, 
we will compare clinical remission and response among those with and without >2 log drop using Fisher’s 
exact test. 
 
Our main statistical and computational tool for comparing the bacterial gut microbiota among different 
groups (e.g. between samples from different time points) is the phylogenetic-based method as 
implemented in the program UniFrac [46, 47], which measures the similarity among the community based 
on phylogenetic distances determined by the 16S rRNA gene sequences of different bacteria. Based on 
these distances, we can cluster the microbiomes using the Principal Coordinate Analysis (PCoA) along 
axes of maximal variance.  The significant principal components can then be compared between two 
groups using the two sample t-tests. Permutations can be used to obtain the p-values. Alternatively, we 
can compare several principal components simultaneously by performing nonparametric permutation test 
for association between the two groups and the microbiome compositions. Specifically, we can randomize 
the labels of the groups and compare all distances between points that both come from the same group 
to all distances between points from different groups using t-tests. In such permutation test, we can obtain 
a nonparametric distribution of the t statistic that takes into account the correlations introduced by the 
pair-wise distance matrix structures. For the fungal community distances, Jaccard and abundance-
weighted Jaccard indices will be calculated. 
8.3.1 
Safety Analysis  
All participants entered into the study at the enrollment visit will be included in the safety analysis. The 
frequencies of AEs by type, body system, severity and relationship to study drug will be summarized. 
SAEs (if any) will be described in detail.  
 
AE incidence will be summarized along with the corresponding exact binomial 95% two-sided confidence 
intervals. 
 
We expect that adverse events will occur because the participants included in this study will have active, 
refractory disease.  Thus, hospitalizations or even life-threatening events may occur.  However, we 
anticipate very few, if any, adverse events that are directly related to the proposed intervention. 
 


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9 
Safety and Adverse Events 
9.1 
Definitions 
9.1.1 
Adverse Event 
An adverse event (AE) is any untoward medical occurrence associated with the use of a drug in humans, 
whether or not considered drug related. 
 
All AEs (including serious AEs) will be noted in the study records and on the case report form with a full 
description including the nature, date and time of onset, determination of non-serious versus serious, 
intensity (mild, moderate, severe), duration, causality, and outcome of the event. 
9.1.2 
Serious Adverse Event 
A serious adverse event (SAE) is an adverse event that, in the view of either the investigator or sponsor, 
results in any of the following outcomes: 
 
• 
death, 
• 
a life-threatening adverse event,  
• 
inpatient hospitalization or prolongation of existing hospitalization, 
• 
a persistent or significant incapacity or substantial disruption of the ability to conduct normal life 
functions, or a congenital anomaly/birth defect 
 
Important medical events that may not result in death, be life-threatening, or require hospitalization may 
be considered a serious adverse drug event when, based upon appropriate medical judgment, they may 
jeopardize the participant and may require medical or surgical intervention to prevent one of the 
outcomes listed in this definition. 
9.2 
Recording of Adverse Events 
At each contact with the participant the Principal Investigator and other study personnel will seek 
information on adverse events by specific questioning and, as appropriate, by examination.  Information 
on all adverse events will be recorded immediately in the adverse event log (AE- CRF).  All clearly related 
signs, symptoms, and abnormal diagnostic procedures results should recorded in the log, though should 
be grouped under one diagnosis. 
 
All adverse events occurring during the period will be recorded.  The clinical course of each event will be 
followed until resolution, stabilization, or until it has been determined that the study intervention or 
participation is not the cause.  Serious adverse events that are still ongoing at the end of the period will 
be followed up to determine the final outcome.  Any serious adverse event that occurs after the study 
period and is considered to be possibly related to the study intervention or study participation will be 
recorded and reported immediately. 
9.3 
Relationship of AE to Study  
The relationship of each AE or SAE to the study intervention will be characterized by the PI, with input 
from the study team as necessary, using one of the following terms in accordance with Penn IRB Policies: 
definitely related, probably related, possibly related, unlikely or unrelated.  
9.4 
Reporting of Adverse Events and Unanticipated Problems 
Adverse events, serious adverse events, unanticipated problems, and adverse reactions will be reported 
to the Penn IRB through the HS-ERA system.  Events that qualify for expedited reporting per Penn IRB 
Reporting Requirements will be reported within 3 or 10 business days, depending on the categories in the 
table below.  All other events that do not fit expedited reporting requirements will be reported at the time 
of Continuing Review submission.  


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9.4.1 
Study SAE Notification to Investigator 
An SAE must be reported to the study investigators by telephone within 24 hours of the event.  An 
SAE Form must be completed by the investigator.  The investigator will keep a copy of this form on 
file at the study site.  Report SAEs by phone or pager to: 
 
James D. Lewis, MD, MSCE 
Phone – (215) 573-5137  
Cell – (856) 906-3173 
 
In the event that Dr. Lewis cannot be reached report SAEs to  
Lindsey Albenberg, DO 
Phone - (215) 590-1680 
 
Or 
 
Unmesha Roy Paladhi 
Phone – (215) 746-7138 
Cell – (267) 788-8417 
 
At the time of the initial report, the following information should be provided: 
• 
Study Name 
• 
Participant number 
• 
A description of the event 
 
• 
Date of onset 
• 
Current status  
• 
The reason why the event is classified 
as serious 
Within the following 48 hours, the investigator must provide further information on the SAE in the form of 
a written narrative.  This should include a copy of the completed SAE Form and any other diagnostic 
information that will assist the understanding of the event.  Significant new information on ongoing SAEs 
should be provided promptly to the study investigator. 
 
9.4.2 
Penn IRB Safety and Expedited Reporting Requirements 
 
Unanticipated Problem/Adverse Event Classification 
Penn IRB Reporting 
Requirements 
All Studies 
Research Related 
Unexpected 
Fatal/Life Threatening 
X 
X 
X 
≤ 3 working days 
X 
X 
 
≤ 10 working days 
Other Events: 
• 
New information showing increased risk to participants 
• 
Unapproved protocol deviation to assure protection of human 
participants 
• 
Protocol deviation that places participants at risk or has the potential 
to occur again 
• 
Any serious or continuing non-compliance 
• 
Breach of confidentiality 
• 
Incarceration of participant 
• 
Withdrawal from marketing for safety concerns of the research drug, 
≤ 10 working days 


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device, or biologic 
 
9.4.3 
Follow-up report 
If an SAE has not resolved at the time of the initial report and new information arises that changes the 
investigator’s assessment of the event, a follow-up report including all relevant new or reassessed 
information (e.g., concomitant medication, medical history) should be submitted to the Penn IRB. The 
investigator is responsible for ensuring that all SAE are followed until either resolved or stable.  
9.4.4 
Investigator Reporting: Notifying the Penn IRB  
The Investigator will promptly notify the Penn IRB of all on-site, unanticipated SAEs that are related to the 
research activity. Other unanticipated problems related to the research involving risk to participants or 
others will also be reported promptly. Written reports will be filed using the HS-ERA system and in 
accordance with the Penn IRB Policies (see Table 9.4.1). External SAEs that are both unexpected and 
related to the study intervention will be reported promptly after the Investigator receives notification.  
9.5 
Medical Monitoring 
The current study has received an Investigational New Drug (IND) Exemption from the Penn IND/IDE 
Support Unit within the Office of Clinical Research. As part of an established safety and monitoring plan, 
the Primary Investigator will monitor adverse events and unanticipated problems during the study to 
monitor ongoing participant safety. A Safety Officer, David S. Goldberg, MD, MSCE (646-242-6349), 
has been identified to provide additional monitoring for the study. This individual is a board-certified 
gastroenterologist in adult practice who is knowledgeable in the natural history and treatment of CD, but 
who is not directly involved in the study and has no conflict of interest, financial or otherwise.  Following 
Penn IRB guidelines, all onsite, unanticipated SAEs will be reported to the Safety Officer promptly (within 
3 working days) in the form of a written narrative. This will include a copy of the completed Serious 
Adverse Event form and any other information that will assist the understanding of the event. Significant 
new information for ongoing serious adverse events should be provided promptly to the study sponsor. 
 
All other adverse events will be reported to the Safety Officer quarterly.  Based on these reports, the 
Safety Officer will have the authority to suspend the study and to convene a Data and Safety Monitoring 
Board (DSMB) as necessary.  We do not feel that a DSMB is necessary at the time of study launch given 
the open-label design and utilization of FDA-approved medications. This monitoring plan, in addition to 
screening for prolonged QTc and kidney and liver dysfunction, has been established to ensure the safety 
of the investigational regimen in this population.  
9.5.1 
Data and Safety Monitoring Plan 
All data and records generated during this study will be kept confidential in accordance with Institutional 
policies and HIPAA regulations, and the Principal Investigator and other site personnel will not use such 
data and records for any purpose other than conducting the study.  Safeguards are described under Data 
Management.  The following groups of people at University of Pennsylvania may have access to this 
information: the research team, medical staff who are directly or indirectly involved in patients’ care, and 
the Penn IRB.  The data shared with the PennCHOP Microbiome Center will be coded.  The results of 
this study may be shown at meetings or published in journals, but we will keep all identifying information 
private in any publication or presentation about this study. 
 


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10 Study Administration, Data Handling and Record Keeping 
10.1 Confidentiality 
Information about study participants will be kept confidential and managed according to the requirements 
of the Health Insurance Portability and Accountability Act of 1996 (HIPAA).  Those regulations require a 
signed participant authorization informing the participant of the following:  
• What protected health information (PHI) will be collected from participants in this study 
• Who will have access to that information and why 
• Who will use or disclose that information 
• The rights of a research participant to revoke their authorization for use of their PHI.  
 
In the event that a participant revokes authorization to collect or use PHI, the investigator, by regulation, 
retains the ability to use all information collected prior to the revocation of participant authorization.  For 
participants that have revoked authorization to collect or use PHI, attempts should be made to obtain 
permission to collect at least vital status (i.e. that the participant is alive) at the end of their scheduled 
study period. 
10.2 Data Collection and Management  
In order to ensure privacy, the collection of stool will be conducted in a private bathroom within the CTRC 
facility or the patient’s home.  Stool and swab samples will be coded, and the processing laboratory will 
receive samples labeled only with the participant ID number and date of collection.  A master patient list 
with identifiers will be maintained by the PI and study team as a password-protected MS Excel 
spreadsheet saved on a secure shared research drive.  Personal health information will be collected; 
however all data will be coded and kept in a secure online REDCap database, separated from identifiers.  
Paper records will be kept in a locked cabinet/desk in the study staff’s office.  
10.3 Records Retention 
Study documents and data will be retained for at least two (2) years after the last participant has 
completed the study.  
11 Study Monitoring, Auditing, and Inspecting 
The investigator will permit study-related monitoring, audits, and inspections by the EC/IRB, the sponsor, 
government regulatory bodies, and University compliance and quality assurance groups of all study 
related documents (e.g. source documents, regulatory documents, data collection instruments, study data 
etc.). The investigator will ensure the capability for inspections of applicable study-related facilities (e.g. 
pharmacy, diagnostic laboratory, etc.).  
 
The Primary Investigator will monitor adverse events and unanticipated problems during the study to 
monitor ongoing participant safety. All SAE’s will be reported to the safety officer within mandated time 
frames. 
 
Participation as an investigator in this study implies acceptance of potential inspection by government 
regulatory authorities and applicable University compliance and quality assurance offices. 
12 Ethical Considerations 
This study is to be conducted in accordance with applicable US government regulations and international 
standards of Good Clinical Practice, and applicable institutional research policies and procedures. 
 
This protocol and any amendments will be submitted to a properly constituted independent Ethics 
Committee (EC) or Institutional Review Board (IRB), in agreement with local legal prescriptions, for formal 
approval of the study conduct.  The decision of the EC/IRB concerning the conduct of the study will be 
made in writing to the investigator and a copy of this decision will be provided to the sponsor before 
commencement of this study.  


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12.1 Risks 
There are potential risks to participants who agree to participate in this trial; however, we believe that the 
risks are relatively small and have been minimized by our design. For participants above the age of 65, 
any additional risk due to Ciprofloxacin should be mitigated through the additional precautions described 
in Section 3.1.2. The participants will have already been refractory to at least one conventional therapy.  
Many of these patients will otherwise be facing escalation of therapy to a medication that is considered 
experimental or will be facing surgery. We believe that the risks associated with this study are significantly 
less than the risks associated with surgery. The risks of each medication will be reviewed in detail as part 
of the consent process.   
 
As the participants’ concurrent therapies will not be discontinued and the intervention portion of this study 
lasts only two weeks, the probability of harm is very low. Participants can be withdrawn from the study at 
any time. Additionally, if a participants’ condition worsens and a rescue therapy is deemed necessary by 
the study team or primary gastroenterologist, the participant will be withdrawn. 
 
We believe that the therapeutic regimen described in this protocol is safe. These are medications that are 
already FDA approved for various indications. We are using standard dosing. Also, many of the 
medications in the proposed regimen are already utilized in children and adults with CD. Finally, in our 
preliminary data, we found a similar regimen to be safe in primates. 
12.2 Benefits 
The primary potential benefit is improvement in disease activity and potential salvaged response to the 
prior treatment regimen (immunomodulator or biologic medication).  Additionally, in terms of indirect 
benefits, participation in this study will improve our understanding of the role of the gut microbiota in the 
inflammatory bowel diseases. The risks of participating in this study are outweighed by the potential 
benefits to participants who have active disease that is refractory to current therapy. Finally, the new 
knowledge about the gut microbiota that stands to be gained presents an additional benefit of this study, 
further contributing to the favorable balance of potential benefits to possible harms. 
12.3 Risk Benefit Assessment 
The risks of participating in this study are small, and these risks are outweighed by the potential benefits 
to participants who have very active disease that is refractory to current therapies.  In addition, the new 
knowledge about the gut microbiota that stands to be gained presents a major benefit of this study, 
outweighs the risks, and justifies the conduct of this study. 
12.4 Informed Consent Process / HIPAA Authorization  
Information about study participants will be kept confidential and managed according to the requirements 
of the Health Insurance Portability and Accountability Act of 1996 (HIPAA). Those regulations require a 
signed participant authorization informing the participant of the following:  
• What protected health information (PHI) will be collected from participants in this study  
• Who will have access to that information and why  
• Who will use or disclose that information  
• The rights of a research participant to revoke their authorization for use of their PHI  
 
In the event that a participant revokes authorization to collect or use PHI, the investigator, by regulation, 
retains the ability to use all information collected prior to the revocation of participant authorization.  
All data related to this trial will be recorded using the patients’ assigned unique study number. Data will be 
reported only in a confidential manner such that the personal identity of any participant will not be 
identifiable. All study data will be maintained under a double locked system, such as a locked closet 
within a locked office or on a password protected computer in a locked office. Data will be entered into a 
secure REDCap database accessed on a password-protected computer. 


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13 Study Finances 
13.1 Funding Source 
This study is funded through a two-year grant from the Broad Medical Research Program at CCFA.  The 
funds for microbiome analysis will come from the Joint Penn-CHOP Center for Digestive, Liver and 
Pancreatic Medicine.   
13.2 Conflict of Interest 
Any investigator who has a conflict of interest with this study (patent ownership, royalties, or financial gain 
greater than the minimum allowable by their institution, etc.) must have the conflict reviewed by a properly 
constituted Conflict of Interest Committee with a Committee-sanctioned conflict management plan that 
has been reviewed and approved by the study sponsor prior to participation in this study. All University of 
Pennsylvania investigators will follow the University conflict of interest policy. 
13.3 Participants Stipends or Payments 
Participants are not provided compensation for their participation in this study. However, in order to defray 
their parking and/ or travel costs, participants will receive $7 per in-person visit. This is equivalent to the 
amount required for parking in the PCAM parking garage for up to 3 hours (which is more than the length 
of each study visit) with validation from the PCAM information desk. 
14 Publication Plan 
Neither the complete nor any part of the results of the study carried out under this protocol, nor any of the 
information provided by the sponsor for the purposes of performing the study, will be published or passed 
on to any third party without the consent of the study sponsor.  Any investigator involved with this study is 
obligated to provide the sponsor with complete test results and all data derived from the study.  
15 References 
1. Lewis, J.D., The utility of biomarkers in the diagnosis and therapy of inflammatory bowel disease. 
Gastroenterology, 2011. 140(6): p. 1817-1826 e2. 
2. Molodecky, N.A., et al., Increasing incidence and prevalence of the inflammatory bowel diseases 
with time, based on systematic review. Gastroenterology, 2012. 142(1): p. 46-54 e42; quiz e30. 
3. Sartor, R.B., Mechanisms of disease: pathogenesis of Crohn's disease and ulcerative colitis. Nat 
Clin Pract Gastroenterol Hepatol, 2006. 3(7): p. 390-407. 
4. Rath, H.C., et al., Different subsets of enteric bacteria induce and perpetuate experimental colitis 
in rats and mice. Infect Immun, 2001. 69(4): p. 2277-85. 
5. Elson, C.O., et al., Experimental models to study molecular mechanisms underlying intestinal 
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6. Harper, P.H., et al., Role of the faecal stream in the maintenance of Crohn's colitis. Gut, 1985. 
26(3): p. 279-84. 
7. Rutgeerts, P., et al., Effect of faecal stream diversion on recurrence of Crohn's disease in the 
neoterminal ileum. Lancet, 1991. 338(8770): p. 771-4. 
8. Dave, M., et al., Opportunistic Infections Due to Inflammatory Bowel Disease Therapy. Inflamm 
Bowel Dis, 2013. 
9. Ben-Horin, S. and Y. Chowers, Review article: loss of response to anti-TNF treatments in Crohn's 
disease. Aliment Pharmacol Ther, 2011. 33(9): p. 987-95. 
10. Hoentjen, F., et al., Antibiotics with a selective aerobic or anaerobic spectrum have different 
therapeutic activities in various regions of the colon in interleukin 10 gene deficient mice. Gut, 
2003. 52(12): p. 1721-7. 
11. Kang, S.S., et al., An antibiotic-responsive mouse model of fulminant ulcerative colitis. PLoS 
Med, 2008. 5(3): p. e41. 
12. Madsen, K.L., et al., Antibiotic therapy attenuates colitis in interleukin 10 gene-deficient mice. 
Gastroenterology, 2000. 118(6): p. 1094-105. 


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13. Khan, K.J., et al., Antibiotic therapy in inflammatory bowel disease: a systematic review and 
meta-analysis. Am J Gastroenterol, 2011. 106(4): p. 661-73. 
14. Wang, S.L., Z.R. Wang, and C.Q. Yang, Meta-analysis of broad-spectrum antibiotic therapy in 
patients with active inflammatory bowel disease. Exp Ther Med, 2012. 4(6): p. 1051-1056. 
15. Ohkusa, T., et al., Newly developed antibiotic combination therapy for ulcerative colitis: a double-
blind placebo-controlled multicenter trial. Am J Gastroenterol, 2010. 105(8): p. 1820-9. 
16. Uehara, T., et al., Efficacy of antibiotic combination therapy in patients with active ulcerative 
colitis, including refractory or steroid-dependent cases. J Gastroenterol Hepatol, 2010. 25 Suppl 
1: p. S62-6. 
17. Terao, S., et al., Antibiotic combination therapy for steroid withdrawal in steroid-dependent 
ulcerative colitis. Digestion, 2011. 83(3): p. 198-203. 
18. Turner, D., et al., Combination of oral antibiotics may be effective in severe pediatric ulcerative 
colitis: A preliminary report. J Crohns Colitis, 2014. 
19. Iliev, I.D., et al., Interactions between commensal fungi and the C-type lectin receptor Dectin-1 
influence colitis. Science, 2012. 336(6086): p. 1314-7. 
20. Barclay, G.R., et al., The effect of dietary yeast on the activity of stable chronic Crohn's disease. 
Scand J Gastroenterol, 1992. 27(3): p. 196-200. 
21. Zwolinska-Wcislo, M., et al., Effect of Candida colonization on human ulcerative colitis and the 
healing of inflammatory changes of the colon in the experimental model of colitis ulcerosa. J 
Physiol Pharmacol, 2009. 60(1): p. 107-18. 
22. Dollive, S., et al., Fungi of the Murine Gut: Episodic Variation and Proliferation during Antibiotic 
Treatment. PLoS One, 2013. 8(8): p. e71806. 
23. Wellmann, W. and F.W. Schmidt, Intestinal lavage in the treatment of Crohn's disease: a pilot 
study. Klin Wochenschr, 1982. 60(7): p. 371-3. 
24. Wellmann, W., P.C. Fink, and F.W. Schmidt, Whole-gut irrigation as antiendotoxinaemic therapy 
in inflammatory bowel disease. Hepatogastroenterology, 1984. 31(2): p. 91-3. 
25. O'Brien, C.L., et al., Impact of colonoscopy bowel preparation on intestinal microbiota. PLoS One, 
2013. 8(5): p. e62815. 
26. Prantera, C., et al., An antibiotic regimen for the treatment of active Crohn's disease: a 
randomized, controlled clinical trial of metronidazole plus ciprofloxacin. Am J Gastroenterol, 1996. 
91(2): p. 328-32. 
27. Sutherland, L., et al., Double blind, placebo controlled trial of metronidazole in Crohn's disease. 
Gut, 1991. 32(9): p. 1071-5. 
28. Ursing, B., et al., A comparative study of metronidazole and sulfasalazine for active Crohn's 
disease: the cooperative Crohn's disease study in Sweden. II. Result. Gastroenterology, 1982. 
83(3): p. 550-62. 
29. McKenna, P., et al., The macaque gut microbiome in health, lentiviral infection, and chronic 
enterocolitis. PLoS Pathogens, 2008. 4(2): p. 8. 
30. Shieh, F.K., et al., MiraLAX-Gatorade bowel prep versus GoLytely before screening colonoscopy: 
an endoscopic database study in a community hospital. J Clin Gastroenterol, 2012. 46(10): p. 
e96-e100. 
31. Harvey, R.F. and Bradshaw, J.M., A simple index of Crohn’s-dease activity. The Lancet, 1980, 
March 8: p. 514. 
32. Vermeire, S., et al., Correlation between the Crohn’s Disease Activity and Harvey–Bradshaw 
Indices in assessing Crohn’s disease severity. Clin Gastroenterol Hepatol, 2010. 8(4): p. 357-363. 
33. Thia, K., et al., Short CDAI: development and validation of a shortened and simplified Crohn's 
disease activity index. Inflamm Bowel Dis, 2011. 17(1): p. 105-11. 
34. Vermeire, S., G. Van Assche, and P. Rutgeerts, Laboratory markers in IBD: useful, magic, or 
unnecessary toys. Gut, 2006. 55: p. 436-31. 
35. Roseth, A.G., et al., Assessment of the neutrophil dominating protein calprotectin in feces. A 
methodologic study. Scandinavian Journal of Gastroenterology, 1992. 27(9): p. 793-8. 
36. Roseth, A.G., E. Aadland, and K. Grzyb, Normalization of faecal calprotectin: a predictor of 
mucosal healing in patients with inflammatory bowel disease. Scandinavian Journal of 
Gastroenterology, 2004. 39(10): p. 1017-20. 


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37. Roseth, A.G., et al., Assessment of disease activity in ulcerative colitis by faecal calprotectin, a 
novel granulocyte marker protein. Digestion, 1997. 58(2): p. 176-80. 
38. Walkiewicz, D., et al., Fecal calprotectin is useful in predicting disease relapse in pediatric 
inflammatory bowel disease. Inflammatory Bowel Diseases, 2008. 14(5): p. 669-673. 
39. Roseth, A.G., et al., Assessment of disease activity in ulcerative colitis by faecal calprotectin, a 
novel granulocyte marker protein. Digestion, 1997. 58(2): p. 176-80. 
40. Lin, J.F., et al., Meta-analysis: fecal calprotectin for assessment of inflammatory bowel disease 
activity. Inflamm Bowel Dis, 2014. 20(8): p. 1407-15. 
41. Sipponen, T., et al., Fecal calprotectin, lactoferrin, and endoscopic disease activity in monitoring 
anti-TNF-alpha therapy for Crohn's disease. Inflamm Bowel Dis, 2008. 14(10): p. 1392-8. 
42. Caporaso, J.G., et al., QIIME allows analysis of high-throughput community sequencing data. Nat 
Methods, 2010. 7(5): p. 335-6. 
43. Su, C., et al., A meta-analysis of the placebo rates of remission and response in clinical trials of 
active Crohn's disease. Gastroenterology, 2004. 126(5): p. 1257-69. 
44. Sandborn, W.J., et al., Adalimumab induction therapy for Crohn disease previously treated with 
infliximab: a randomized trial. Ann Intern Med, 2007. 146(12): p. 829-38. 
45. Sandborn, W.J., et al., Vedolizumab as induction and maintenance therapy for Crohn's disease. 
N Engl J Med, 2013. 369(8): p. 711-21. 
46. Lozupone, C., M. Hamady, and R. Knight, UniFrac--an online tool for comparing microbial 
community diversity in a phylogenetic context. BMC Bioinformatics, 2006. 7(371). 
47. Lozupone, C. and R. Knight, UniFrac: a new phylogenetic method for comparing microbial 
communities. Applied & Environmental Microbiology, 2005. 71(12): p. 8228-35. 
48. Ciprofloxacin. 
Drugs@FDA. 
Food 
and 
Drug 
Administration 
Web 
site. 
http://www.accessdata.fda.gov/drugsatfda_docs/label/2009/019537s073,020780s030lbl.pdf. 
Accessed February 18, 2015.  
49. Vancocin (Vancomycin hydrochloride). DailyMed. U.S. National Library of Medicine, NIH Web 
site. 
http://dailymed.nlm.nih.gov/dailymed/drugInfo.cfm?setid=a078d9c2-f89c-4f9f-8ded-
60ffb2983c3f&. Accessed February 18, 2015. 
50. Neomycin 
sulfate. 
DailyMed. 
U.S. 
National 
Library 
of 
Medicine, 
NIH 
Web 
site. 
http://dailymed.nlm.nih.gov/dailymed/drugInfo.cfm?setid=777dbfab-f83e-4738-ae1e-
78619a9f82a7. Accessed April 1, 2015. 
51. Diflucan 
(fluconazole). 
Drugs@FDA. 
Food 
and 
Drug 
Administration 
Web 
site. 
http://www.accessdata.fda.gov/drugsatfda_docs/label/2014/019949s060,020090s044lbl.pdf. 
Accessed April 1, 2015.  
52. GoLytely™. 
Drugs@FDA. 
Food 
and 
Drug 
Administratino 
Web 
site. 
http://www.accessdata.fda.gov/drugsatfda_docs/label/2013/019011s025lbl.pdf. Accessed April 
20, 2015.  
 
 


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16 Appendix A: Adverse Reactions 
16.1 Ciprofloxacin 
Common reactions (>5%) 
Occasional Reactions (1% to 5%) 
Rare but Serious Reactions (<1%) 
  
Nausea, diarrhea, abnormal liver function 
tests, vomiting, rash 
Body: headache, abdominal 
pain/discomfort, foot pain, pain, pain in 
extremities 
  
 
Cardiovascular: palpitation, atrial flutter, 
ventricular ectopy, syncope, hypertension, 
angina pectoris, myocardial infarction, 
cardiopulmonary arrest, cerebral 
thrombosis, phebitis, tachycardia, 
migraine, hypotension 
  
 
Central nervous system: restlessness, 
dizziness, lightheadedness, insomnia, 
nightmares, hallucinations, manic reaction, 
irritability, tremor, ataxia, convulsive 
seizures, grand mal convulsion, lethargy, 
drowsiness, weakness, malaise, anorexia, 
phobia, depersonalization, depression, 
paresthesia, abnormal gain 
  
 
Gastrointestinal: painful oral mucosa, 
oral candidiasis, dysphagia, intestinal 
perforation, gastrointestinal bleeding, 
cholestatic jaundice, hepatisis 
  
 
Hemic/Lymphatic: lymphadenopathy, 
petechia 
  
 
Metabolic/Nutritional: amylase increase, 
lipase increase, hyperglycemia, 
hypoglycemia 
  
 
Musculoskeletal*: arthalgia or back pain, 
joint stiffness, achiness, neck or chest 
pain, flare up of gout, muscle weakness 
  
 
Renal/Urogenital: interstitial nephritis, 
nephritis, renal failure, polyuria, urinary 
retention, urethral bleeding, vaginitis, 
acidosis, breast pain 
  
 
Respiratory: dyspnea, epitaxis, laryngeal 
or pulmonary edema, hiccough, 
hemoptysis, bronchospasm, pulmonary 
embolism 


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Skin/Hypersensitivity: allergic reaction, 
pruritis, urticaria, 
photosensitivity/phototoxicity reaction, 
flushing, fever, chills, angioedema, edema 
of the face, neck, lips, conjunctivae or 
hands, cutaneous candidiasis, 
hyperpigmentation, erythema nodosum, 
sweating 
  
 
Senses: blurred vision, disturbed vision 
(changes in color perception, 
overbrightness of lights), decreased visual 
acuity, diplopia, eye pain, tinnitus, hearing 
loss, bad taste, chromatopsia 
 
16.2 Vancomycin 
Common Reactions (>5%) 
Occasional Reactions (1% to 5%) 
Rare but Serious Reactions (<1%) 
Body: pyrexia, edema 
peripheral, fatigue 
Renal: nephrotoxicity including renal 
failure, renal impairment, increased blood 
creatinine 
Ototoxicity: hearing loss (associated with 
intravenous administration), vertigo, 
dizziness, tinnitus 
Gastrointestinal: nausea, 
abdminal pain, vomiting, 
diarrhea, flatulence 
  
Hematopoetic: reversible neutropenia 
(associated with intraveous 
administration), thrombocytopenia 
Infections: urinary tract 
infection 
  
Miscellaneous: anaphylaxis, drug fever, 
chills, nausea, eosinphilia, rash, Stevens-
Johnson syndrome, toxic epidermal 
necolysis, vasculitis 
Metabolism: hypokalemia 
  
  
Musculoskeletal: back pain 
  
  
Nervous system: headache 
  
 
 
16.3 Neomycin 
Common Reactions (>5%) 
Occasional Reactions (1% to 5%) 
Rare but Serious Reactions (<1%) 
  
  
Neurological: ototoxicity, respiratory 
paralysis, neuromuscular blockage 
  
  
Renal: nephrotoxicity, including 
numbness, skin tingling, muscle twitching 
and convulsions 
  
  
Musculoskeletal: aggravation of muscle 
weakness in patients with myasthenia 
gravis or parkinsonism 


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Miscellaneous: fungal overgrowth, 
malabsorption syndrome for various 
substances, including fat, nitrogen, 
cholesterol, carotene, glucose, xylose, 
lactose, sodium, calcium, cyanocobalamin 
and iron 
 
16.4 Fluconazole 
Common Reactions (>5%) 
Occasional Reactions (1% to 5%) 
Rare Reactions (<1%) 
  
Body: skin rash, headache 
Hepatic: hepatotoxicity with no relation to 
daily dose, duration of therapy, age, or sex 
  
Gastrointestinal: nausea, vomiting, 
diarrhea, abdominal pain 
Immunological: anaphylaxis, including 
angioedema, face edema and pruritus 
  
  
  
  
  
  
  
  
  
Cardiovascular: prolonged QT interval, 
torsade de pointes 
  
Central Nervous System: dizziness, 
seizures 
  
Hematopoietic and Lymphatic: 
leukopenia, including neutropenia and 
agranulocytosis, thrombocytopenia 
  
Metabolic: hypercholesterolemia, 
hypertriglyceridemia, hypokalemia 
  
Gastrointestinal: Cholestasis, dry mouth, 
hepatocellular damage, dyspepsia 
  
Musculoskeletal: myalgia 
  
Skin: acute generalized exanthematous-
pustulosis, drug eruption, increased 
sweating, exfoliative skin disorders 
including Stevens-Johnson syndrome and 
toxic epidermal necrolysis, alopecia 
  
Miscellaneous: taste perversion, insomnia, 
paresthesia, somnolence, tremor, vertigo 
 
16.5 Miralax (PEG 3350) 
Common Reactions (>5%) 
Occasional Reactions (1% to 5%) 
Rare but Serious Reactions (<1%) 
Nausea, abdominal fullness, 
bloating 
Abdominal cramps, vomiting, anal 
irritation 
Immunological: urticaria, rhinorrhea, 
dermatitis, anaphylaxis 
  
  
In patients > 60 years-old: upper GI 
bleeding from Mallory-Weiss tear, 
esophageal perforation, asystole, sudden 
dyspnea with pulmonary edema, 
"butterfly-like" infiltrates on chest X-ray 
after vomiting and aspirating PEG 


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Miscellaneous: cardiac arrhythmia, tonic-
clonic seizures and/or generalized loss of 
consciousness associated with electrolyte 
abnormalities 
 
16.6 Promethazine 
(frequencies are not defined by the literature) 
Common Reactions 
Rare but Serious Reactions 
CNS: drowsiness/sedation and confusion 
(use 
caution 
with 
performing 
tasks 
requiring 
mental 
alertness 
such 
as 
operating machinery or driving) 
Cardiac: cardiac conduction abnormalities; abnormal 
blood pressure (use with caution for people who have 
history of stroke, heart disease, and concurrent use of 
BP meds)  
Derm: 
photosensitivity 
and 
skin 
pigmentation (slate gray), avoid prolonged 
sun exposure 
Neuro: slow abnormal movements, increased rigidity, 
tongue protrusion, restlessness, mental status change, 
fever, 
muscle 
rigidity, 
autonomic 
instability, 
hallucinations, seizures 
Misc: constipation, dry mouth, runny nose, 
blurred 
vision, 
urinary 
retention, 
temperature regulation 
Local: injection site reactions (burning, edema, 
erythema, pain), abscess, local tissue destruction, clots 
 
Misc: cholestatic jaundice, hormone imbalance, 
abnormal platelets, increased risk for infection, loss of 
breathing (only seen in infants), allergic reaction  
 
 


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17 Appendix B: Investigational Drug Service Documentation 
 
UniversityofPennsylvaniaͲINVESTIGATIONALDRUGSERVICEͲPerelmanSchoolofMedicine
Main:  3600 Spruce St (Maloney Bldg, Ground Floor) | Satellite:  51 N. 39th St (1 Mutch Bldg) | Philadelphia, PA 19104
Protocol Title:
An Open-Label Pilot Study of Fundamental Modification of the Gut Microbiota in the Treatment of Refractory Crohn’s Disease             REVISED 
JULY 2015 VERSION 3
Investigator:
Albenberg, Lindsey, DO
Sponsor:
Sponsor ID:
pending
Subjects:
Total of 20 outpatients to be enrolled
Detail/Narrative:
For each subject, IDS would (a) purchase commercial vancomycin for injection and compound oral syringes, to administer as 500mg QID on Days 1-14), 
packaged as 14 'daily' sets of 4; (b) dispense a 4-day course of ciprofloxacin, packaged as individual 'AM' and 'PM' bottles together with the vancomycin 
syringes; c) dispense  neomycin daily for 3 days; (d) prepare and dispense a single 8.3oz bottle of Miralax(R) ready to drink, along with one 20mg dose of 
bisacodyl; then (e) mail everything to the subject's home, 15 days prior to scheduled study visit 1 in CTRC.  Quantities below are based on 20 completed 
courses plus enough overage for 2 additional courses (for loss, replacement, repeating missed doses, etc); if not needed then overall costs may be less.  For 
FLUCONAZOLE, supplies & manufacturing assume enough for 11 active and 11 placebo courses, encapsulated for blinding purposes, based on a 400mg 
once/daily dose.
Actual enrollment may vary; as a service center the IDS applies user fees for dispensing or drug preparation at the time those expenses actually occur.
Activity
Cost
Units
Total
*NOTEͲTHISISANAPPROXIMATIONOFCOSTSBASEDONCURRENTPRICES,NOTA'CONTRACT'*Estimate#
1508
Date Printed
7/21/2015
Per-subject cost (this is just an 'average' cost assuming a subject who completes the entire study) ............................
$335.70
TOTAL assuming all subjects above complete treatment (note - for budget guidance only - actual enrollment may vary) ............................
$18,082.00
Protocol And Inventory Maintenance (Per MONTH)
$25.50
18.00
$459.00
$1,155.00
1
$1,155.00
Study Initiation (procedures, product formulation, randomization scheme, planning, meetings, 
etc.)
Materials:
$61.00
3.00
Manufacturing (Per 100 Caps): placebo fluconazole
$183.00
$3.00
1.00
Drug Purchase
$3.00
$13.00
616.00
Drug Purchase
$8,008.00
$23.00
4.00
Drug Purchase
$92.00
$119.00
4.00
Drug Purchase
$476.00
$137.00
10.00
Drug Purchase
$1,370.00
$12.00
22.00
Drug Purchase
$264.00
$61.00
3.00
Manufacturing (Per 100 Caps): blinded fluconazole
$183.00
Preparation and Dispensation
Prepare/dispense fluconazole (14 day course, 14 bottles); collect back used bottles to 
document compliance.
$29.55
20.00
$591.00
Prepare/dispense neomycin (3-day course, as 9 bottles); collect back used bottles to document 
compliance.
$20.85
20.00
$417.00
Prepare/dispense Miralax™ (8.3oz) with dosing cup and a 20mg dose of bisacodyl
$20.85
20.00
$417.00
Compound 1,120mL of oral vancomycin suspension (500mg/20mL concentration), then draw up 
into 56 individual oral syringes, hand-label and package into 14 separate ziploc bags (labeled 
as Day 1 through Day 14).  Includes cost of suspending agents $(119/gallon), syringes 
($92/100), labour, etc.
$184.25
20.00
$3,685.00
UPS shipping (estimated - note this is a pass-through expense)
$12.00
20.00
$240.00
Prepare/dispense ciprofloxacin (4-day course as 8 bottles)
$20.85
20.00
$417.00
Study Closure (copies, record archival, return or destroy supplies, etc.)
$122.00
1
$122.00
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UniversityofPennsylvaniaͲINVESTIGATIONALDRUGSERVICEͲPerelmanSchoolofMedicine
Main:  3600 Spruce St (Maloney Bldg, Ground Floor) | Satellite:  51 N. 39th St (1 Mutch Bldg) | Philadelphia, PA 19104
Study Initiation costs (everything that occurs BEFORE first enrollment - this cost occurs regardless of enrollment) ...
$1,155.00
DRUG PURCHASE COSTS (PASS-THROUGH EXPENSE) - subject to change, prices not guaranteed .............
$10,213.00
Business Admin. address and phone:
Business Admin. Signature
Date:
Account Number (26-digits - call if you need to use a different format/system)
Business Admin. E-Mail
Prepared by: Kenneth Rockwell Jr, PharmD, MS
Date: 4/15/2015
Additional Information:
Drug purchases are a pass-through expense at time of purchase, separate from user fees and prices are not guaranteed.  Purchases listed above include 
vancomycin injection ($13/1gm vial x 616 vials), ciprofloxacin 750mg tabs ($23/btl of 50 x 4 btls), neomycin 500mg tabs ($119/100 x 4 btls), fluconazole 200mg tabs 
($137/30 x 10 btls), Miralax® ($12 ea x 22 jars, 8.3oz each), bisacodyl 5mg tabs ($3/100 x 1 btl)
NOTE: Business manager should complete & return to IDS before start.  Account# needed before ANY PURCHASES.  If project not funded yet, an alternate account should be 
provided.  This ESTIMATE is subject to change if actual IDS costs or sponsor requirements change.  The IDS is a service center subject to OMB-A21 and required to adjust its fees 
periodically to reflect actual cost of services.  Reduced monthly fees may apply for materials stored after close of a trial
Business Admin. Embossment Below:                                                                                             IRB #:
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18 Appendix C: CCFA-BMRP Sponsor Guidelines 
 


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1. 
Sheehan, D., C. Moran, and F. Shanahan, The microbiota in inflammatory bowel disease. J 
Gastroenterol, 2015. 50(5): p. 495-507. 
2. 
Chehoud, C., et al., Fungal Signature in the Gut Microbiota of Pediatric Patients With 
Inflammatory Bowel Disease. Inflamm Bowel Dis, 2015. 
3. 
Shen, T.C., et al., Engineering the gut microbiota to treat hyperammonemia. J Clin Invest, 2015. 
125(7): p. 2841-50. 
4. 
Dethlefsen, L. and D.A. Relman, Incomplete recovery and individualized responses of the human 
distal gut microbiota to repeated antibiotic perturbation. Proc Natl Acad Sci U S A, 2011. 108 
Suppl 1: p. 4554-61. 
5. 
Albenberg, L., et al., Correlation Between Intraluminal Oxygen Gradient and Radial Partitioning of 
Intestinal Microbiota in Humans and Mice. Gastroenterology, 2014. 
6. 
Vermeire, S., G. Van Assche, and P. Rutgeerts, Laboratory markers in IBD: useful, magic, or 
unnecessary toys. Gut, 2006. 55: p. 436-31. 
7. 
Roseth, A.G., E. Aadland, and K. Grzyb, Normalization of faecal calprotectin: a predictor of 
mucosal healing in patients with inflammatory bowel disease. Scandinavian Journal of 
Gastroenterology, 2004. 39(10): p. 1017-20. 
8. 
Gaya, D.R., et al., Faecal calprotectin in the assessment of Crohn's disease activity. Qjm, 2005. 
98(6): p. 435-41. 
9. 
Walkiewicz, D., et al., Fecal calprotectin is useful in predicting disease relapse in pediatric 
inflammatory bowel disease. Inflammatory Bowel Diseases, 2008. 14(5): p. 669-673. 
10. 
Dollive, S., et al., Fungi of the Murine Gut: Episodic Variation and Proliferation during Antibiotic 
Treatment. PLoS One, 2013. 8(8): p. e71806. 
11. 
Caporaso, J.G., et al., QIIME allows analysis of high-throughput community sequencing data. Nat 
Methods, 2010. 7(5): p. 335-6. 
12. 
Hoffmann, C., et al., Archaea and fungi of the human gut microbiome: correlations with diet and 
bacterial residents. PLoS One, 2013. 8(6): p. e66019.