Personalized AZithromycin/metronidAZole, in Combination With Standard Induction Therapy, to Achieve a Fecal Microbiome Community Structure and Metagenome Changes Associated With Sustained Remission in Pediatric Crohn's Disease (CD): a Pilot Study
Phase: PHASE2
Conditions: Crohn Disease, Pediatric Crohns Disease
Sponsor: University of North Carolina, Chapel Hill

 
 
 Personalized AZithromycin/metronidAZole, in combination with 
standard induction therapy, to achieve a fecal microbiome community 
structure and metagenome changes associated with sustained 
remission in pediatric Crohn’s Disease (CD): a pilot study 
 
 
 
NCT number NCT04186247 
Document Date 10/21/2021 
 

Personalized AZithromycin/metronidAZole, in 
combination with standard induction therapy, to 
achieve a fecal microbiome community structure and 
metagenome changes associated with sustained 
remission in pediatric Crohn’s Disease (CD): a pilot study  
The PAZAZ Study 
 
Protocol Number: 140041 
National Clinical Trial (NCT) Identified Number:  NCT04186247 
Sponsor Investigator: Johan Van Limbergen, MD, FRCPCH, PhD 
Monitor: Alimentiv B.V. (formerly Robarts Clinical Trials Inc.) 
Funded by: Crohn’s and Colitis Foundation of America 
Version Number:  v.1.9 
October 12, 2021 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
ii 
Summary of Changes from Previous Version: 
Version 
Date 
Affected 
Section(s) 
Summary of Revisions Made 
Rationale 
1.9 
October 
2021 
6.3 
Use CastorEDC randomization 
function instead of an additional 
separate program (SPSS) 
To use only one program (CastorEDC) as eCRF 
and randomization tool to minimize mistakes   
1.8 
March 
2021 
4.1 
Adding study timepoint window of 
+/- 5 days in case needed 
 
To facilitate planning of study visits 
1.8 
March 
2021 
6.3, 10.1.3, 
10.1.4 
Changed IWK health center to 
Amsterdam UMC, location AMC as 
principal receiver of trial data and 
biological samples 
 
PI moved to Amsterdam and trial will be 
coordinated from Amsterdam UMC, location 
AMC 
1.8 
March 
2021 
1.1 
Updated participating sites  
North Carolina Children’s Hospital (US), Sheba 
Medical Centre (Israel), Location VUMC of 
Amsterdam UMC (The Netherlands) will no 
longer be participating. 
1.8 
March 
2021 
10.1.5 
Changed medical monitor to Prof. 
Anthony Otley 
Prof. Antony Otley (local PI IWK Canada) will be 
the new medical monitor as former medical 
monitor prof. Francisco Sylvester (UNC) will no 
longer be involved in the study.  
1.8 
March 
2021 
10.1.9.1 
Changed electronic data capture 
system to Castor 
Castor EDC is license is provided by the 
Amsterdam UMC, the coordinating center of this 
trial.  
1.8 
 
Updated trial monitor name to 
Alimentiv B.V. 
 
Alimentiv B.V. is the new name of the PAZAZ 
trial monitor, formerly known as Robarts Clinical 
Trial Inc. 

 
iii 
October 
2020 
Addendum 
for the 
Netherlands 
Feasibility study clarification 
Required by National Ethics Review Board in the 
Netherlands October 2020 
May 
2021 
Addendum 
for the 
Netherlands 
Specifying chosen medicinal 
products for the AmsterdamUMC. 
Metronidazole 500 mg, Metronidazole 250 mg, 
Azitromycin 250 mg will be the medicinal 
products used in the AmsterdamUMC. 

  
 
ii 
Table of Contents 
STATEMENT OF COMPLIANCE ............................................................................................................................. 1 
1 
PROTOCOL SUMMARY ............................................................................................................................... 1 
1.1 
Synopsis ................................................................................................................................................ 1 
1.2 
Schema ................................................................................................................................................. 4 
1.3 
Schedule of Activities (SoA) ............................................................................................................... 5 
2 
INTRODUCTION ............................................................................................................................................ 6 
2.1 
Study Rationale .................................................................................................................................... 6 
2.2 
Background........................................................................................................................................... 7 
2.3 
Risk/Benefit Assessment .................................................................................................................... 9 
2.3.1 
Known Potential Risks ..................................................................................................... 9 
2.3.2 
Known Potential Benefits .............................................................................................. 12 
2.3.3 
Assessment of Potential Risks and Benefits .............................................................. 13 
3 
OBJECTIVES AND OUTCOME VARIABLES .......................................................................................... 13 
4 
STUDY DESIGN ........................................................................................................................................... 14 
4.1 
Overall Design .................................................................................................................................... 14 
4.2 
Scientific Rationale for Study Design.............................................................................................. 16 
4.3 
Justification for Dose ......................................................................................................................... 16 
4.4 
End of Study Definition ..................................................................................................................... 16 
5 
STUDY POPULATION ................................................................................................................................ 16 
5.1 
Inclusion Criteria ................................................................................................................................ 16 
5.2 
Exclusion Criteria ............................................................................................................................... 17 
5.3 
Lifestyle Considerations .................................................................................................................... 18 
5.4 
Screen Failures .................................................................................................................................. 18 
5.5 
Strategies for Recruitment and Retention ...................................................................................... 18 
6 
STUDY INTERVENTION ............................................................................................................................ 19 
6.1 
Study Intervention(s) Administration ............................................................................................... 19 
6.1.1 
Study Intervention Description ..................................................................................... 19 
6.1.2 
Dosing and Administration ............................................................................................ 20 
6.2 
Preparation/Handling/Storage/Accountability ................................................................................ 20 
6.2.1 
Acquisition and accountability ...................................................................................... 20 
6.2.2 
Formulation, Appearance, Packaging, and Labeling ................................................ 21 
6.2.3 
Product Storage and Stability ....................................................................................... 21 
6.2.4 
Preparation ...................................................................................................................... 22 
6.3 
Measures to Minimize Bias: Randomization and Blinding ........................................................... 22 
6.4 
Study Intervention Compliance ........................................................................................................ 22 
6.5 
Concomitant Therapy ........................................................................................................................ 23 
6.5.1 
Rescue Medicine ............................................................................................................ 23 
7 
STUDY INTERVENTION DISCONTINUATION AND PARTICIPANT 
DISCONTINUATION/WITHDRAWAL .................................................................................................................... 23 
7.1 
Discontinuation of Study Intervention ............................................................................................. 23 
7.2 
Participant Discontinuation/Withdrawal from the Study ............................................................... 24 
7.3 
Lost to Follow-Up ............................................................................................................................... 24 
8 
STUDY ASSESSMENTS AND PROCEDURES ..................................................................................... 25 
8.1 
Efficacy Assessments ....................................................................................................................... 25 
8.2 
Safety and Other Assessments ....................................................................................................... 26 
8.3 
Adverse Events and Serious Adverse Events ............................................................................... 26 

  
 
iii 
8.3.1 
Definition of Adverse Events (AE) ............................................................................... 26 
8.3.2 
Definition of Serious Adverse Events (SAE) .............................................................. 26 
8.3.3 
Classification of an Adverse Event .............................................................................. 27 
8.3.4 
Time Period and Frequency for Event Assessment and Follow-Up ....................... 27 
8.3.5 
Adverse Event Reporting .............................................................................................. 28 
8.3.6 
Serious Adverse Event Reporting ............................................................................... 28 
8.3.7 
Reporting Events to Participants ................................................................................. 29 
8.3.8 
Events of Special Interest ............................................................................................. 29 
8.3.9 
Reporting of Pregnancy ................................................................................................ 29 
8.4 
Unanticipated Problems .................................................................................................................... 29 
8.4.1 
Definition of Unanticipated Problems (UP) ................................................................. 29 
8.4.2 
Unanticipated Problem Reporting ................................................................................ 30 
8.4.3 
Reporting Unanticipated Problems to Participants ................................................... 30 
9 
STATISTICAL CONSIDERATIONS .......................................................................................................... 30 
9.1 
Statistical Hypotheses ....................................................................................................................... 30 
9.2 
Sample Size Determination .............................................................................................................. 31 
9.3 
Populations for Analyses .................................................................................................................. 31 
9.4 
Statistical Analyses............................................................................................................................ 32 
9.4.1 
General Approach .......................................................................................................... 32 
9.4.2 
Analysis of the Primary Efficacy Outcome variable(s) .............................................. 32 
9.4.3 
Analysis of the Secondary Outcome variable(s) ....................................................... 32 
9.4.4 
Safety Analyses .............................................................................................................. 33 
9.4.5 
Baseline Descriptive Statistics ..................................................................................... 33 
9.4.6 
Planned Interim Analyses ............................................................................................. 33 
9.4.7 
Sub-Group Analyses ..................................................................................................... 33 
9.4.8 
Tabulation of Individual participant Data .................................................................... 34 
9.4.9 
Exploratory Analyses ..................................................................................................... 34 
10 
SUPPORTING DOCUMENTATION AND OPERATIONAL CONSIDERATIONS .............................. 34 
10.1 
Regulatory, Ethical, and Study Oversight Considerations ........................................................... 34 
10.1.1 
Informed Consent Process ........................................................................................... 34 
10.1.2 
Study Discontinuation and Closure ............................................................................. 35 
10.1.3 
Confidentiality and Privacy ........................................................................................... 35 
10.1.4 
Future Use of Stored Specimens and Data ............................................................... 36 
10.1.5 
Key Roles and Study Governance .............................................................................. 36 
10.1.6 
Safety Oversight ............................................................................................................. 37 
10.1.7 
Clinical Monitoring .......................................................................................................... 37 
10.1.8 
Quality Assurance and Quality Control ....................................................................... 38 
10.1.9 
Data Handling and Record Keeping ............................................................................ 38 
10.1.10 
Protocol Deviations ........................................................................................................ 39 
10.1.11 
Publication and Data Sharing Policy ........................................................................... 39 
10.1.12 
Conflict of Interest Policy .............................................................................................. 40 
10.2 
Additional Considerations ................................................................................................................. 40 
10.3 
Abbreviations ...................................................................................................................................... 41 
10.4 
Protocol Amendment History ........................................................................................................... 43 
11 
REFERENCES 
44 

PAZAZ 
Version 1.9 
Protocol 140041 
Oct 12, 2021 
 
 
 
1 
STATEMENT OF COMPLIANCE 
The trial will be conducted in accordance with International Conference on Harmonisation Good Clinical 
Practice (ICH GCP), applicable United States (US) Code of Federal Regulations (CFR), and Crohn’s & 
Colitis Foundation Terms and Conditions of Award. The Principal Investigator will assure that no 
deviation from, or changes to the protocol will take place without prior agreement from the 
Investigational New Drug (IND) or Investigational Device Exemption (IDE) sponsor, funding agency and 
documented approval from the Institutional Review Board (IRB), except where necessary to eliminate an 
immediate hazard(s) to the trial participants. All personnel involved in the conduct of this study have 
completed Human Subjects Protection and ICH GCP Training. 
 
The protocol, informed consent form(s), recruitment materials, and all participant materials will be 
submitted to the IRB for review and approval.  Approval of both the protocol and the consent form must 
be obtained before any participant is enrolled.  Any amendment to the protocol will require review and 
approval by the IRB before the changes are implemented to the study.  All changes to the consent form 
will be IRB approved; a determination will be made regarding whether a new consent needs to be 
obtained from participants who provided consent, using a previously approved consent form. 
1 
 
PROTOCOL SUMMARY 
1.1 
SYNOPSIS  
Title: 
PAZAZ: Personalized azithromycin/metronidazole, in combination with 
standard induction therapy, to achieve a fecal microbiome community 
structure and metagenome changes associated with sustained remission 
in pediatric Crohn’s Disease (CD)-- a pilot study 
 
Study Description: 
This is a multi-center, randomized, controlled open-label add-on design 
trial pilot study to evaluate the efficacy of personalized adjunctive 
antibiotic (azithromycin + metronidazole) therapy in pediatric subjects 
with mild to moderate Crohn’s disease (CD) who have a relapse-associated 
microbiome profile.  The study hypothesis is that adjunctive antibiotic 
therapy will improve clinical response to standard of care (SOC) induction 
therapy in a subgroup of CD patients with a relapse-associated 
microbiome profile.  This is an add-on design trial for subjects already 
receiving SOC induction therapy; there will be no placebos.  
 
Prior to starting SOC induction therapy at week 0, subjects will provide a 
baseline stool sample that will be screened for microbiome profiles 
associated with risk of relapse according to an established statistical 
model.   
At week 4, subjects with a relapse-associated microbiome will be 
randomized into either a control arm that will continue to receive SOC 
induction therapy for an additional 8 weeks, or a treatment arm that will 
receive adjunctive antibiotic therapy in addition to continuing to receive 
SOC induction therapy for an additional 8 weeks.  Subjects who do not 
have a relapse-associated microbiome will enter a separate control arm 
that will continue to receive SOC induction therapy and will have data 
collected for exploratory objectives. Subjects who are not in clinical 

PAZAZ 
Version 1.9 
Protocol 140041 
Oct 12, 2021 
 
 
 
2 
remission by week 4 will receive antibiotic therapy regardless of 
microbiome signature at baseline. Subjects will be monitored for an 
additional 40 weeks after the treatment period (52 weeks total). 
 
Objectives: 
 
Primary Objective:  To evaluate the potential efficacy of personalized 
adjunctive antibiotic therapy in maintaining remission in pediatric subjects 
undergoing SOC induction therapy for mild to moderate Crohn’s disease. 
 
Secondary Objective: To evaluate longitudinal (clustered and with respect 
to baseline) changes in disease activity indices (PCDAI) components and 
inflammatory markers in stool and blood. 
 
 
Exploratory Objectives:  To investigate relationship between changes in 
subject microbiome composition and changes in disease activity over time. 
 
 
Outcome variables: 
Primary Outcome variable: Sustained remission defined as no need of re-
induction for clinical flare (new course of nutritional therapy, need to start 
steroids), steroid dependence, biologic (anti-TNF) use, and/or intestinal 
surgery by 12 months. 
Secondary Outcome variables: Longitudinal (clustered and with respect to 
baseline) changes in disease activity indices (PCDAI: 0-100) components 
and inflammatory markers in stool and blood at each study visit up to 12 
months. Longitudinal (clustered and with respect to baseline) changes in 
patient-reported outcomes (PRO) by 12 months. 
Exploratory Outcome variable: Longitudinal (clustered and with respect to 
baseline) changes in fecal microbiome taxonomic composition or in total 
gene (metagenome) content.  The changes will be analyzed for association 
with changes in disease activity (e.g. relapse or sustained remission) over 
time up to 12 months. 
 
 
Study Population: 
Children aged 3 to 17 years (inclusive) with mild to moderately active 
Crohn’s disease who will receive standard of care induction therapy.  
 
Phase: 
2 
 
Description of 
Sites/Facilities Enrolling 
Participants: 
This is a multi-center trial with eight participating sites in Canada, the 
U.S.A. the Netherlands, and Israel.  The sites are pediatric 
gastroenterology clinics located at: 
1. Dalhousie University, IWK Health Centre, Halifax, Nova Scotia 
(Canada) 
2. University of California San Francisco, Benioff Children’s Hospital, 
San Francisco, CA 
3. University of Pittsburgh Medical Center, Children’s Hospital of 
Pittsburgh, Pittsburgh, PA 
4. Amsterdam University Medical Centers, Academic Medical Center. 
Amsterdam, The Netherlands 

PAZAZ 
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3 
5. Wolfson Medical Centre, University of Tel Aviv, Tel Aviv, Israel 
 
Description of Study 
Intervention: 
Antibiotics will be administered orally for an 8-week period.  Azithromycin 
will be administered at a dose of 7.5mg/kg to a maximum of 500mg/day 
for 5 consecutive days per week for the first 4 weeks and then 3 
consecutive days/week for 4 weeks. Metronidazole will be administered 
10mg/kg twice daily to a maximum of 1000mg/day for 8 weeks. 
 
Study Duration: 
3 
years 
 
 
Participant Duration: 
The duration of study participation is 52 weeks (12 months) 
 
 
 
 
 
 

PAZAZ 
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4 
1.2 
SCHEMA 
Visit 1   
Screening 
 
 
Visit 2 
Week 0 
 
 
 
Visit 3 
Week 4 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Visit 4 
Week 12 
 
 
Visit 5 
Week 24 
 
 
Visit 6 
Week 36 
 
 
Visit 7 
Week 52 
 
 
 
*see text 4.1 for guidance 
Perform baseline assessments. 
Refer to Section 1.3, Schedule of Activities 
Start standard of care (SOC) induction therapy  
Follow-up assessments of study outcome variables and safety 
Refer to Section 1.3, Schedule of Activities 
Final Assessments 
Refer to Section 1.3, Schedule of 
Group C 
Group A1  
If in clinical remission, define patient groups based on microbiome analysis results 
Group A: ≥30% posterior probability relapse-associated 
Group B: <30% posterior probability relapse-associated 
Group C: patients not in clinical remission 
Group A2  
 
Administer study interventions: 
Group A1: CDED+PEN, MTX s.c. 15mg/m2* 
Group A2: CDED+PEN  +  8 weeks azithromycin/metronidazole,  MTX s.c. 15mg/m2* 
Group B: CDED+PEN,  MTX s.c. 15mg/m2* 
Group C: CDED+PEN  +  8 weeks azithromycin/metronidazole,  MTX s.c. 15mg/m2* 
Follow-up assessments of study outcome variables and safety 
Refer to Section 1.3, Schedule of Activities 
Follow-up assessments of study outcome variables and safety 
Refer to Section 1.3, Schedule of Activities 
Total N:  Obtain informed consent. Screen potential participants by inclusion and 
exclusion criteria; obtain history, document. 
 
Group B 
Randomize 

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5 
1.3 
SCHEDULE OF ACTIVITIES (SOA) 
 
 
Procedures 
Visit 1 Screening 
≤30 days prior to week 0 
Visit2 
 Week 0 
Study Visit3  
Week 4 
Study Visit 4 
Week 12 
Study Visit 5 
Week 24 
Study Visit6 
Week 36 
Final Study Visit 7 
Week 52 
Informed consent 
X 
 
 
 
 
 
 
Demographic data 
X 
 
 
 
 
 
 
Medical history 
X 
X 
X 
X 
X 
X 
X 
Review inclusion/exclusion criteria 
X 
X 
 
 
 
 
 
Concomitant medication review  
X 
X 
X 
X 
X 
X 
X 
Physical exam (including height and 
weight) 
X 
X 
X 
X 
X 
X 
X 
Medication calendar distribution 
and teaching  
 
X 
 
 
 
 
 
Blood sample a 
X 
Xb 
X 
X 
X 
X 
X 
Calculate PCDAI score (0-100) 
X 
Xb 
X 
X 
X 
X 
X 
Stool sample (16S rRNA) 
X 
Xb 
X 
X 
X 
X 
X 
Stool sample (FCP) 
X 
Xb 
X 
X 
X 
X 
X 
Stool sample (C. difficile infection 
testing) c 
X 
 
 
 
 
 
 
Urine pregnancy test d 
X 
 
X 
 
 
 
 
Adverse event review and 
evaluation 
 
 
X 
X 
X 
X 
X 
Randomization 
 
 
X 
 
 
 
 
ECG, if applicable e 
 
 
X 
 
 
 
 
Administer antibiotic, if applicable 
 
 
X 
 
 
 
 
Teaching for antibiotic medication 
calendar, if applicable 
 
 
X 
 
 
 
 
Complete Case Report Forms 
(CRFs) 
X 
X 
X 
X 
X 
X 
X 
IMPACT (ages 9 and older) 
 
X 
 
X 
X 
 
X 
A window of study timepoint +/- 5 days is allowed for all visits/questionnaires/sampling. 
a: Includes hematology and clinical chemistry Albumin, C reactive protein (CRP), hematocrit (HCT), erythrocyte sedimentation 
rate (ESR), alanine transaminase (ALT), aspartate transaminase (AST), creatinine, and bilirubin. Total blood volume collected 
at each visit will be 5ml. 
b: The timing of the Screening and Week 0 visits will depend on the start of therapy. Week 0 blood and stool samples will not 
be repeated if provided during the screening visit, provided that the stool samples were collected prior to bowel prep. 
C: If performed for clinical indication, as per standard of care 
d: Only applicable for post-menarchal female participants initiating methotrexate 

PAZAZ 
Version 1.9 
Protocol 140041 
Oct 12, 2021 
 
 
 
6 
Procedures 
Visit 1 Screening 
≤30 days prior to week 0 
Visit2 
 Week 0 
Study Visit3  
Week 4 
Study Visit 4 
Week 12 
Study Visit 5 
Week 24 
Study Visit6 
Week 36 
Final Study Visit 7 
Week 52 
e: All patients randomized to group A2 and C will receive an ECG exam both prior to and at 2-4 weeks after the 
initiation of the study drug 
 
 
2 
 
INTRODUCTION 
 
2.1 
STUDY RATIONALE  
 
Crohn’s disease (CD) is a major phenotype of inflammatory bowel disease (IBD) characterized by 
transmural (often granulomatous and patchy) inflammation throughout the gastrointestinal (GI) tract, 
most often involving the ileum and colon.  Chronic inflammation within the GI tract can cause 
progressive tissue damage leading to serious complications requiring surgery, including intra-abdominal 
abscesses, fistulas, and intestinal strictures.1  CD is highly heterogeneous with respect to age of onset, 
disease location (i.e. anatomical extent) and behavior (i.e. inflammatory or stricturing/penetrating 
disease).  Disease course is also highly variable among patients with some experiencing chronically 
active severe disease while other have intermittent periods of clinical remission and disease 
exacerbation (i.e. ‘flares’). Patient responses to therapy are also highly variable, but the reasons for this 
are not fully understood.  
 
Current treatments for CD aim to not only control symptoms but to maintain clinical remission and 
mucosal healing (deep remission).2 Corticosteroids, enteral nutrition, thiopurines, methotrexate and 
biologics (such as monoclonal antibodies against tumour necrosis factor-α: ‘anti-TNF’) are effective to 
modulate inflammatory activity, but surgery is still frequently required.3 
 
The exact cause of CD is unclear, but current thinking holds that disease results from a defective or 
inappropriate activation of the mucosal immune system response to commensal gut microbiota, 
collectively termed the gut microbiome.1  Certain bacteria can adhere to and invade epithelial cells of 
the inflamed mucosa and granulomas to replicate inside macrophage phagolysosomes.4  Numerous CD 
susceptibility genes are involved in pathways that govern innate immunity, recognition of bacterial 
pathogens, and handling of intracellular bacteria.5  Diversion of the fecal stream can lead to clinical 
improvement in medically refractory Crohn’s colitis.6  
 
The field of microbiome research has grown exponentially over the past several years, catalyzed by the 
US-led Human Microbiome Project (HMP)7 and the European-led MetaHIT8 project. Studies using next-
generation sequencing and new bioinformatics approaches have begun to characterize fundamental 
differences in the gut microbiome in children (and adults) with CD versus healthy controls. 9-11 The ileum 
and colon is densely populated with a variety of metabolically active bacteria that interact with the host 
immune system.  The collective genomic content of the microbiome—the metagenome—has been 

PAZAZ 
Version 1.9 
Protocol 140041 
Oct 12, 2021 
 
 
 
7 
estimated to contain at least 100-times more genes than the human genome. The influence of the 
microbiome on disease pathogenesis and progression in CD is a significant area of research interest, 
since a breakdown in the balance between protective and harmful bacteria (termed ‘dysbiosis’) is the 
current prevailing hypothesis for the development of CD.12 Animal studies have shown that bacterial 
load and the composition of bacterial communities can influence both the site and degree of 
inflammation in the GI tract.13,14  Several studies applying microbial profiling and in-depth sequencing 
techniques to the collective DNA content of gut microbiota (i.e. microbiome) of CD patients have shown 
distinct microbiome profiles associated with different clinical outcomes or responses to treatment.3,15-19 
 
The probable role for bacteria in triggering disease activity implies that antibiotics could have a role in 
CD therapy.   However, while several meta-analyses support the use of antibiotics in controlling luminal 
inflammation, results of individual trials are heterogeneous.2,20 Recently, a combination of 
azithromycin/metronidazole has been found to be superior to metronidazole alone for induction of 
remission and improvement in fecal calprotectin. 21However, individual responses to this antibiotic 
treatment were variable, showing the percentage of clinical response to equal that of remission.  This 
“all or nothing” phenomenon suggests that the effect of antibiotics may depend on the type of 
microbiota involved and their susceptibility to antibiotics.  Antibiotics used in previous trials such as 
rifaximin are non-absorbable and would be less effective against bacteria that have already translocated 
into tissue.21-23 
 
Recently, the Crohn’s Disease Exclusion diet was shown to be associated with comparable efficacy but 
superior tolerance and maintenance of remission than exclusive enteral nutrition in a randomized 
controlled trial.24 
 
The purpose for the proposed pilot trial is to determine whether personalized, microbiome-informed 
administration of an azithromycin/metronidazole antibiotic can improve clinical response to nutritional 
induction therapy and prolong remission compared to induction therapy alone. 
 
 
2.2 
BACKGROUND   
 
Current role of antibiotics in pediatric Crohn’s disease treatment 
The most recently reported consensus guidelines of European Crohn’s and Colitis Organization 
(ECCO)/European Society of Pediatric gastroenterology, Hepatology and Nutrition (ESPGHAN) on the 
medical management of pediatric Crohn’s disease in 2014 include the following on antibiotics: 
 
“Antibiotics, such as metronidazole or ciprofloxacin, are recommended in the treatment of 
perianal fistulizing disease (EL 3 (pediatrics) EL1 (adults)) 80% agreement…In more severe 
perianal fistulizing disease, antibiotics should be used as adjuvant (EL3)) 88% agreement 
 
Practice points: 1. In perianal disease, metronidazole/ciprofloxacin-based treatments have a 
good short-term response and may offer a bridge to immunosuppressive medications; 
2. Usual daily doses for metronidazole are 10–20 mg/kg, and for ciprofloxacin 20 mg/kg;  
3. Azithromycin and rifaximin may be useful for induction of remission in children with mild to 
moderate luminal inflammatory pediatric CD; 4. There is no evidence to recommend the use of 
anti-mycobacterial antibiotics.”2 
 

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8 
Antibiotics such as metronidazole or ciproflaxin are regularly used in CD standard care to treat 
penetrating complications such as perianal fistulizing disease or abdominal abscesses in children and 
adults.2 Multiple trials have shown that metronidazole at a dosage of 10-20mg/kg/day may improve or 
heal perianal CD and the drug is now widely used for this indication in children and adults with CD.2 The 
most recent guidelines published by the World Gastroenterology Organization support the use of 
antibiotics in perianal disease, fistulizing, and bacterial overgrowth secondary to stricturing disease.25 
There is also extensive evidence regarding antibiotic use in post-operative CD management.25  
Long-term metronidazole use (20mg/kg/day for 3 months) has been shown to prevent recurrence of 
Crohn’s after ileal resection in adults with CD.26 
 
Potential role for antibiotics in Crohn’s disease treatment 
The probable role of bacteria in triggering disease activity in CD has led to the use of antibiotic therapy 
in mild to moderate luminal disease but previous trials have produced mixed results.27,28 29 In a recent 
systematic review including 10 randomized controlled trials (RCT) (1160 patients), there was a 
statistically significant effect of antibiotics being superior to placebo. Different antibiotics were 
administered (anti-tuberculosis therapy, macrolides, fluroquinolones, 5-nitroimidazoles, and rifaximin) 
either alone or in combination.29  Antibiotics are proposed to work by reducing bacterial load, limiting 
bacterial translocation, and reducing the concentration of adherent bacteria in the lumen and mucosa.25 
Ideally, antibiotics used to treat CD should be active in the lumen, biofilms, and inside host cells in order 
to target adherent and invasive bacteria thought to provoke disease. Unlike quinolones, rifaximin or 
azoles, azithromycin (a macrolide antibiotic) has excellent tissue penetration, is able to maintain 
relatively high luminal concentrations and is effective against biofilms.30,31  
 
Macrolide antibiotics are also known to have anti-inflammatory and immune modulating effects and 
have been explored in several clinical trials for their effects in patients with chronic lung diseases.32,33  A 
recent multicentre, double-blind, randomized, parallel-group, placebo-controlled trial 
(ACTRN12610000383066) in children aged 1-8 with either non-cystic-fibrosis bronchiectasis or chronic 
suppurative lung disease found that once-weekly azithromycin for up to 24 months decreased 
pulmonary exacerbations.34 The dose used was 30mg/kg per week and was well-tolerated with no 
serious adverse events being attributed to the intervention. However, antibiotic use was also 
accompanied by increased carriage of azithromycin-resistant bacteria, of which the clinical 
consequences are uncertain.34  
 
Azithromycin + metronidazole-based therapy for inducing remission in pediatric Crohn’s disease (CD) 
Levine and Turner conducted a retrospective analysis of 32 children with active CD who were treated 
with an 8 week course of combined azithromycin and metronidazole identical to the dosage proposed in 
this trial: azithromycin was given 7.5–10 mg/kg, once daily (maximal dose: 500 mg), for five consecutive 
days/ week for 4 weeks, and 3 times a week for the following 4 weeks, in conjunction with 
metronidazole. Azithromycin-based therapy was applied due to its effect in inducing apoptosis (down 
regulation of anti-apoptotic transcription factor Bcl-xL), efficacy against biofilms and intracellular 
bacteria. Clinical remission was observed in 21/32 (66%) patients and 54% of children with elevated CRP 
at baseline normalized their C-reactive protein (CRP).35  
 
More recently, Levine et al. performed a randomized controlled trial (NCT01596894) allocating children 
with mild to moderately active disease into groups receiving azithromycin + metronidazole therapy 
(7.5mg/kg azithromycin once daily for five consecutive days/ week for 4 weeks, and 3 times a week for 
the following 4 weeks, in conjunction with 20mg/kg/day of metronidazole) or metronidazole alone daily 
for 8 weeks.21  The results of this trial show that remission rates with azithromycin + metronidazole 

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therapy are better than with metronidazole alone, with a 66% clinical remission.  There were no drug-
related serious adverse events (SAEs) in the study. 
 
 
Personalized adjunctive antibiotic therapy in pediatric Crohn’s disease treatment 
We have recently shown that the use of immunomodulators and the need for treatment escalation to 
anti-TNF or surgery are comparable after 6 years of follow-up, following induction of remission using 
either exclusive enteral nutrition (EEN) or steroids, both of which are used as ‘standard-of-care’ 
induction therapy in pediatric CD.36 We also showed that a baseline (B) fecal microbiome signature with 
predominance of Proteobacteria, does not correct with nutritional therapy by week 12 and is associated 
with recurrence of disease.37 We have recently shown, in an independent cohort of CD patients, that 
remission due to nutritional therapy corrects features of the dysbiosis described in the study by Gevers 
et al. in the RISK-CCFA cohort of treatment naïve pediatric CD.38 
 
We have recently shown that the Crohn’s Disease Exclusion Diet (CDED) + Partial Enteral Nutrition (PEN) 
has similar efficacy to EEN but has significantly better tolerance and sustained remission. Dietary 
induced remission with CDED+PEN by 12 weeks resulted in sustained improvement in Proteobacteria. 
 
We hypothesize that the risk of early flare after achieving clinical remission using standard-of-care 
nutritional induction therapy in CD patients with a relapse-associated (i.e. Proteobacteria-rich) 
microbiome signature can be reduced via personalized adjunctive induction therapy with antibiotics 
(azithromycin + metronidazole). 
 
 
2.3 
RISK/BENEFIT ASSESSMENT   
 
2.3.1 KNOWN POTENTIAL RISKS  
 
Drug Risks:  
Azithromycin39 (see Attachments for complete product package insert) 
In clinical trials, most of the reported side effects were mild to moderate in severity and were reversible 
upon discontinuation of the drug. Potentially serious side effects of angioedema and cholestatic 
jaundice were reported rarely. Most of the side effects leading to discontinuation were related to the 
gastrointestinal tract, e.g., nausea, vomiting, diarrhea, or abdominal pain.  
 
In adult studies, side effects that occurred with a frequency of 1% or less included the following: 
 
Cardiovascular: Palpitations, chest pain. 
Gastrointestinal: Dyspepsia, flatulence, vomiting, melena and cholestatic jaundice. 
Genitourinary: Monilia, vaginitis and nephritis. 
Nervous System: Dizziness, headache, vertigo and somnolence. 
General: Fatigue. 
Allergic: Rash, pruritus, photosensitivity and angioedema. 
 
The side effects in pediatric patients are comparable to those seen in adults, with different incidence 
rates for the dosage regimens recommended in pediatric patients. In pediatric studies, side effects that 
occurred with a frequency of 1% or less included the following: 

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Cardiovascular: Chest pain. 
Gastrointestinal: Dyspepsia, constipation, anorexia, enteritis, flatulence, gastritis, jaundice, loose stools 
and oral moniliasis. 
Hematologic and Lymphatic: Anemia and leukopenia. 
Nervous System: Headache (otitis media dosage), hyperkinesia, dizziness, agitation, nervousness and 
insomnia. 
General: Fever, face edema, fatigue, fungal infection, malaise and pain. 
Allergic: Rash and allergic reaction. 
Respiratory: Cough increased, pharyngitis, pleural effusion and rhinitis. 
Skin and Appendages: Eczema, fungal dermatitis, pruritus, sweating, urticaria and vesiculobullous rash. 
Special Senses: Conjunctivitis 
 
Metronidazole40 (see Attachments for complete product package insert) 
The following reactions have been reported during treatment with metronidazole: 
 
Central Nervous System: The most serious adverse reactions reported in patients treated with 
metronidazole have been convulsive seizures, encephalopathy, aseptic meningitis, optic and peripheral 
neuropathy, the latter characterized mainly by numbness or paresthesia of an extremity. Since 
persistent peripheral neuropathy has been reported in some patients receiving prolonged 
administration of metronidazole, patients should be specifically warned about these reactions and 
should be told to stop the drug and report immediately to their physicians if any neurologic symptoms 
occur. In addition, patients have reported headache, syncope, dizziness, vertigo, incoordination, ataxia, 
confusion, dysarthria, irritability, depression, weakness, and insomnia (see WARNINGS). 
Gastrointestinal: The most common adverse reactions reported have been referable to the 
gastrointestinal tract, particularly nausea, sometimes accompanied by headache, anorexia, and 
occasionally vomiting; diarrhea; epigastric distress; abdominal cramping; and constipation. 
 
Mouth: A sharp, unpleasant metallic taste is not unusual. Furry tongue, glossitis, and stomatitis have 
occurred; these may be associated with a sudden overgrowth of Candida which may occur during 
therapy. 
 
Dermatologic: Erythematous rash and pruritus. 
 
Hematopoietic: Reversible neutropenia (leukopenia); rarely, reversible thrombocytopenia. 
 
Cardiovascular: Flattening of the T-wave may be seen in electrocardiographic tracings. 
 
Hypersensitivity: Urticaria, erythematous rash, Stevens-Johnson Syndrome, toxic epidermal necrolysis, 
flushing, nasal congestion, dryness of the mouth (or vagina or vulva), and fever. 
 
Renal: Dysuria, cystitis, polyuria, incontinence, and a sense of pelvic pressure. Instances of darkened 
urine have been reported by approximately one patient in 100,000. Although the pigment which is 
probably responsible for this phenomenon has not been positively identified, it is almost certainly a 
metabolite of metronidazole and seems to have no clinical significance. 
 

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Other: Proliferation of Candida in the vagina, dyspareunia, decrease of libido, proctitis, and fleeting joint 
pains sometimes resembling “serum sickness.” Rare cases of pancreatitis, which generally abated on 
withdrawal of the drug, have been reported. 
 
Patients with CD are known to have an increased incidence of gastrointestinal and certain extra-
intestinal cancers. There have been some reports in the medical literature of breast and colon cancer in 
Crohn’s disease patients who have been treated with metronidazole at high doses for extended periods 
of time. A cause and effect relationship has not been established.  
 
 
Methotrexate:  
The most frequently reported adverse reactions include ulcerative stomatitis, leukopenia, nausea, and 
abdominal distress. Other frequently reported adverse effects are malaise, undue fatigue, chills and 
fever, dizziness and decreased resistance to infection. 
 
Other adverse reactions that have been reported with methotrexate are listed below by organ system. 
Alimentary System: gingivitis, pharyngitis, stomatitis, anorexia, nausea, vomiting, diarrhea, 
hematemesis, melena, gastrointestinal ulceration and bleeding, enteritis, pancreatitis.  
 
Blood and Lymphatic System Disorders: suppressed hematopoiesis, anemia, aplastic anemia, 
pancytopenia, leukopenia, neutropenia, thrombocytopenia, agranulocytosis, eosinophilia, 
lymphadenopathy and lymphoproliferative disorders (including reversible). Hypogammaglobulinemia 
has been reported rarely.  
 
Cardiovascular: pericarditis, pericardial effusion, hypotension, and thromboembolic events (including 
arterial thrombosis, cerebral thrombosis, deep vein thrombosis, retinal vein thrombosis, 
thrombophlebitis, and pulmonary embolus).  
 
Central Nervous System: headaches, drowsiness, blurred vision, transient blindness, speech impairment 
including dysarthria and aphasia, hemiparesis, paresis and convulsions have also occurred following 
administration of methotrexate. Following low doses, there have been occasional reports of transient 
subtle cognitive dysfunction, mood alteration or unusual cranial sensations, leukoencephalopathy, or 
encephalopathy.  
 
Hepatobiliary Disorders: hepatotoxicity, acute hepatitis, chronic fibrosis and cirrhosis, hepatic failure, 
decrease in serum albumin, liver enzyme elevations.  
 
Infection: There have been case reports of sometimes fatal opportunistic infections in patients receiving 
methotrexate therapy for neoplastic and non-neoplastic diseases. Pneumocystis carinii pneumonia was 
the most common opportunistic infection. There have also been reports of infections, pneumonia, 
Cytomegalovirus infection, including cytomegaloviral pneumonia, sepsis, fatal sepsis, nocardiosis; 
histoplasmosis, cryptococcosis, Herpes zoster, H. simplex hepatitis, and disseminated H. simplex.  
 
Musculoskeletal System: stress fracture.  
 
Ophthalmic: conjunctivitis, serious visual changes of unknown etiology.  
 

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Pulmonary System: respiratory fibrosis, respiratory failure, alveolitis, interstitial pneumonitis deaths 
have been reported, and chronic interstitial obstructive pulmonary disease has occasionally occurred.  
 
Skin: erythematous rashes, pruritus, urticaria, photosensitivity, pigmentary changes, alopecia, 
ecchymosis, telangiectasia, acne, furunculosis, erythema multiforme, toxic epidermal necrolysis, 
Stevens-Johnson syndrome, skin necrosis, skin ulceration and exfoliative dermatitis 
Urogenital System: severe nephropathy or renal failure, azotemia, cystitis, hematuria, 
proteinuria; defective oogenesis or spermatogenesis, transient oligospermia, menstrual 
dysfunction, vaginal discharge, and gynecomastia; infertility, abortion, fetal death, fetal defects. 
 
Other rarer reactions related to or attributed to the use of methotrexate such as nodulosis, 
vasculitis, arthralgia/myalgia, loss of libido/impotence, diabetes, osteoporosis, sudden death, 
lymphoma, including reversible lymphomas, tumor lysis syndrome, soft tissue necrosis and 
osteonecrosis. Anaphylactoid reactions have been reported. 
 
Blood Draws: Phlebotomy can cause discomfort, bruising, a small risk of infection, or thrombosis. 
Standard aseptic technique will be employed to prevent infection. All blood draws will take place at the 
time of routine blood test monitoring.  The volume of blood required to perform the clinical tests 
described in this study is 5 ml.  A total of 30 ml is expected to be collected over the 52-week study 
period.  These volumes are well below the recommended blood draw volume limits for pediatric study 
participants—5 ml/kg in a single day and 9.5 ml/kg over an 8 week period—as described by NIH 
guidelines.  
 
Stool Sample: There is no specific risk to the subject in obtaining a stool sample though bringing a stool 
sample from home may be inconvenient. 
 
Breach of confidentiality: There is a risk that someone not authorized to view participant information, 
including identifiable information, will gain access to this information. Clinical sites will take measures to 
prevent this from happening and will comply with local regulations. 
 
 
2.3.2 KNOWN POTENTIAL BENEFITS  
 
A retrospective uncontrolled analysis of pediatric subjects with active CD showed that combined 
azithromycin-metronidazole therapy may be effective in inducing remission.35  A recent randomized 
controlled trial (NCT01596894) allocating children with mild to moderately active CD into groups 
receiving azithromycin + metronidazole therapy (7.5mg/kg azithromycin once daily for five consecutive 
days/ week for 4 weeks, and 3 times a week for the following 4 weeks, in conjunction with 20mg/kg/day 
of metronidazole) or metronidazole alone daily for 8 weeks, showed that remission rates with 
azithromycin + metronidazole therapy are better than with metronidazole alone, with a 66% clinical 
remission. 21 
 
This study may not result in any direct benefit to participants. However, results of this study could help 
determine whether personalized adjunct antibiotic therapy can improve the efficacy of current standard 
treatment protocols for children and adolescents with CD.  
 
 

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2.3.3 ASSESSMENT OF POTENTIAL RISKS AND BENEFITS  
 
The available information suggests that the present clinical study has an acceptable risk-benefit ratio.   
 
Approximately 50% of pediatric patients with CD will relapse within a year of starting induction therapy 
and require repeated courses of induction therapy and/or treatment escalation.41,42   Subjects in the 
antibiotic treatment arm of this trial will continue to receive standard therapy and are at comparable 
risk of relapse as subjects in the control arms. In group C of subjects not achieving remission with 
nutritional induction therapy alone, there will be no randomization and antibiotics will be given as 
additional induction treatment. The patient, parents and treating physician are free at all times to 
choose any treatment outside of the study. 
 
The risk of rare, but serious, heart-related side effects from the antibiotic drug azithromycin are 
minimized by screening patients for heart rhythm irregularities prior to administering antibiotics.  
Metronidazole presents minimal risk to the subjects and is routinely used in this pediatric population. 
The safety monitoring practices employed by this protocol are adequate to protect the subjects’ safety 
and detect all anticipated adverse events. 
 
Subjects in the antibiotic intervention arm may experience direct health benefit through the study 
treatment by reducing their risk of disease flare thus avoiding additional intestinal damage as well as 
additional immune suppression or surgery.  There will be no additional direct benefit for subjects in the 
reference arms because they will be receiving standard care.  The knowledge gained from this trial could 
lead to improved treatment approaches for a subgroup of patients who have a relapse-associated 
microbiome profile.  
 
3 
OBJECTIVES AND OUTCOME VARIABLES 
   
OBJECTIVES 
OUTCOME VARIABLES 
JUSTIFICATION FOR 
OUTCOME 
VARIABLES 
Primary 
 
 
To evaluate the potential efficacy 
of personalized adjunctive 
antibiotic therapy in maintaining 
clinical remission in pediatric 
subjects undergoing SOC induction 
therapy for mild to moderate 
Crohn’s disease who have a 
relapse-associated microbiome 
profile 
 
 
Proportion of subjects in sustained 
remission at 52 weeks after starting 
standard of care induction therapy  
 
Sustained remission is defined PCDAI 
less than or equal to 10 AND  no need 
of re-induction for clinical flare (new 
course of EEN, need to restart 
steroids), no steroid dependence, no 
biologic (anti-TNF) use, and no 
intestinal surgery by 12 months. 
 
 
We will aim to 
demonstrate 
superiority of 
adjunctive antibiotic 
therapy + SOC over 
SOC alone in patients 
with a relapse-
associated 
microbiome.  Group A1 
and A2 will be 
compared and 
sustained remission is 
the principal analysis 
variable. 
Secondary 
 
 

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OBJECTIVES 
OUTCOME VARIABLES 
JUSTIFICATION FOR 
OUTCOME 
VARIABLES 
To evaluate the potential efficacy 
of personalized adjunctive 
antibiotic therapy in improving 
PRO, components of established 
disease activity measures in 
remission, as well as ‘biochemical’ 
remission in pediatric subjects 
undergoing SOC induction therapy 
for mild to moderate Crohn’s 
disease who have a relapse-
associated microbiome profile 
 
 
Longitudinal (clustered and with 
respect to baseline) changes in 
disease activity indices (PCDAI) 
components, and inflammatory 
markers in blood and stool:  
 
 
PCDAI score (0-100) 
 
Normalization of CRP (mg/dL)  
 
Fecal calprotectin (FCP) 
(microgram/g)  
 
 
Longitudinal (clustered and with 
respect to baseline) changes PRO: 
 
IMPACT (self-report, ages 9-17 
years) 
Multiple secondary 
variables are needed to 
describe clinically 
relevant treatment 
benefits.  Depending 
on the results of the 
microbiome analysis, 
either groups A2 and B 
will be pooled and 
compared with group 
A1 or groups A1 and B 
will be pooled and 
compared with group 
A2 (section 9.4.3).   
Tertiary/Exploratory  
 
 
To investigate relationship 
between changes in subject 
microbiome composition and 
changes in disease activity over 
time. 
Longitudinal (clustered and with 
respect to baseline) changes in fecal 
microbiome taxonomic composition 
or in total gene (metagenome) 
content.  The changes will be 
analyzed for association with changes 
in disease activity (e.g. relapse or 
sustained remission) over time.  
Not applicable; these 
changes are 
exploratory and not 
intended to 
demonstrate clinical 
benefit of treatment 
 
 
4 
STUDY DESIGN  
 
4.1 
OVERALL DESIGN 
The primary hypothesis tested is that administering adjunctive antibiotic therapy to patients with a 
relapse-associated gut microbiome profile will help to sustain clinical response to standard induction 
therapy for mild to moderately active Crohn’s disease.   
 
To test this hypothesis, patients with a relapse-associated microbiome profile will be identified via 
baseline stool sample analysis and will be randomly allocated to the treatment arm that will receive 
antibiotics (azithromycin + metronidazole) on top of SOC induction therapy, or a control arm that will 
receive SOC induction therapy alone.  Patients not achieving clinical remission (PCDAI greater than 10 at 
week 4), can receive antibiotics (azithromycin/metronidazole), will not be randomized, and will continue 
to be followed until 52 weeks. This pilot trial is intended to be a smaller version of a larger trial, and will 
be used to evaluate feasibility including standard deviation of the outcome measures, adherence, and 
baseline microbiome profiling. 
 

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Standard of care (SOC) induction therapy, as started by the responsible physician, for this study will be 
defined as Crohn’s Disease Exclusion diet nutritional therapy (CDED Phase 1 from 0-6 weeks and Phase 2 
from 6-12 weeks), in accordance with the recently published RCT for the CDED.24  
 
This is a 52-week, multicenter, randomized, controlled, open-label add-on design clinical trial pilot trial 
to test the feasibility of a larger trial evaluating the efficacy of adjuvant antibiotic therapy in pediatric 
patients with mild to moderate CD.  This study will not be blinded.   
 
A total of 20 subjects between the ages of 3 and 17 years inclusive will be enrolled in eight 
investigational sites located in Canada, the United States, the Netherlands and Israel. 
 
Subjects will be stratified based on microbiome profile before they are randomized.  A baseline stool 
sample provided at or within 30 days of week 0 will be processed for DNA sequencing and analyzed by a 
blinded bioinformatician at Dalhousie University.  At week 4, subjects will be assigned to Group A or B 
based on posterior probability of having a ‘relapse-associated’ microbiome according to an established 
statistical model.  If patients have not achieved clinical remission, they will be assigned to group C to 
receive antibiotic for 8 weeks. Subjects in Group A will be randomly assigned to continue SOC therapy 
alone (Group A1) or to receive add-on antibiotic therapy in addition to SOC (Group A2) for the next 8 
weeks (week 4-12). Subjects in Group B will continue on SOC therapy alone for the next 8 weeks. 
 
At week 0, a medication calendar will be distributed to all subjects and patients/families will receive 
teaching on how to complete it for SOC therapy. At week 4, subjects randomized to Group A2 will be 
given additional instructions on how to complete the calendar for antibiotic treatment. The calendar will 
be returned to study staff at week 12 to be used as a measure of compliance and adherence. 
Pediatric patients with mild to moderate CD are normally started on an immunomodulator during 
induction therapy to help maintain remission.  Although methotrexate is not FDA approved for this 
purpose, in this study, subjects will receive 15mg/m2 subcutaneous (s.c.) methotrexate (MTX) as 
maintenance therapy (max. 25mg weekly), consistent with several current international guidelines as 
standard of care46, unless there are contraindications, or oral MTX is preferred and/or the patient/family 
refuses immunomodulatory therapy.47 However, the use of MTX is not a condition to take part in this 
study. The use of MTX is only prescribed under guidance of the treating physician.  
Because methotrexate depletes folic acid, patients receiving methotrexate may also receive folic acid 
supplementation under the guidance of the treating gastroenterologist.  Reflecting the variability of 
maintenance options in current clinical care 21, the recommendation of MTX s.c. as maintenance therapy 
is intended to help mitigate the confounding effect of different types or lack of immunomodulatory 
therapy but is not intended to trigger patients being excluded from or leaving the study, e.g. if a treating 
physician prefers to continue azathioprine, then MTX will not be started concurrently.  Details of 
immunomodulatory therapy will be recorded on a study CRF for concomitant medications.  
Participants will complete the following PRO questionnaire at week 0, 12, 24 and 52 study visits:  
1. IMPACT (self-report, ages 9-17 years): 
https://mirapeds.ca/miraresearch/impact/  
If needed, a study timepoint window of  +/- 5 days is allowed for all visits/questionnaires/sampling. 
 
 

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4.2 
SCIENTIFIC RATIONALE FOR STUDY DESIGN 
 
The experimental treatment arm (Group A2) will receive an add-on therapy given on top of existing SOC 
induction therapy and the control groups will receive SOC induction therapy alone.  This will be a 
superiority study to demonstrate that add-on antibiotic therapy is superior to existing standard of care. 
 
The control arms (Group A1 and B) are stratified based on microbiome profile.  This will allow us to 
confirm our previous observations that subjects with a relapse-associated microbiome profile are at an 
increased risk of relapse after completing induction therapy.  The primary objective will be to compare 
the rates of sustained remission between Groups A1 and A2 to determine whether add-on antibiotic 
therapy improves outcomes for patients with a relapse-associated microbiome profile.  Group C will 
allow patients to receive additional antibiotic for a separate indication of induction of remission, as this 
group has not received clinical remission on nutritional therapy alone by week 4. 
 
 
4.3 
JUSTIFICATION FOR DOSE 
 
Azithromycin and metronidazole will be administered orally as a tablet as according to their FDA-
approved labels, or as a suspension if required (see Attachments). Both azithromycin and metronidazole 
are available in 250mg tablets, doses will be given in multiple of 125mg (1/2 tablets). 
 
Azithromycin will be administered at a dose of 7.5mg/kg to a maximum of 500mg/day for 5 consecutive 
days per week for the first 4 weeks and then 3 consecutive days/week for 4 weeks. Metronidazole will 
be administered 10mg/kg twice daily to a maximum of 1000mg/day for 8 weeks. There will be no 
placebos administered in this study. These dosages are based on the recent RCT of azithromycin + 
metronidazole.21 
 
4.4 
END OF STUDY DEFINITION 
A participant is considered to have completed the study if he or she has completed all phases of the 
study including the last visit or the last scheduled procedure shown in the Schedule of Activities (SoA), 
Section 1.3. 
 
The end of the study is defined as completion of the last visit or procedure shown in the SoA in the trial 
globally. 
 
5 
STUDY POPULATION 
 
5.1 
INCLUSION CRITERIA 
 
In order to be eligible to participate in this study, an individual must meet all of the following criteria: 
1. Provision of signed and dated informed consent form (and assent form, as applicable) 
2. Stated willingness to comply with all study procedures and availability for the duration of the 
study  
3. Male or female, aged 3 to 17 years 
4. Diagnosed with CD according to standard clinical and histological criteria, within 36 months of 
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5. Exhibiting mild to moderate symptoms of active disease, as determined by a PCDAI score >10 (or 
>7.5 excluding the height item) and ≤37.5 
6. Evidence of active inflammation based on either: fecal calprotectin level >=250 microgram/g 
(local laboratory or pre-arranged sponsor testing) within 30 days prior to week 0 visit; or 
according to accepted endoscopic and histologic evidence obtained during an endoscopy 
procedure completed within 30 days prior to Week 0 Visit.  
5.2 
EXCLUSION CRITERIA 
An individual who meets any of the following criteria will be excluded from participation in this study: 
 
1. Current or previous use of anti-TNF or other biologic therapy  
2. Presence of stricturing, penetrating (intestinal or perianal) and/or fistulizing CD. 
3. Pregnancy or lactation 
4. Have undergone intestinal resection 
5. Laboratory diagnosis of Clostridium Difficile Infection (CDI), if performed for clinical indication 
6. Treatment with another investigational drug or other intervention within 30 days before week 0 
7. Risk factors for arrhythmia including history of prolonged QTc, hypokalemia or 
hypomagnesemia, resting bradycardia, or concurrent treatment with other drugs with potential 
for QT prolongation. 
8. History of Cockayne syndrome 
9. Prior diagnosis of any hematologic condition/blood dyscrasia which may result in leukopenia 
(even if leukocyte count is normal at screening) 
10. Known allergy or intolerance to azithromycin or metronidazole 
11. Subjects who received IV anti-infective within 35 days prior to week 0 visit or oral anti-infectives 
within 14 days prior to the week 0 visit. 
12. Subject on oral aminosalicylates who has not been on stable doses for greater than, or 
discontinued within, at least 14 days prior to week 0. 
13. Subject on cyclosporine, tacrolimus or mycophenolate mofetil. Stable doses (no change within 
14 days prior to week 0) of Azathioprine, 6-mercaptopurine or MTX are not a reason for 
exclusion. 
14. Subject who received fecal microbial transplantation within 35 days prior to week 0 visit. 
15. Screening laboratory and other analyses show any of the following abnormal results: 
o AST, ALT > 2 X upper limit of the reference range (as determined locally at each site) 
o Urea, Creatinine > 1.5X upper limit of the reference range (as determined locally at each 
site) 
o White blood cell (WBC) count < 3.0 X 109/L 
o Total bilirubin >= 20 micromol/liter (1.17mg/dl); except for subjects with isolated 
elevation of indirect bilirubin relating to Gilbert syndrome 
o Hemoglobin < 80 gram/liter 
o Platelets < 100,000/L 
 
Note: The laboratory markers of e.g. AST and ALT are deliberately specified relative to upper limit of 
normal as these values (and units used) differ between the different labs within the USA and across the 
study sites in the USA, Canada and Europe. 
 
 

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5.3 
LIFESTYLE CONSIDERATIONS 
 
Not applicable. 
 
5.4 
SCREEN FAILURES 
 
Screen failures are defined as participants who consent to participate in the clinical trial but are not 
subsequently randomly assigned to the study intervention or entered in the study. A minimal set of 
screen failure information is required to ensure transparent reporting of screen failure participants, to 
meet the Consolidated Standards of Reporting Trials (CONSORT) publishing requirements and to 
respond to queries from regulatory authorities. Minimal information includes demography, screen 
failure details, eligibility criteria, and any serious adverse event (SAE). 
 
Individuals who do not meet the criteria for participation in this trial (screen failure) because of 
incomplete clinical data or recent Clostridium difficile infection positivity may be rescreened. 
Rescreened participants should be assigned the same participant number as for the initial screening. 
 
 
5.5 
STRATEGIES FOR RECRUITMENT AND RETENTION 
 
Potential participants will be identified by their treating gastroenterologist within 1 of 8 participating 
centres across the U.S.A., Canada, the Netherlands, and Israel. The study sites are pediatric 
gastroenterology clinics and participants will be recruited from patients who are undergoing diagnostic 
procedures and receiving medical treatment for Crohn’s disease, as per standard of care.  
 
For this pilot study, up to 5 participants from each site will be recruited until a total of 20 subjects. The 
treating gastroenterologist will ask the patient and their parent/guardian if they are interested in 
hearing more about the study and will obtain their verbal consent to be approached by a member of the 
study staff.  The study staff member will explain the study and conduct and informed consent discussion 
with the potential participant and their parent/guardian.  The treating gastroenterologist will be 
available to address any questions or concerns that the potential participant and their parent/guardian 
may have about participating in the study.  
 
Study visits and procedures are timed to coincide with regularly scheduled follow-up visits and collection 
of biospecimens that would occur as per standard of care for children and adolescents being treated for 
active Crohn’s disease.  This design is expected to minimize the burden of study participation for the 
subject and their parent/guardian and to improve the adherence to the study visit schedule. If needed, a  
study timepoint window of +/- 5 days is allowed for all visits/questionnaires/sampling. 
 
Direct incentives will not be offered to participants or their parent/guardian.  
 
 

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6 
STUDY INTERVENTION 
6.1 
STUDY INTERVENTION(S) ADMINISTRATION 
 
6.1.1 STUDY INTERVENTION DESCRIPTION 
 
The antibiotics to be used in this study are azithromycin and metronidazole.   Both drugs are FDA-
approved and commercially available, most recent product labels are available at the following URLs:  
 
Azithromycin: 
https://www.accessdata.fda.gov/drugsatfda_docs/label/2017/050670s032,050710s046,050711s043,05
0784s030lbl.pdf 
 
Metronidazole: 
https://www.accessdata.fda.gov/drugsatfda_docs/label/2015/012623s066lbl.pdf 
 
Azithromycin (also Zithromax, Zmax) is a macrolide antibacterial drug indicated for mild to moderate 
infections caused by designated, susceptible bacteria: 
• 
 Acute bacterial exacerbations of chronic bronchitis in adults 
• 
 Acute bacterial sinusitis in adults  
• 
 Uncomplicated skin and skin structure infections in adults  
• 
 Urethritis and cervicitis in adults  
• 
 Genital ulcer disease in men  
• 
 Acute otitis media in pediatric patients  
• 
 Community-acquired pneumonia in adults and pediatric patients 
• 
 Pharyngitis/tonsillitis in adults and pediatric patients 
 
Metronidazole (also Flagyl) is an oral formulation of the synthetic nitroimidazole antimicrobial, 2-
methyl-5-nitro-1H-imidazole-1-ethanol indicated for infections strongly suspected to be caused by 
bacteria, particularly in the treatment of serious infections caused by susceptible anaerobic bacteria.  
 
 
Symptomatic or asymptomatic Trichomoniasis (T. vaginalis) infection in females and male and 
asymptomatic sexual partners. T 
 
Acute intestinal amebiasis (amebic dysentery) and amebic liver abscess. 
 
Anaerobic bacterial infections, in a mixed aerobic and anaerobic infection, antimicrobials 
appropriate for the treatment of the aerobic infection should be used in addition to 
metronidazole. 
o INTRA-ABDOMINAL INFECTIONS, including peritonitis, intra-abdominal abscess, and liver 
abscess, caused by Bacteroides species including the B. fragilis group (B. fragilis, B. 
distasonis, B. ovatus, B. thetaiotaomicron, B. vulgatus), Clostridium species, 
Eubacterium species, Peptococcus species, and Peptostreptococcus species. 
o SKIN AND SKIN STRUCTURE INFECTIONS caused by Bacteroides species including the B. 
fragilis group, Clostridium species, Peptococcus species, Peptostreptococcus species, 
and Fusobacterium species. 
o GYNECOLOGIC INFECTIONS, including endometritis, endomyometritis, tubo-ovarian 
abscess, and postsurgical vaginal cuff infection, caused by Bacteroides species including 

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the B. fragilis group, Clostridium species, Peptococcus species, Peptostreptococcus 
species, and Fusobacterium species. 
o BACTERIAL SEPTICEMIA caused by Bacteroides species including the B. fragilis group and 
Clostridium species. 
o BONE AND JOINT INFECTIONS, (as adjunctive therapy), caused by Bacteroides species 
including the B. fragilis group. 
o CENTRAL NERVOUS SYSTEM (CNS) INFECTIONS, including meningitis and brain abscess, 
caused by Bacteroides species including the B. fragilis group. 
o LOWER RESPIRATORY TRACT INFECTIONS, including pneumonia, empyema, and lung 
abscess, caused by Bacteroides species including the B. fragilis group. 
o ENDOCARDITIS caused by Bacteroides species including the B. fragilis group. 
 
6.1.2 DOSING AND ADMINISTRATION 
 
Dosage 
Azithromycin will be administered at a dose of 7.5mg/kg to a maximum of 500mg/day for 5 consecutive 
days per week for the first 4 weeks and then 3 consecutive days/week for 4 weeks. 
 
Metronidazole will be administered 10mg/kg twice daily (20mg/kg/day) to a maximum of 1000mg/day 
for 8 weeks.  
Patients who suspect they are intolerant to the medications are advised to contact the study 
coordinator regarding any new symptoms (predefined as new onset of emesis, significant nausea, 
abdominal pain or diarrhoea).  
 
At the discretion of the responsible physician, patients can be instructed to reduce the dose of 
metronidazole by 25% to 15mg/kg/day and divide the frequency to 3 times daily. Any dosage change 
will be appropriately documented in the CRF. 
 
Formulations 
Azithromycin and metronidazole are both available in 250mg tablets.  Azithromycin is also commercially 
available as an oral suspension.  Metronidazole is not commercially available as an oral suspension, but 
a suspension can be prepared by a pharmacy from 250mg tablets. 
 
 
6.2 
PREPARATION/HANDLING/STORAGE/ACCOUNTABILITY 
  
 
6.2.1 ACQUISITION AND ACCOUNTABILITY 
 
The investigational drugs will be acquired at each site independently, and labeled according to FDA 
labeling requirements found in 21CFR 312.6. The investigational Pharmacy at each site will dispense 
study drugs following enrollment and randomization according to study protocol. For Azithromycin 250 
mg in tablets and suspension are available. Metronidazole is available in 250 mg tablets and pharmacies 
can also prepare an extemporaneous compound of the Metronidazole liquid (to be refrigerated). 
  

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Each of the participating study sites will dispense and label study drugs according to local guidelines. For 
example, at UNC, Vestigo software is used for the management of inventory and accountability as well 
as labeling the product with an outpatient label.  Return of unused study drug and accountability on 
those returns will be performed using this same software system. 
  
Subjects will be seen in dedicated outpatient clinics during this study and the local study coordinator will 
be delivering the appropriately dosed and labelled study drugs to the child/parent and explaining the 
medication and use and storage to them. 
 
 
 
6.2.2 FORMULATION, APPEARANCE, PACKAGING, AND LABELING 
 
Azithromycin and metronidazole are both available in 250mg tablets.  Azithromycin is also commercially 
available as an oral suspension as 100 mg/5 mL.  Metronidazole is not commercially available as an oral 
suspension, but a suspension can be prepared by a pharmacy from 250mg tablets. 
 
Both drugs are FDA-approved and commercially available, most recent product labels are available at 
the following URLs:  
 
https://www.accessdata.fda.gov/drugsatfda_docs/label/2017/050670s032,050710s046,050711s043,05
0784s030lbl.pdf 
 
https://www.accessdata.fda.gov/drugsatfda_docs/label/2015/012623s066lbl.pdf 
 
The supplier of the drug may differ from the label provided, but the route, administration, and product 
information will be the same. 
 
 
6.2.3 PRODUCT STORAGE AND STABILITY 
 
Azithromycin tablets should be stored between 15o to 30 o C (59 o to 86 o F). Metronidazole tablets 
should be stored below 77°F (25°C) and protected from light. 
 
Azithromycin for oral suspension is supplied to provide 100 mg/5 mL or 200 mg/5 mL suspension.  Dry 
powder should be stored below 30°C (86°F). Constituted suspension should be stored between 5° to 
30°C (41° to 86°F) and discarded when full dosing is completed. 
 
Metronidazole is not commercially available as an oral suspension, but a suspension can be prepared by 
a pharmacy from 250mg tablets (to be refrigerated). 
 
Both drugs are FDA-approved and commercially available, most recent product labels are available at 
the following URLs:  
 
https://www.accessdata.fda.gov/drugsatfda_docs/label/2017/050670s032,050710s046,050711s043,05
0784s030lbl.pdf 
 
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The supplier of the drug may differ from the label provided, but the route, administration, and product 
information will be the same. 
 
 
6.2.4 PREPARATION 
Azithromycin and metronidazole are both available in 250mg tablets.  Azithromycin is also commercially 
available as an oral suspension as 100 mg/5 mL.  Metronidazole is not commercially available as an oral 
suspension, but a suspension can be prepared by a pharmacy from 250mg tablets. Pharmacies at each 
of the participating study sites will dispense and label study drugs according to local policies. 
 
Both drugs are FDA-approved and commercially available, most recent product labels are available at 
the following URLs:  
 
https://www.accessdata.fda.gov/drugsatfda_docs/label/2017/050670s032,050710s046,050711s043,05
0784s030lbl.pdf 
 
https://www.accessdata.fda.gov/drugsatfda_docs/label/2015/012623s066lbl.pdf 
 
The supplier of the drug may differ from the label provided, but the route, administration, and product 
information will be the same. 
 
 
6.3 
MEASURES TO MINIMIZE BIAS: RANDOMIZATION AND BLINDING 
 
We will randomly allocate the subjects who are identified as having a relapse-associated microbiome 
into experimental treatment or control arms. The randomization process and creation of randomization 
envelopes are performed by the study coordinator at the Amsterdam UMC, location AMC. To ensure 1:1 
randomization, blocks of 2 will be generated by using CastorEDC for patients randomized to group A. 
The same number of patients will be randomly allocated in the cohort groups A1 and A2.     
 
Once the temporary ID has been matched with the participant’s Study ID, a randomization pdf is created 
for the Study ID number, with the treatment group assignment and emailed to the local study-
coordinator. The study coordinator will then prepare a sealed randomization envelope with a card 
stating whether they will take antibiotics or not to take antibiotics. The study coordinator will give this 
envelope to the participant & family to open this envelope in the presence of the local site-study 
coordinator. The physician will be informed of the outcome of the randomization. A prescription will 
then be issued by the treating physician. The local site-study coordinator will verify that each patient 
understands the instructions. 
 
This study will not be blinded. 
 
6.4 
STUDY INTERVENTION COMPLIANCE 
 
Subjects in the antibiotic treatment arm will be asked to complete a medication calendar to record the 
dose and any problems encountered while administering the dose (e.g. vomiting). Study staff will 
monitor adherence by counting tablets returned at the 12 week visit.  

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6.5 
CONCOMITANT THERAPY 
 
All subjects in this study are asked to continue their standard of care (SOC) treatment for CD as 
prescribed by their treating gastroenterologist.  
 
For this protocol, a prescription medication is defined as a medication that can be prescribed only by a 
properly authorized/licensed clinician. Medications to be reported in the Case Report Form (CRF) are 
concomitant prescription medications (e.g. antacid medication, including additional clinically indicated 
antibiotics or anti-fungals), over-the-counter medications and supplements.  
 
Patients taking over-the-counter prebiotics and/or probiotics will be counseled by their treating 
gastroenterologist at the time of enrolment and recommended to stop taking pre/probiotics by the 
week 0 study visit, and to refrain from using probiotics for the duration of the study (Week 52). If 
patients choose to continue using pre-and/or pro-biotics, this will be recorded on the CRF. 
 
 
6.5.1 RESCUE MEDICINE 
 
Not applicable. 
 
7 
STUDY INTERVENTION DISCONTINUATION AND PARTICIPANT 
DISCONTINUATION/WITHDRAWAL 
 
7.1 
DISCONTINUATION OF STUDY INTERVENTION 
 
Discontinuation of the antibiotic treatment between weeks 4 and 12 does not mean discontinuation of 
the study.  A participant who discontinues the antibiotic intervention will be moved into the Group A 
control group (Group A1) and receive SOC, but these patients will not be included in the analysis of 
Group A1 to avoid selection bias as a patient who discontinues treatment in week 11 may differ from a 
patient who discontinued in week 5. Any patients discontinuing treatment will therefore be reported 
separately. 
The remaining study procedures should be completed as indicated by the study protocol.  If a clinically 
significant finding is identified (including, but not limited to changes from baseline) after enrollment, the 
investigator or qualified designee will determine if any change in participant management is needed. 
Any new clinically relevant finding will be reported as an adverse event (AE).  Specific criteria for 
discontinuation due to known or potential AE associated with the study intervention are provided in 
section 7.2. 
 
 

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7.2 
PARTICIPANT DISCONTINUATION/WITHDRAWAL FROM THE STUDY 
 
Participants are free to withdraw from participation in the study at any time for any reason if they wish 
to do so without any consequences.   An investigator may discontinue or withdraw a participant from 
the study for the following reasons: 
 
 
Pregnancy 
 
Significant study intervention non-compliance  
 
Occurrence of a clinical AE known to be associated with study intervention including new onset 
peripheral neuropathy, new onset seizures, new onset clinically significant leukopenia, new-
onset QT prolongation, evidence of hepatotoxicity, allergic reaction.  Criteria for stopping 
investigational therapy due to potential hepatotoxicity include any of the following laboratory 
abnormalities:  
o ALT or AST >8xULN  
o ALT or AST >5xULN for more than 2 weeks  
o ALT or AST >3xULN and (TBL >2xULN or INR >1.5)  
o ALT or AST >3xULN with the appearance of fatigue, nausea, vomiting, right upper 
quadrant pain or tenderness, fever, rash, and/or eosinophilia (>5%) 
 
Should any of these laboratory abnormalities occur, the participant will discontinue investigational 
therapy and undergo appropriate evaluation for other causes of abnormal liver function tests. 
The participant must be followed until resolution or stabilization and the results of the 
evaluation for alternate causes of abnormal liver enzymes will be documented in the Reason for 
Withdrawal Case Report Form (CRF) 
 
If any other clinical adverse event, laboratory abnormality, or other medical condition or 
situation occurs such that continued participation in the study would not be in the best interest 
of the participant 
 
Disease progression which requires discontinuation of the study intervention 
 
If the participant meets an exclusion criterion (either newly developed or not previously 
recognized) that precludes further study participation 
The reason for participant discontinuation or withdrawal from the study will be recorded on the Reason 
for Withdrawal CRF. Subjects who withdraw during the pilot study will try to be replaced.  
7.3 
LOST TO FOLLOW-UP 
 
A participant will be considered lost to follow-up if he or she fails to return for 2 consecutive scheduled 
visits as well as the Week 52 follow-up visit, and is unable to be contacted by the study site staff.  
 
The following actions must be taken if a participant fails to return to the clinic for a required study visit: 
 
The site will attempt to contact the participant and reschedule the missed visit and counsel the 
participant on the importance of maintaining the assigned visit schedule and ascertain if the 
participant wishes to and/or should continue in the study. 
 
Before a participant is deemed lost to follow-up, the investigator or designee will make every 
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a certified letter to the participant’s last known mailing address or local equivalent methods). 
These contact attempts should be documented in the participant’s medical record or study file.  
 
Should the participant continue to be unreachable, he or she will be considered to have 
withdrawn from the study with a primary reason of lost to follow-up. 
 
 
8 
STUDY ASSESSMENTS AND PROCEDURES 
 
8.1 
EFFICACY ASSESSMENTS  
History and Physical exam – These will be performed as per the standard of clinical care including height 
(height velocity), weight and perianal inspection. The primary outcome variable will be whether 
sustained remission at 52 weeks after starting standard of care induction therapy is achieved. Sustained 
remission is not defined by PCDAI alone; but rather by PCDAI ≤ 10 at week 52 AND not having need of 
re-induction for clinical flare (new course of EEN, need to start steroids), no steroid dependence, no 
biologic (anti-TNF) use, and no intestinal surgery by 12 months. As secondary outcome variables of 
efficacy, longitudinal (clustered and with respect to baseline) changes in disease activity indices 
components, and inflammatory markers in blood and stool, will be collected including the PCDAI score 
(0-100), normalization of CRP (<0.5mg/dL or the local reference range as applicable), fecal calprotectin 
(microgram/g) and Delta-fecal calprotectin before and after the study intervention. In addition, 
longitudinal (clustered and with respect to baseline) changes of Patient-reported outcome will be 
collected (IMPACT (self-report, ages 9-17 years). 
Blood samples – Blood samples will be drawn as per the standard of clinical care including erythrocyte 
sedimentation rate (ESR), C-reactive protein, albumin, alanine transaminase (ALT), aspartate 
transaminase (AST), bilirubin, Urea (Blood Urea Nitrogen (BUN) and Creatinine,)and complete blood 
count (including hematocrit). These blood results, provided by the local clinical labs, will be required to 
calculate the disease activity index and monitor for toxicity due to methotrexate. 
 
Stool samples – For assessment of inclusion criteria for Screening, stool samples will be obtained for 
local testing for the presence of toxigenic C. difficile.  This sample may be a pre-consent sample collected 
as standard of care. Study stool samples will be collected at each study visit and split into two aliquots; 
one for Fecal calprotectin testing and one for microbiome analysis at the central laboratory Dalhousie 
University (Integrated Microbiome Resource) as coordinated from Amsterdam UMC.   
 
Special assays - DNA sequencing and microbiome analysis: Identification of favorable vs unfavorable 
(Proteobacteria-rich) signature: the stool for analysis will be collected at baseline for screening (in 
children undergoing endoscopic investigations as decided by the responsible physician, stool will be 
obtained before the bowel prep for diagnostic endoscopy or starting from 48 hours after an endoscopy), 
in a dedicated kit (e.g. DNAGenotek) with solution buffer. Analysis of the V4-V5 16S region by a blinded 
bioinformatician (labelling the patients using Bayesian analysis (posterior probability >30% of carrying 
the Proteobacteria-rich, unfavorable signature) within 2-3 weeks, in order to allow participants to be 
randomized to either adjuvant antibiotics or no additional treatment, by the end of week 4 from 
baseline.  
 
 

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8.2 
SAFETY AND OTHER ASSESSMENTS 
 
Electrocardiograms (ECGs): ECG is for screening purposes only to identify prolongation of QT (QTc) 
corrected for age appropriate norms. If there is prolongation, as identified by each responsible 
physician, then the responsible physician is responsible for communicating this finding with the patient 
and family and arranging appropriate follow-up with cardiology. Patients found to have QT prolongation 
will not be treated with investigational therapy and appropriate guidance will be given regarding 
avoiding other medication with the potential of QTc prolongation (e.g. avoiding ondansetron). Further, 
all patients randomized to group A2 will receive a follow-up ECG 2-4 weeks after the initiation of the 
study drug to ensure that new-onset QT prolongation is not occurring as a result of azithromycin 
treatment.  If clinically significant QT prolongation is detected at follow-up ECG, patients should 
discontinue investigational therapy. If the subject is not in clinical remission at week 4, then the 
responsible physician will decide whether azithromycin/metronidazole can be started as additional 
induction therapy. 
 
Blood samples: Blood drawn as per the standard of clinical care will be tested for alanine transaminase 
(ALT), aspartate transaminase (AST), bilirubin, BUN and Creatinine by the local clinical labs to monitor 
for toxicity due to methotrexate. 
 
Urine pregnancy tests:  A urine pregnancy test will be administered at screening and repeated at week 4 
prior to the initiation of methotrexate in post-menarchal female patients.  These tests will be used to 
prevent exposure of participants to potential embryotoxic/teratogenic effects of methotrexate. 
Counselling and reinforcement of these potential risks and the importance of pregnancy prevention will 
be included at every study visit.  Post-menarchal patients will be counselled about the use of birth 
control to prevent unplanned pregnancy while taking methotrexate during this study period. 
   
 
8.3 
ADVERSE EVENTS AND SERIOUS ADVERSE EVENTS 
 
 
8.3.1 DEFINITION OF ADVERSE EVENTS (AE) 
 
Adverse event means any untoward medical occurrence associated with the use of an intervention in 
humans, whether or not considered intervention-related (21 CFR 312.32 (a)). 
 
 
8.3.2 DEFINITION OF SERIOUS ADVERSE EVENTS (SAE)  
 
An adverse event (AE) or suspected adverse reaction is considered "serious" if, in the view of either the 
investigator or sponsor, it 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 serious when, based upon appropriate medical judgment, they may jeopardize the 

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participant and may require medical or surgical intervention to prevent one of the outcomes listed in 
this definition.  
 
8.3.3 CLASSIFICATION OF AN ADVERSE EVENT 
 
8.3.3.1 SEVERITY OF EVENT 
 
For adverse events (AEs) not included in the protocol defined grading system, the following guidelines 
will be used to describe severity.  
 
• 
Mild – Events require minimal or no treatment and do not interfere with the participant’s daily 
activities.  
• 
Moderate – Events result in a low level of inconvenience or concern with the therapeutic 
measures. Moderate events may cause some interference with functioning. 
• 
Severe – Events interrupt a participant’s usual daily activity and may require systemic drug 
therapy or other treatment. Severe events are usually potentially life-threatening or 
incapacitating.  Of note, the term “severe” does not necessarily equate to “serious”. 
 
 
8.3.3.2 RELATIONSHIP TO STUDY INTERVENTION 
All adverse events (AEs) must have their relationship to study intervention assessed by the clinician who 
examines and evaluates the participant based on temporal relationship and his/her clinical judgment. 
The degree of certainty about causality will be graded using the categories below. In a clinical trial, the 
study product must always be suspect.  
 
• 
Related – The AE is known to occur with the study intervention, there is a reasonable possibility 
that the study intervention caused the AE, or there is a temporal relationship between the study 
intervention and event. Reasonable possibility means that there is evidence to suggest a causal 
relationship between the study intervention and the AE. 
• 
Not Related – There is not a reasonable possibility that the administration of the study 
intervention caused the event, there is no temporal relationship between the study intervention 
and event onset, or an alternate etiology has been established. 
 
 
8.3.3.3 EXPECTEDNESS  
 
The investigator will be responsible for determining whether an adverse event (AE) is expected or 
unexpected.  An AE will be considered unexpected if the nature, severity, or frequency of the event is 
not consistent with the risk information previously described for the study intervention. 
8.3.4 TIME PERIOD AND FREQUENCY FOR EVENT ASSESSMENT AND FOLLOW-UP 
 
The occurrence of an adverse event (AE) or serious adverse event (SAE) may come to the attention of 
study personnel during study visits of a study participant presenting for medical care, or upon review by 
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All AEs including local and systemic reactions not meeting the criteria for SAEs will be captured on the 
appropriate case report form (CRF). Information to be collected includes event description, time of 
onset, clinician’s assessment of severity, relationship to study product (assessed only by those with the 
training and authority to make a diagnosis), and time of resolution/stabilization of the event. All AEs 
occurring while on study must be documented appropriately regardless of relationship. All AEs will be 
followed to adequate resolution. 
 
Any medical condition that is present at the time that the participant is screened will be considered as 
baseline and not reported as an AE. However, if the study participant’s condition deteriorates at any 
time during the study, it will be recorded as an AE.  
 
Changes in the severity of an AE will be documented to allow an assessment of the duration of the event 
at each level of severity to be performed. AEs characterized as intermittent require documentation of 
onset and duration of each episode. 
 
The study clinician will record all reportable events with start dates occurring any time after informed 
consent is obtained until 7 (for non-serious AEs) or 30 days (for SAEs) after the last day of study 
participation.  At each study visit, the investigator will inquire about the occurrence of AE/SAEs since the 
last visit.  Events will be followed for outcome information until resolution or stabilization.] 
 
 
8.3.5 ADVERSE EVENT REPORTING  
 
Details of all adverse events reported spontaneously by subjects or observed by the investigator or 
study clinician will be recorded in appropriate adverse event report forms and reported to the principal 
investigator and the responsible medical doctor. All adverse events observed will be recorded 
separately. 
 
8.3.6 SERIOUS ADVERSE EVENT REPORTING  
 
The study clinician will immediately report to the sponsor any serious adverse event, whether or not 
considered study intervention related, including those listed in the protocol or investigator brochure and 
must include an assessment of whether there is a reasonable possibility that the study intervention 
caused the event. Study outcome variables that are serious adverse events (e.g., all-cause mortality) 
must be reported in accordance with the protocol unless there is evidence suggesting a causal 
relationship between the study intervention and the event (e.g., death from anaphylaxis). In that case, 
the investigator must immediately report the event to the sponsor. 
 
All serious adverse events (SAEs) will be followed until satisfactory resolution or until the site 
investigator deems the event to be chronic or the participant is stable. Other supporting documentation 
of the event may be requested by the Data Coordinating Center (DCC)/study sponsor and should be 
provided as soon as possible. 
 
As per C.05.014 (1) of the FDR, the study sponsor will be responsible for notifying FDA/Health 
Canada/CCMO (Netherlands) of any unexpected fatal or life-threatening suspected adverse reaction as 
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information.  Within 8 days after having initially informed FDA/Health Canada/CCMO of the fatal or life-
threatening SAE, the sponsor will submit as complete a report as possible.  Follow-up reports of fatal or 
life-threatening reactions must include an assessment of the importance and implication of the findings, 
including relevant previous experience with the same or similar drugs.  
 
Where the SAE is neither fatal nor life-threatening, the sponsor will inform FDA/Health Canada/CCMO 
(Netherlands) within 15 days after becoming aware of the information. 
 
SUSAR´s that have occurred in all participating countries should be reported by the sponsor. Further 
steps will be taken according to local legislation.  
 
 
 
8.3.7 REPORTING EVENTS TO PARTICIPANTS  
 
Participants will be notified directly be a study staff member. 
 
8.3.8 EVENTS OF SPECIAL INTEREST  
 
Not applicable 
 
8.3.9 REPORTING OF PREGNANCY  
 
If a participant becomes pregnant while taking the antibiotic intervention, they will discontinue and will 
be withdrawn from the study.  
 
Animal reproduction studies have been performed in rats and mice at doses up to moderately 
maternally toxic dose concentrations (i.e., 200 mg/kg/day) (2-4 times the human daily dose) and no 
evidence of harm to the fetus due to azithromycin was found.39  Reproduction studies for metronidazole 
have been performed in rates, rabbits, and mice at doses similar to the maximum recommended human 
dose and have found no evidence of harm to the fetus due to metronidazole.40  However, there are no 
adequate or well-controlled studies of azithromycin or metronidazole use in pregnant women.   
  
 
8.4 
UNANTICIPATED PROBLEMS 
 
 
8.4.1 DEFINITION OF UNANTICIPATED PROBLEMS (UP) 
 
The Office for Human Research Protections (OHRP) considers unanticipated problems involving risks to 
participants or others to include, in general, any incident, experience, or outcome that meets all of the 
following criteria: 
 
• 
Unexpected in terms of nature, severity, or frequency given (a) the research procedures that are 
described in the protocol-related documents, such as the Institutional Review Board (IRB)-
approved research protocol and informed consent document; and (b) the characteristics of the 
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• 
Related or possibly related to participation in the research (“possibly related” means there is a 
reasonable possibility that the incident, experience, or outcome may have been caused by the 
procedures involved in the research); and 
• 
Suggests that the research places participants or others at a greater risk of harm (including 
physical, psychological, economic, or social harm) than was previously known or recognized. 
 
 
8.4.2  UNANTICIPATED PROBLEM REPORTING  
The investigator will report unanticipated problems (UPs) to the reviewing Institutional Review Board 
(IRB) and to the Data Coordinating Center (DCC)/lead principal investigator (PI). The UP report will 
include the following information: 
 
• 
Protocol identifying information: protocol title and number, PI’s name, and the IRB project 
number; 
• 
A detailed description of the event, incident, experience, or outcome;  
• 
An explanation of the basis for determining that the event, incident, experience, or outcome 
represents an UP;  
• 
A description of any changes to the protocol or other corrective actions that have been taken or 
are proposed in response to the UP. 
 
To satisfy the requirement for prompt reporting, UPs will be reported using the following timeline:   
 
• 
UPs that are serious adverse events (SAEs) will be reported to the IRB and to the DCC/study 
sponsor within 7 calendar days of the investigator becoming aware of the event.  
• 
Any other UP will be reported to the IRB and to the DCC/study sponsor within <insert timeline in 
accordance with policy> of the investigator becoming aware of the problem.  
• 
All UPs should be reported to appropriate institutional officials (as required by an institution’s 
written reporting procedures), the supporting agency head (or designee), and the Office for 
Human Research Protections (OHRP) within <insert timeline in accordance with policy> of the 
IRB’s receipt of the report of the problem from the investigator. 
 
 
8.4.3 REPORTING UNANTICIPATED PROBLEMS TO PARTICIPANTS  
 
Participants will be notified directly by a study staff member. 
 
9 
STATISTICAL CONSIDERATIONS  
 
9.1 
STUDY OUTCOME VARIABLES 
 
The primary outcome evaluation:  
 
The primary efficacy outcome variable is being in sustained remission defined as PCDAI ≤ 10 at 52 weeks 
from start of therapy AND without steroid dependence and without the need for repeat induction 
therapy, anti-TNFα, or surgery by 52 weeks from start of therapy. 

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The secondary outcome evaluations at each study visit include: 
 
Mean change in PCDAI score from baseline to weeks 4, 12, 24, 36, and 52. 
 
Mean change in Inflammatory markers: CRP and fecal calprotectin from baseline to weeks 4, 12, 
24, 36, and 52. 
 
Mean change in PRO score (IMPACT questionnaire) from baseline to weeks 4, 12, 24, 36, and 52. 
 
Improvement (yes vs no) of disease activity (change in PCDAI < 0) rate at weeks 4, 12, 24, 36, and 
52. 
 
Improvement (yes vs no) of C-Reactive Protein (change in CRP < 0 mg/dL) and fecal calprotectin 
(change in FCP < 0 μg/g) rate at weeks 4, 12, 24, 36, and 52. 
 
Normalization (yes vs no) of disease activity (PCDAI < 10) rate at weeks 4, 12, 24, 36, and 52. 
 
Normalization (yes vs no) of C-Reactive Protein (CRP; < 0.5 mg/dL) and fecal calprotectin (FCP; < 
250 μg/g) rate at weeks 4, 12, 24, 36, and 52. 
 
 
9.2 
SAMPLE SIZE DETERMINATION 
 
Based on published literature, 50% of children will require repeat induction/treatment escalation by the 
end of the first year after diagnosis.43-45 25 children carrying the high-risk microbiome signature will 
need to be randomized to each study arm (A1 or A2) (10% drop out assumed) in the larger trial following 
on from this pilot trial,  assuming 80% power, to reduce the risk of flaring from 80% to 40%. These 
calculations are based on assumptions of relapse risk as no prospective microbiome data are available to 
inform this risk prediction of an unfavourable disease course.  
In this pilot trial, 20 children will be enrolled to assess feasibility. The clinical data about relapse risk 
were not used to determine this sample size for the pilot trial. The 20 children of the pilot study will be 
allocated to either group A or group B, based on baseline microbiome data, unless the subject does not 
achieve clinical remission at week 4. If the subject does not achieve clinical remission (PCDAI less than or 
equal to 10) then the responsible physician can decide to start azithromycin/metronidazole for 8 weeks 
as additional induction therapy in Group C.  Randomization will determine whether patients in group A, 
will be given SOC (A1) or SOC+antibiotics (A2). 
 
9.3 
POPULATIONS FOR ANALYSES 
 
 
Intention-to-Treat (ITT) Analysis Dataset will include all randomized participants: we will 
compare group A1 vs. group A2 for the primary analyses. In secondary/exploratory outcome 
analyses, we will compare the effects of treatment with antibiotics on the microbiome to assess 
whether indeed the signature of group A (A2) can be modified to move closer to group B. 
If this is the case, then (A2+B) vs. A1, will be compared. If the microbiome shifts associated with 
antibiotic use do not make A2 more similar to group B, then we will compare A1+B vs. A2 to 
assess whether there is an additional immune-regulatory effect of azithromycin (in combination 
with metronidazole) to influence disease flare risk. Group C samples will be analysed as a 
separate group in an observational study arm. 
 
 
Modified Intention-to-Treat Analysis Dataset will include participants who took the antibiotic 
intervention for at least 48 hours and had at least 6 months of follow-up outcome data. 

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 
Safety Analysis Dataset will include participants who took at least 1 week of study intervention 
for whom safety analyses will be conducted 
 
 
Per-Protocol Analysis Dataset will include participants in the full analysis (ITT) set who took at 
least 2 weeks of study intervention during the treatment period and complied with the protocol 
sufficiently to ensure that these data would be likely to represent the effects of study 
intervention according to the underlying scientific model  
 
 
9.4 
STATISTICAL ANALYSES 
 
9.4.1 GENERAL APPROACH 
 
All summary tables will be presented by treatment group (Group A1, A2, and B) and, where applicable, 
by study visits. For descriptive statistics, continuous variables will be summarized with the mean, 
median, standard deviation, minimum and maximum.  Categorical variables will be presented as counts 
and proportions expressed as a percentage.  In addition, summary figures will be generated for 
longitudinal data to display change in mean or proportion over time. 
 
Missing values in continuous variables will not be imputed.  Missing values in categorical variables will 
be imputed as non-responders. 
 
All tests will be two-sided and considered significant at P<0.05.  Hypothesis tests that are not statistically 
significant will be deemed inconclusive.  No adjustments to the test-wise error rate will be made.   
 
 
 
 
9.4.2 ANALYSIS OF THE PRIMARY OUTCOME VARIABLES 
 
The primary outcome variable is sustained remission defined as PCDAI less than or equal to 10 at 52 
weeks AND no need of re-induction for clinical flare (new course of nutritional therapy, need to restart 
steroids), steroid dependence, biologic (anti-TNF/ustekinumab/vedolizumab) use, and/or intestinal 
surgery by 12 months. The outcome variable is categorical and measured at a single outcome variable 
(52 weeks).  The number and percentage of patients with a relapse-associated gut microbiome profile 
will be summarized by group and the difference in sustained remission rate will be compared using a 
Fisher’s exact test (comparing group A1 with A2).   
 
 
The analysis of the primary efficacy parameter will be based on the ITT population.  A sensitivity analysis 
using the mITT population will also be conducted for confirmatory purposes. 
 
9.4.3 ANALYSIS OF THE SECONDARY OUTCOME VARIABLE(S) 
 
In secondary outcome analyses, the effects of treatment with antibiotics on the microbiome will be 
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If this is the case, then groups A2 and B will be pooled and compared against A1. If the microbiome 
shifts associated with antibiotic use do not make A2 more similar to group B, then groups A1 and B will 
be pooled and compared against A2 to assess whether there is an additional immune-regulatory effect 
of azithromycin (in combination with metronidazole) to influence disease flare risk. 
Data for secondary outcome variable analysis are independent of the primary outcome variable and will 
be collected at each study visit. 
Continuous secondary outcomes will be summarized at each visit for both the observed values and the 
changes from baseline.  A linear mixed model with timepoint, group, their interaction and baseline as 
covariates will be used to assess the difference in means between groups at each timepoint.  An AR(1) 
covariance structure will be assumed and the Kenward-Roger degrees of freedom approximation will be 
utilized.  Least squares estimates for the population mean and standard deviation will be provided at each 
assessment timepoint by group.  Missing values will not be imputed.  Due to the exploratory nature of the 
assessments, no correction for multiplicity will be made. 
Categorical secondary outcomes variables will be analyzed similarly to the primary outcome (Section 
9.4.2). 
The analysis of the secondary efficacy parameters will be based on the ITT population. 
 
 
 
9.4.4 SAFETY ANALYSES 
 
This study is not evaluating a formal safety outcome variable.  
 
9.4.5 DEMOGRAPHIC AND BASELINE DESCRIPTIVE STATISTICS 
 
Demographic and baseline characteristics will be based on the Intent-to-Treat (ITT) population.  For 
evaluations which are performed at both the screening and baseline visits (Visit 2), baseline values are 
to be taken from the baseline visit. 
 
Baseline characteristics to be compared between treatments will include age, gender, age at diagnosis, 
disease location and behavior (classified using the Paris classification scheme), disease activity (PCDAI 
score), CRP values, FCP values, and anthropometrics including height and weight z-score. 
 
9.4.6 PLANNED INTERIM ANALYSES  
 
Not applicable 
 
9.4.7 SUB-GROUP ANALYSES 
 
A priori subgroup analyses are unlikely to be powered sufficiently in this pilot trial. We will assess the 
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ileo-colonic involvement of the lower GI tract) although these are often influenced by disease severity at 
diagnosis and their effects may be confounded with the effects of the disease severity.  All results will be 
considered exploratory. 
 
9.4.8 TABULATION OF INDIVIDUAL PARTICIPANT DATA 
 
Participant data will be recorded individually by measure and for each time point. 
 
9.4.9 EXPLORATORY ANALYSES 
 
Exploratory analyses will involve the microbiome sequence data obtained from stool samples collected 
during the study.  Changes in microbiome taxonomic composition and genome (metagenome) 
composition will be compared to changes in disease activity measured at each study visit, while taking 
into account other clinical phenotypic data. 
 
10 SUPPORTING DOCUMENTATION AND OPERATIONAL CONSIDERATIONS 
 
10.1 REGULATORY, ETHICAL, AND STUDY OVERSIGHT CONSIDERATIONS 
 
 
10.1.1 INFORMED CONSENT PROCESS 
  
 
10.1.1.1 CONSENT/ASSENT AND OTHER INFORMATIONAL DOCUMENTS PROVIDED TO 
PARTICIPANTS 
Consent forms describing in detail the study intervention, study procedures, and risks are given to the 
participant and written documentation of informed consent is required prior to starting 
intervention/administering study intervention.  The following consent materials are submitted with this 
protocol: 
 
Information and Authorization Form 
Information and Consent Form 
Assent Form 
 
10.1.1.2 CONSENT PROCEDURES AND DOCUMENTATION 
Informed consent is a process that is initiated prior to the individual’s agreeing to participate in the 
study and continues throughout the individual’s study participation. Consent forms will be Institutional 
Review Board (IRB)-approved and the participant will be asked to read and review the document. The 
investigator will explain the research study to the participant and answer any questions that may arise. 
A verbal explanation will be provided in terms suited to the participant’s comprehension of the 
purposes, procedures, and potential risks of the study and of their rights as research participants.  
Participants will have the opportunity to carefully review the written consent form and ask questions 
prior to signing. The participants should have the opportunity to discuss the study with their family or 
surrogates or think about it prior to agreeing to participate. The participant will sign the informed 
consent document prior to any procedures being done specifically for the study. Participants must be 
informed that participation is voluntary and that they may withdraw from the study at any time, without 

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prejudice. A copy of the informed consent document will be given to the participants for their records. 
The informed consent process will be conducted and documented in the source document (including the 
date), and the form signed, before the participant undergoes any study-specific procedures. The rights 
and welfare of the participants will be protected by emphasizing to them that the quality of their 
medical care will not be adversely affected if they decline to participate in this study. 
10.1.2 STUDY DISCONTINUATION AND CLOSURE 
This study may be temporarily suspended or prematurely terminated if there is sufficient reasonable 
cause.  Written notification, documenting the reason for study suspension or termination, will be 
provided by the suspending or terminating party to study participants, investigator, funding agency, the 
Investigational New Drug (IND) sponsor and regulatory authorities.  If the study is prematurely 
terminated or suspended, the Principal Investigator (PI) will promptly inform study participants, the 
Institutional Review Board (IRB), and sponsor and will provide the reason(s) for the termination or 
suspension.  Study participants will be contacted, as applicable, and be informed of changes to study 
visit schedule. 
  
Circumstances that may warrant termination or suspension include, but are not limited to: 
 
Determination of unexpected, significant, or unacceptable risk to participants 
 
Demonstration of efficacy that would warrant stopping    
 
Insufficient compliance to protocol requirements 
 
Data that are not sufficiently complete and/or evaluable 
 
Determination that the primary outcome variable has been met 
 
Determination of futility 
 
The study may resume once concerns about safety, protocol compliance, and data quality are 
addressed, and satisfy the sponsor, IRB and/or FDA/Health Canada. 
  
 
10.1.3 CONFIDENTIALITY AND PRIVACY  
Participant confidentiality and privacy is strictly held in trust by the participating investigators, their 
staff, and the sponsor(s) and their interventions. This confidentiality is extended to cover testing of 
biological samples and genetic tests in addition to the clinical information relating to participants. 
Therefore, the study protocol, documentation, data, and all other information generated will be held in 
strict confidence. No information concerning the study or the data will be released to any unauthorized 
third party without prior written approval of the sponsor.  
 
All research activities will be conducted in as private a setting as possible. 
 
The study monitor, other authorized representatives of the sponsor, representatives of the Institutional 
Review Board (IRB), regulatory agencies or pharmaceutical company supplying study product may 
inspect all documents and records required to be maintained by the investigator, including but not 
limited to, medical records (office, clinic, or hospital) and pharmacy records for the participants in this 
study. The clinical study site will permit access to such records. 
 
The study participant’s contact information will be securely stored at each clinical site for internal use 
during the study. At the end of the study, all records will continue to be kept in a secure location for as 
long a period as dictated by the reviewing IRB, Institutional policies, or sponsor requirements. 

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Study participant research data, which is for purposes of statistical analysis and scientific reporting, will 
be transmitted to and stored at the Amsterdam UMC, location AMC (Amsterdam, The Netherlands). This 
will not include the participant’s contact or identifying information. Rather, individual participants and 
their research data will be identified by a unique study identification number. The study data entry and 
study management systems used by clinical sites and by the Amsterdam UMC research staff will be 
secured and password protected. At the end of the study, all study databases will be de-identified and 
archived at the Amsterdam UMC, location AMC . 
 
To further protect the privacy of study participants, a Certificate of Confidentiality will be issued by the 
National Institutes of Health (NIH).  This certificate protects identifiable research information from 
forced disclosure. It allows the investigator and others who have access to research records to refuse to 
disclose identifying information on research participation in any civil, criminal, administrative, legislative, 
or other proceeding, whether at the federal, state, or local level. By protecting researchers and 
institutions from being compelled to disclose information that would identify research participants, 
Certificates of Confidentiality help achieve the research objectives and promote participation in studies 
by helping assure confidentiality and privacy to participants. 
 
 
10.1.4 FUTURE USE OF STORED SPECIMENS AND DATA  
Data collected for this study will be analyzed and stored at the Amsterdam UMC, location AMC . After 
the study is completed, the de-identified, archived data will be transmitted to and stored at the 
Amsterdam UMC, location AMC for use by other researchers including those outside of the study. 
Permission to transmit data to the Amsterdam UMC will be included in the informed consent.  
 
With the participant’s approval and as approved by local Institutional Review Boards (IRBs), de-
identified biological samples will be stored at the Amsterdam UMC with the same goal as the sharing of 
data with the Amsterdam UMC. These samples could be used to research the causes of inflammatory 
bowel diseases (IBD), its complications and other conditions for which individuals with IBD are at 
increased risk, and to improve treatment. The Amsterdam UMC will also be provided with a code-link 
that will allow linking the biological specimens with the phenotypic data from each participant, 
maintaining the blinding of the identity of the participant. 
 
During the conduct of the study, an individual participant can choose to withdraw consent to have 
biological specimens stored for future research. However, withdrawal of consent with regard to 
biospecimen storage may not be possible after the study is completed.  
 
When the study is completed, access to study data and/or samples will be provided through the 
Amsterdam UMC, location AMC. 
 
10.1.5 KEY ROLES AND STUDY GOVERNANCE 
 
Principal Investigator 
Medical Monitor 
Johan Van Limbergen MD FRCPCH PhD 
Anthony Otley MD MSc FRCPC 
Amsterdam University Medical Centers – 
location AMC  
IWK Health Centre, Halifax 

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Division of Pediatric Gastroenterology & 
Nutrition 
Room H7-228 
Meibergdreef 9, 1105 AZ Amsterdam 
Division of Gastroenterology & Nutrition, 
Department of Pediatrics 
5850 University Avenue 
Halifax, NS, Canada B3K 6R8 
+31 20 566 3053 
902-470-8225 
j.e.vanlimbergen@amsterdamumc.nl 
Anthony.Otley@Dal.Ca 
 
The study oversight committee has been convened by Crohn’s Colitis Foundation and consists of 
internationally renowned pediatric IBD experts. For conduct of the study in Canada, a separate Health 
Canada application will be submitted. For participating centres in the Netherlands, local ethics 
committee applications will be filed. In Israel, conduct of the study has been submitted to the ministry 
of Health and will follow guidelines of Good Clinical Practice (GCP). 
 
 
10.1.6 SAFETY OVERSIGHT 
 
Safety oversight will be under the direction of a Data and Safety Monitoring Board (DSMB) composed of 
individuals with the appropriate expertise, including experts in clinical care and trials in pediatric 
gastroenterology and inflammatory bowel disease. Members of the DSMB should be independent from 
the study conduct and free of conflict of interest, or measures should be in place to minimize perceived 
conflict of interest.  The DSMB will meet at least semiannually to assess safety and efficacy data on each 
arm of the study. The DMSB will operate under the rules of an approved charter that will be written and 
reviewed at the organizational meeting of the DSMB. At this time, each data element that the DSMB 
needs to assess will be clearly defined. The DSMB will provide its input to the study sponsor Dr. Johan 
Van Limbergen as well to Dr. Francisco Sylvester. 
 
 
10.1.7 CLINICAL MONITORING 
 
Clinical site monitoring is conducted to ensure that the rights and well-being of trial participants are 
protected, that the reported trial data are accurate, complete, and verifiable, and that the conduct of 
the trial is in compliance with the currently approved protocol/amendment(s), with International 
Conference on Harmonisation Good Clinical Practice (ICH GCP), and with applicable regulatory 
requirement(s).  
 
• 
Centralized monitoring will be arranged by the PI in Amsterdam by means of review by an 
accredited study monitor, who is not a member of the research team, of the electronically 
recorded CRF (using RedCap) data from this pilot trial, after each site has enrolled (to ensure an 
initial assessment of each site’s data quality) and at the end of the study. Local monitoring will 
be ensured, as indicated by the central study monitor on an as-needed basis, or by targeted 
review of copies of study documents shared electronically with the central monitor.  
• 
Independent audits will not be conducted for this pilot trial.  
 
 
 
 

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10.1.8 QUALITY ASSURANCE AND QUALITY CONTROL 
 
Each clinical site will perform internal quality management of study conduct, data and biological 
specimen collection, documentation and completion.  An individualized quality management plan will be 
developed to describe a site’s quality management. 
 
Quality control (QC) procedures will be implemented beginning with the data entry system and data QC 
checks that will be run on the database will be generated. Any missing data or data anomalies will be 
communicated to the site(s) for clarification/resolution and will be documented in the study database. 
 
Following written Standard Operating Procedures (SOPs), the monitors will verify that the clinical trial is 
conducted and data are generated and biological specimens are collected, documented (recorded), and 
reported in compliance with the protocol, International Conference on Harmonisation Good Clinical 
Practice (ICH GCP), and applicable regulatory requirements (e.g., Good Laboratory Practices (GLP), Good 
Manufacturing Practices (GMP)).  
 
The investigational site will provide direct access to all trial related sites, source data/documents, and 
reports for the purpose of monitoring and auditing by the sponsor, and inspection by local and 
regulatory authorities. 
 
The reasons for missing data values will be investigated and documented in the study database. 
 
 
10.1.9 DATA HANDLING AND RECORD KEEPING  
 
10.1.9.1 DATA COLLECTION AND MANAGEMENT RESPONSIBILITIES  
Data collection is the responsibility of the clinical trial staff at the site under the supervision of the site 
investigator. The investigator is responsible for ensuring the accuracy, completeness, legibility, and 
timeliness of the data reported. 
 
All source documents should be completed in a neat, legible manner to ensure accurate interpretation 
of data.   
 
Hardcopies of the study visit worksheets will be provided for use as source document worksheets for 
recording data for each participant enrolled in the study.  Data recorded in the electronic case report 
form (eCRF) derived from source documents should be consistent with the data recorded on the source 
documents.  
 
Clinical data (including adverse events (AEs), concomitant medications, and expected adverse reactions 
data) and clinical laboratory data will be entered into a study-specific Castor (Research Electronic Data 
Capture) database (www.castoredc.com). The data system includes password protection and internal 
quality checks, such as automatic range checks, to identify data that appear inconsistent, incomplete, or 
inaccurate. Clinical data will be entered directly from the source documents. 
 
 
 

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10.1.9.2 STUDY RECORDS RETENTION  
 
The sponsor will retain and maintain all record pertaining to the clinical trial for a period of 25 years [as 
per C.05.012 (4) of the FDR] 
 
10.1.10 
PROTOCOL DEVIATIONS  
A protocol deviation is any noncompliance with the clinical trial protocol, International Conference on 
Harmonisation Good Clinical Practice (ICH GCP), or Manual of Procedures (MOP) requirements. The 
noncompliance may be either on the part of the participant, the investigator, or the study site staff. As a 
result of deviations, corrective actions are to be developed by the site and implemented promptly.  
 
These practices are consistent with ICH GCP:  
• 
4.5 Compliance with Protocol, sections 4.5.1, 4.5.2, and 4.5.3  
• 
5.1 Quality Assurance and Quality Control, section 5.1.1  
• 
5.20 Noncompliance, sections 5.20.1, and 5.20.2.  
 
It is the responsibility of the site investigator to use continuous vigilance to identify and report 
deviations within 30 working days of identification of the protocol deviation, or within 5 working days of 
the scheduled protocol-required activity.  All deviations must be addressed in study source documents, 
reported to Crohn’s & Colitis Foundation Program Official and Dr. Johan Van Limbergen.  Protocol 
deviations must be sent to the reviewing Institutional Review Board (IRB) per their policies. The site 
investigator is responsible for knowing and adhering to the reviewing IRB requirements. Further details 
about the handling of protocol deviations will be included in the MOP.  
 
 
10.1.11 
PUBLICATION AND DATA SHARING POLICY 
This study will be conducted in accordance with the following publication and data sharing policies and 
regulations: 
 
National Institutes of Health (NIH) Public Access Policy, which ensures that the public has access to the 
published results of NIH funded research. It requires scientists to submit final peer-reviewed journal 
manuscripts that arise from NIH funds to the digital archive PubMed Central upon acceptance for 
publication. 
 
This study will comply with the NIH Data Sharing Policy and Policy on the Dissemination of NIH-Funded 
Clinical Trial Information and the Clinical Trials Registration and Results Information Submission rule. As 
such, this trial will be registered at ClinicalTrials.gov, and results information from this trial will be 
submitted to ClinicalTrials.gov. In addition, every attempt will be made to publish results in peer-
reviewed journals.  Data from this study may be requested by other researchers for 5 years after the 
completion of the primary outcome variable by contacting Dr. Johan Van Limbergen (Amsterdam UMC).  
 
In addition, this study will comply with the NIH Genomic Data Sharing Policy, which applies to all NIH-
funded research that generates large-scale human or non-human genomic data, as well as the use of these 
data for subsequent research. Large-scale data include genome-wide association studies (GWAS), single 
nucleotide polymorphisms (SNP) arrays, and genome sequence, transcriptomic, epigenomic, and gene 
expression data. 
 

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10.1.12 
CONFLICT OF INTEREST POLICY 
 
The independence of this study from any actual or perceived influence, such as by the pharmaceutical 
industry, is critical.  Therefore, any actual conflict of interest of persons who have a role in the design, 
conduct, analysis, publication, or any aspect of this trial will be disclosed and managed. Furthermore, 
persons who have a perceived conflict of interest will be required to have such conflicts managed in a 
way that is appropriate to their participation in the design and conduct of this trial.  The study 
leadership in conjunction with the Crohn’s & Colitis Foundation has established policies and procedures 
for all study group members to disclose all conflicts of interest and will establish a mechanism for the 
management of all reported dualities of interest. 
 
 
 
 
 
 
 
10.2 ADDITIONAL CONSIDERATIONS 
 
 
 

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10.3 ABBREVIATIONS 
 
AE 
Adverse Event 
ALT 
Alanine Transaminase 
ANCOVA 
Analysis of Covariance 
AST 
Aspartate Transaminase 
CD 
Crohn’s Disease 
CFR 
Code of Federal Regulations 
CLIA 
Clinical Laboratory Improvement Amendments 
CMP 
Clinical Monitoring Plan 
COC 
Certificate of Confidentiality 
CONSORT 
Consolidated Standards of Reporting Trials 
CRF 
Case Report Form 
CRP 
C-reactive Protein 
DCC 
Data Coordinating Center 
DHHS 
Department of Health and Human Services 
DSMB 
Data Safety Monitoring Board 
DRE 
Disease-Related Event 
EC 
Ethics Committee 
ECG 
Electrocardiogram 
eCRF 
Electronic Case Report Forms 
EEN 
Exclusive Enteral Nutrition 
ESR 
Erythrocyte sedimentation rate 
FCP 
Fecal calprotectin 
FDR 
Food and Drug Regulations 
FFR 
Federal Financial Report 
GCP 
Good Clinical Practice 
GLP 
Good Laboratory Practices 
GMP 
Good Manufacturing Practices 
GWAS 
Genome-Wide Association Studies 
HIPAA 
Health Insurance Portability and Accountability Act  
HRQOL 
Health-Related Quality of Life 
IB 
Investigator’s Brochure 
IBD 
Inflammatory bowel disease 
ICH 
International Conference on Harmonisation  
ICMJE 
International Committee of Medical Journal Editors 
IDE 
Investigational Device Exemption 
IMPACT 
IBD Quality-of-Life Questionnaire 
IND 
Investigational New Drug Application 
IRB 
Institutional Review Board 
ISM 
Independent Safety Monitor 
ISO 
International Organization for Standardization 
ITT 
Intention-To-Treat 
LSMEANS 
Least-squares Means 
MedDRA 
Medical Dictionary for Regulatory Activities 
MOP 
Manual of Procedures 
MSDS 
Material Safety Data Sheet 
MTX 
Methotrexate 
NCT 
National Clinical Trial 
NIH  
National Institutes of Health 

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NIH IC 
NIH Institute or Center 
OHRP 
Office for Human Research Protections 
PCDAI 
Pediatric Crohn’s Disease Activity Index 
PI 
Principal Investigator 
PRO 
Patient-Reported Outcome 
QA 
Quality Assurance 
QC 
Quality Control 
REDCap 
Research Electronic Data Capture 
SAE 
Serious Adverse Event 
SAP 
Statistical Analysis Plan 
SMC 
Safety Monitoring Committee 
SOA 
Schedule of Activities 
SOC 
Standard of Care 
SOP 
Standard Operating Procedure 
TNF 
Tumor Necrosis Factor 
UP 
Unanticipated Problem 
US 
United States 
 
 
 

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10.4 PROTOCOL AMENDMENT HISTORY 
 
Version 
Date 
Description of Change  
Brief Rationale 
 
August 2019 
Only CDED+PEN as induction therapy 
Open label antibiotics 
(azithromycin/metronidazole) if not in 
clinical remission by week 4 
Publication of CDED RCT and 
reduction of patients in pilot study 
to 20 
Addendum  
October 2020 
Feasibility study clarification 
Required by National Ethics 
Review Board in the Netherlands 
October 2020 
1.8 
 
March 2021 
Adding study timepoint window of +/- 5 
days in case needed 
 
To facilitate planning of study 
visits 
1.8 
 
March 2021 
Changed IWK health centre to 
Amsterdam UMC, location AMC as 
principal receiver of trial data and 
biological samples 
 
PI moved to Amsterdam and trial 
will be coordinated from 
Amsterdam UMC, location AMC 
1.8 
 
March 2021 
Updated participating sites  
North Carolina Children’s Hospital 
(US), Sheba Medical Centre 
(Israel), Location VUMC of 
Amsterdam UMC (The 
Netherlands) will no longer be 
participating. 
1.8 
 
March 2021 
Changed medical monitor to Prof. 
Anthony Otley 
Prof. Antony Otley (local PI IWK 
Canada) will be the new medical 
monitor as former medical monitor 
prof. Francisco Sylvester (UNC) 
will no longer be involved in the 
study.  
1.8 
May 2021 
Updated trial monitor name to Alimentiv 
B.V. 
 
Alimentiv B.V. is the new name of 
the PAZAZ trial monitor, formerly 
known as Robarts Clinical Trial 
Inc. 
Addendum 
May 2021 
Specifying chosen medicinal products 
for the AmsterdamUMC. 
Metronidazole 500 mg, 
Metronidazole 250 mg, 
Azitromycin 250 mg will be the 
medicinal products used in the 
AmsterdamUMC. 
1.9 
October 2021 
Use CastorEDC randomization function 
instead of an additional separate program 
(SPSS) 
To use only one program 
(CastorEDC) as eCRF and 
randomization tool to minimize 
mistakes   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Diet and Partial Enteral Nutrition (CDED+ PEN) vs Exclusive Enteral Nutrition (EEN)-Microbiome Changes of a 
Randomized Clinical Trial (RCT) in Pediatric CD: Remission is Associated with Similar Structural and Functional 
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Dunn KA, Moore-Connors J, MacIntyre B, et al. The Gut Microbiome of Pediatric Crohn's Disease Patients 
Differs from Healthy Controls in Genes That Can Influence the Balance Between a Healthy and Dysregulated 
Immune Response. Inflammatory bowel diseases 2016;22:2607-18. 
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Dunn KA, Moore-Connors J, MacIntyre B, et al. Early Changes in Microbial Community Structure Are 
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diseases 2016;22:2853-62. 
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the gut microbiome in pediatric Crohn’s disease. Cell host & microbe 2015;18:489-500. 
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maintenance of remission in Crohn's disease. The Cochrane Library 2017. 
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the induction of remission in mild to moderate paediatric Crohn's disease : a randomised controlled trial. Gut 2018. 
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Sustained Remission in a Randomized Controlled Trial. Gastroenterology 2019;157:440-50.e8. 
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disease. The American journal of gastroenterology 2000;95:725-9. 
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Wang X, Cai Y, Xing H, et al. Increased therapeutic efficacy of combination of azithromycin and 
ceftazidime on Pseudomonas aeruginosa biofilm in an animal model of ureteral stent infection. BMC microbiology 
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remission following nutritional treatment of Pediatric Crohn' Disease. Inflammatory Bowel Diseases 2016;22:2853-
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multicenter propensity score study. Gut 2015;64:1898-904 
 
12 ADDENDUM FOR THE NETHERLANDS (OCTOBER 2020, UPD. MAY 2021) 
This addendum contains specific information regarding the pilot trial of the PAZAZ study and additional 
information for The Netherlands.  
PAZAZ PILOT STUDY 
Aim 
The aim of this pilot study is feasibility for the protocol of a multinational microbiome-randomisation trial in which 
early microbiome diagnostic procedure helps with personalizing treatment in children with mild to moderate CD.  
Rationale for Pilot study 
We want to investigate the workability of a protocol with specific timeframes for analysis that have to be met in 
order to conduct the trial as described. This is especially important for logistics concerning baseline stool sample 
analysis, in which baseline stool samples have to be sent from every site to Halifax (Canada) to be analyzed and 
interpreted by a central analyst. Results have to be available at week 4 at the local study centers in order to 
perform correct treatment allocation.  
Objectives and outcome variables 
Adapted from section 3 
Objectives 
Outcome variables 
Justification of outcome 
variables and remarks 
Primary 
To evaluate the feasibility of a 
multicenter trial on different 
continents with treatment allocation 
at week 4 depending on stool sample 
results at baseline 
Proportion of subjects that are 
successfully randomized in 
randomization procedure, 
proportion of patients per 
treatment arm, proportion of 
subjects that complete 1 year 
endpoint  
For a potential RCT on full-scale we 
first need to investigate whether 
the protocol is feasible in terms of 
logistics and randomisation plan.  
To evaluate the potential efficacy of 
personalized adjunctive antibiotic 
therapy in maintaining clinical 
remission in pediatric subjects 
undergoing SOC induction therapy 
for mild to moderate Crohn’s disease 
who have a relapse-associated 
microbiome profile  
 
Proportion of subjects in 
sustained remission at 52 weeks 
after starting standard of care 
induction therapy  
 
Sustained remission is defined 
PCDAI less than or equal to 10 
AND no need of re-induction for 
clinical flare (new course of EEN, 
We will aim to demonstrate 
superiority of adjunctive antibiotic 
therapy + SOC over SOC alone in 
patients with a relapse-associated 
microbiome. Group A1 and A2 will 
be compared and sustained 
remission is the principal analysis 
variable.  
 

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need to restart steroids), no 
steroid dependence, no biologic 
(anti-TNF) use, and no intestinal 
surgery by 12 months.  
We understand that the number of 
patients recruited is not enough to 
draw conclusions regarding 
efficacy of the interventions. The 
protocol was written to meet the 
requirements of clear primary and 
secondary endpoints with a 
detailed analytical plan after 
scientific review and subsequent 
approval by the Institutional 
Review Board of the University of 
North Carolina.  
Exploratory 
To evaluate the potential efficacy of 
personalized adjunctive antibiotic 
therapy in improving PRO, 
components of established disease 
activity measures in remission, as 
well as ‘biochemical’ remission in 
pediatric subjects undergoing SOC 
induction therapy for mild to 
moderate Crohn’s disease who have 
a relapse-associated microbiome 
profile  
Longitudinal (clustered and with 
respect to baseline) changes in 
disease activity indices (PCDAI) 
components, and inflammatory 
markers in blood and stool:  
 PCDAI score (0-100)  
 Normalization of CRP (mg/dL)  
 Fecal calprotectin (FCP) 
(microgram/g)  
 
Longitudinal (clustered and with 
respect to baseline) changes PRO:  
 IMPACT (self-report, ages 9-17 
years)  
 
Multiple secondary variables are 
needed to describe clinically 
relevant treatment benefits. 
Depending on the results of the 
microbiome analysis, either groups 
A2 and B will be pooled and 
compared with group A1 or groups 
A1 and B will be pooled and 
compared with group A2 (section 
9.4.3).  
To investigate relationship between 
changes in subject microbiome 
composition and changes in disease 
activity over time.  
Longitudinal (clustered and with 
respect to baseline) changes in 
fecal microbiome taxonomic 
composition or in total gene 
(metagenome) content. The 
changes will be analyzed for 
association with changes in 
disease activity (e.g. relapse or 
sustained remission) over time.  
These changes are exploratory and 
not intended to demonstrate 
clinical benefit of treatment  
Design 
This is a 52-week, multicenter, randomized, controlled, open-label add-on design clinical trial pilot trial to test the 
feasibility of a larger trial evaluating the efficacy of adjuvant antibiotic therapy in pediatric patients with mild to 
moderate CD. This study will not be blinded.  
A total of 20 subjects between the ages of 3 and 17 years inclusive will be enrolled in eight investigational sites 
located in Canada, the United States, the Netherlands and Israel.  
For a full description of the study design see section 4.1 and 4.2 of this protocol. 
Analyses 

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Feasibility will be assessed by the proportion (relative to the number of enrolled patients at visit 2 - week 0)  of 
patients that are successfully randomized in randomization procedure following microbiome analysis, proportions 
of patients per treatment arm, proportion of subjects that complete 1 year endpoint. 
Description of analysis is described in section 9.4. 
 
 
Development of full scale trial 
If results from this pilot trial show adequate feasibility of the protocol, there will be no substantial changes to the 
protocol before resubmitting this protocol for approval for a full scale trial. If this will be the case, then data of 
subjects included in the pilot trial can also be included in the full scale trial. 
If results from subject’s baseline stool sample are not available at randomization (week4), we will have to redesign 
the randomization timepoint. Also, if proportions of subjects within treatment arms vary strongly from expected, 
we will have to redesign the study procedure.    
Additional remarks 
This protocol was approved by IWK Health Centre (Halifax, Canada), North Carolina Children’s Hospital (Chapel Hill, 
North Carolina, USA), Wolfson Medical Centre (Tel Aviv, Israel), Sheba Medical Centre (Tel Aviv, Israel).  The FDA 
(USA), Health Canada and Ministry of Health (Israel) have reviewed the protocol and approved it.  
 
The scientific review board of the University of North Carolina requested changes in the protocol to be made based 
on a full scale trial (endpoints, power analysis, statistical analysis).  
 
SPECIFIC INFORMATION FOR THE NETHERLANDS 
Study procedures 
A window of study timepoint +/- 5 days is allowed for all visits/questionnaires/sampling. 
Data sharing and storage 
Data of participants will be stored until 25 years after the end of the study. Adequate and accurate records of each 
study visit will be maintained to enable the conduct of the study to be fully documented.  
Paperwork as needed in the trial master file (TMF) concerning the participants are stored in a locked office at the 
Amsterdam University Medical Centre (AUMC), location AMC. A database with coded information and examination 
results will be maintained and stored on the research server of the Amsterdam University Medical Centre. The key 
for identifying the data is saved on a different server at the Amsterdam University Medical Centre. 
Only pseudonymised information and material will be shared between study sites and the AMC and/or other 
centers where patient material will be analysed within the study.  

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Recruitment and Informed consent 
Participants attending the outpatient clinic of the Amsterdam UMC, location AMC and VUmc, whom are suspected 
of or have been diagnosed with CD will be eligible to participate in this study. The treating physician  will inform 
parents and children about the study when they visit the outpatient clinic of the Amsterdam UMC. A member of 
the research team will provide more detailed information about the study and will give the information leaflet and 
informed consent form. Children and parents are then asked for their signed informed consent in order to be able 
to participate in the study. Informed consent will be obtained from the participant (depending on the participants 
age) and their legal representatives conform WGBO article 7. After counseling, informed consent (a copy of the 
signed informed consent form is given to the participants and this is noted in the file) is signed by both children 
and parents (if child is 12 – 15 years old). In daily practice, the subject is often joined by one parent. The other 
parent is allowed to sign at home before or after the visit. As long as both signatures are collected, it is acceptable. 
In situations where only one parent/legal guardian is known or if one parent is deceased, the absence of the 
second signature is  explained on a Note-to-File. A copy of the signed informed consent form is given to the 
participant. 
The local investigator of each participating centre will maintain a list of appropriately qualified persons to whom 
he/she has delegated significant trial-related tasks. 
Objection by minors or incapacitated subjects  
This study will follow the conduct code concerning resistance in minors who participate in clinical trials as defined 
by the Dutch Paediatric Society. Informed consent will be asked from parents/guardians of children < 12 years. In 
children/adolescents >= 12 years informed consent will be asked from the parents-guardians and the 
children/adolescents. 
Medicinal products 
In the AmsterdamUMC, preparation, packaging and labeling of medicinal products will be done by the hospital’s 
Trial Pharmacy. The choice of medicinal products will be: Metronidazole 500 mg, Metronidazole 250 mg, 
Azitromycin 250 mg.