The Effect of Riboflavin Supplementation on Faecalibacterium Prausnitzii in Crohn's Disease
Phase: NA
Conditions: Crohn Disease
Sponsor: University Medical Center Groningen

 
 
 
RESEARCH PROTOCOL RISE-UP study 
 
The effect of Riboflavin 
SupplemEntation on FaecalibacteriUm 
Prausnitzii in Crohn’s disease 
RISE-UP Study 
Protocol ID: NL-48186.042.14 
 March 2016 
 
 

The effect of riboflavin supplementation on Faecalibacterium prausnitzii in Crohn’s 
disease 
RISE-UP Study – UMCG 
March 2016 – Version 4 
 
 
3 
 
PROTOCOL TITLE: The effect of riboflavin supplementation on Faecalibacterium 
prausnitzii in Crohn’s disease  
 
 
Protocol ID 
CCMO: NL-48186.042.14 
 
Short title 
Riboflavin in Crohn’s disease 
 
Title on information 
for subjects sheet 
Het effect van een vitamine B2 supplement op de 
ziekte van Crohn 
 
Version 
4.0 
 
Date 
Maart 2016 
 
Coordinating investigator/project 
leader 
 
 
 
 
 
 
Main coordinating investigator 
Prof. dr. G. Dijkstra, gastroenterologist 
Department Gastroenterology and Hepatology 
UMC Groningen 
 
Dr. H.J.M. Harmsen, microbiologist 
Medical Microbiology  
UMC Groningen 
 
Drs. J.Z.H. von Martels, PhD-candidate 
Department Gastroenterology and Hepatology 
UMC Groningen  
Hanzeplein 1 
9713 GZ Groningen 
The Netherlands 
E-mail: j.z.h.von.martels@umcg.nl 
 
Principal investigator (in Dutch: 
hoofdonderzoeker/ uitvoerder) 
Prof. dr. G. Dijkstra, gastroenterologist 
Department Gastroenterology and Hepatology 
UMC Groningen 
Room: S.3.133; internal postal code BB41 
Hanzeplein 1 
9713 GZ Groningen 
The Netherlands 
Tel: +31-50-3612620 (19968) 
Fax +31-50-3619306 
E-mail: gerard.dijkstra@umcg.nl 
 
Sponsor (in Dutch: 
Investigator initiated research 
heeft opmaak toegepast: Nederlands
heeft opmaak toegepast: Engels (V.S.)
heeft opmaak toegepast: Nederlands
heeft opmaak toegepast: Nederlands
heeft opmaak toegepast: Nederlands

The effect of riboflavin supplementation on Faecalibacterium prausnitzii in Crohn’s 
disease 
RISE-UP Study – UMCG 
March 2016 – Version 4 
 
 
4 
verrichter/opdrachtgever) 
UMC Groningen 
Hanzeplein 1 
9713 GZ Groningen 
The Netherlands 
 
Subsidising party 
Not applicable 
 
Independent expert 
Dr. J.J. Koornstra, gastroenterologist 
Department Gastroenterology and Hepatology  
UMC Groningen 
Room: T3.234 
Hanzeplein 1 
9713 GZ Groningen 
The Netherlands 
Tel: +31-50-3612620 (12823) 
Fax +31-50-3619306 
Email: j.j.koornsta@med.umcg.nl 
 
Laboratory sites  
Laboratory dr. H.J.M. Harmsen, microbiologist 
Medical Microbiology  
UMC Groningen 
Hanzeplein 1 
9713 GZ Groningen 
Tel: 050-361 3480 
 
Laboratory prof. dr. G. Dijkstra, gastroenterologist 
Gastroenterology  
UMC Groningen   
Hanzeplein 1 
9713 GZ Groningen 
Tel: 050-3612620 (19968) 
 
Department of Laboratory medicine  
UMC Groningen 
Hanzeplein 1 
9700 RB Groningen 
Tel: 050-3611576 
 
Pharmacy  
 
Department of Clinical Pharmacy 
UMC Groningen 
Secretary 3.065 ‘De Brug’ 
Tel: 050-3614087 
 
heeft opmaak toegepast: Nederlands
heeft opmaak toegepast: Nederlands

The effect of riboflavin supplementation on Faecalibacterium prausnitzii in Crohn’s 
disease 
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PROTOCOL SIGNATURE SHEET 
 
 
Name 
Signature 
Date 
Main coordinating investigator: 
  Drs. J.Z.H. von Martels, PhD-candidate 
  Department Gastroenterology and Hepatology 
  UMCG 
 
 
Principal investigator: 
Prof. dr. G. Dijkstra, gastroenterologist 
Department Gastroenterology and Hepatology 
UMCG 
 
 
Head of Department: 
Prof. dr. J.H. Kleibeuker, gastroenterologist 
Department of Gastroenterology and Hepatology 
UMCG 
 
 
 
 
 
 
 

The effect of riboflavin supplementation on Faecalibacterium prausnitzii in Crohn’s 
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TABLE OF CONTENTS 
 
1. 
INTRODUCTION AND RATIONALE ............................................................................... 11 
2. 
OBJECTIVES .................................................................................................................. 14 
3. 
STUDY DESIGN ............................................................................................................. 14 
4. 
STUDY POPULATION .................................................................................................... 17 
4.1 
Population (base) ................................................................................................... 17 
4.2 
Inclusion criteria ...................................................................................................... 17 
4.3 
Exclusion criteria .................................................................................................... 18 
4.4 
Sample size calculation .......................................................................................... 18 
5. 
TREATMENT OF SUBJECTS ........................................................................................ 20 
5.1 
Investigational product/treatment ........................................................................... 20 
5.2 
Use of co-intervention (if applicable) ...................................................................... 20 
5.3 
Escape medication (if applicable) ........................................................................... 20 
6. 
INVESTIGATIONAL PRODUCT ..................................................................................... 20 
6.1 
Name and description of investigational  product(s) ............................................... 20 
6.2 
Summary of findings from non-clinical studies ....................................................... 20 
6.3 
Summary of findings from clinical studies .............................................................. 20 
6.4 
Summary of known and potential risks and benefits .............................................. 20 
6.5 
Description and justification of route of administration and dosage ....................... 21 
6.6 
Dosages, dosage modifications and method of administration .............................. 21 
6.7 
Preparation and labelling of Investigational Medicinal Product .............................. 21 
6.8 
Drug accountability ................................................................................................. 21 
7. 
NON-INVESTIGATIONAL PRODUCT ............................................................................ 21 
7.1 
Name and description of non-investigational product(s) ........................................ 21 
7.2 
Summary of findings from non-clinical studies ....................................................... 21 
7.3 
Summary of findings from clinical studies .............................................................. 21 
7.4 
Summary of known and potential risks and benefits .............................................. 22 
7.5 
Description and justification of route of administration and dosage ....................... 23 
7.6 
Dosages, dosage modifications and method of administration .............................. 23 
7.7 
Preparation and labelling of Non Investigational Medicinal Product ....................... 23 
7.8 
Drug accountability ................................................................................................. 24 
8. 
METHODS ...................................................................................................................... 24 
8.1 
Study parameters/endpoints ................................................................................... 24 
8.1.1 
Main study parameter/endpoint .......................................................................... 24 
8.1.2 
Secondary study parameters/endpoints (if applicable) ....................................... 24 
8.1.3 
Other study parameters (if applicable) ............................................................... 25 
8.2 
Randomisation, blinding and treatment allocation .................................................. 25 
8.3 
Study procedures ................................................................................................... 25 
8.4 
Withdrawal of individual subjects ............................................................................ 27 
8.4.1 
Specific criteria for withdrawal (if applicable) ...................................................... 27 
8.5 
Replacement of individual subjects after withdrawal .............................................. 27 
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8.6 
Follow-up of subjects withdrawn from treatment .................................................... 28 
8.7 
Premature termination of the study ........................................................................ 28 
9. 
SAFETY REPORTING .................................................................................................... 28 
9.1 
Section 10 WMO event ........................................................................................... 28 
9.2 
AEs, SAEs and SUSARs ........................................................................................ 28 
9.2.1 
Adverse events (AEs) ......................................................................................... 28 
9.2.2 
Serious adverse events (SAEs) .......................................................................... 29 
9.2.3 
Suspected unexpected serious adverse reactions (SUSARs) ........................... 29 
9.3 
Annual safety report ............................................................................................... 30 
9.4 
Follow-up of adverse events ................................................................................... 31 
9.5 
Data Safety Monitoring Board (DSMB) ................................................................... 31 
10. 
STATISTICAL ANALYSIS .......................................................................................... 31 
10.1 
Primary study parameter(s) .................................................................................... 31 
10.2 
Secondary study parameter(s) ............................................................................... 31 
10.3 
Other study parameters .......................................................................................... 32 
10.4 
Interim analysis (if applicable) ................................................................................ 32 
11. 
ETHICAL CONSIDERATIONS ................................................................................... 32 
11.1 
Regulation statement .............................................................................................. 32 
11.2 
Recruitment and consent ........................................................................................ 32 
11.3 
Objection by minors or incapacitated subjects (if applicable) ................................. 33 
11.4 
Benefits and risks assessment, group relatedness ................................................ 33 
11.5 
Compensation for injury .......................................................................................... 33 
11.6 
Incentives (if applicable) ......................................................................................... 34 
12. 
ADMINISTRATIVE ASPECTS, MONITORING AND PUBLICATION ......................... 34 
12.1 
Handling and storage of data and documents ........................................................ 34 
12.2 
Monitoring and Quality Assurance .......................................................................... 34 
12.3 
Amendments .......................................................................................................... 35 
12.4 
Annual progress report ........................................................................................... 35 
12.5 
End of study report ................................................................................................. 35 
12.6 
Public disclosure and publication policy ................................................................. 35 
13. 
STRUCTURED RISK ANALYSIS ............................................................................... 36 
13.1 
Potential issues of concern ..................................................................................... 36 
13.2 
Synthesis ................................................................................................................ 36 
14. 
REFERENCES ........................................................................................................... 41 
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LIST OF ABBREVIATIONS AND RELEVANT DEFINITIONS  
 
ABR 
ABR form, General Assessment and Registration form, is the application 
form that is required for submission to the accredited Ethics Committee 
(In Dutch, ABR = Algemene Beoordeling en Registratie) 
AE 
Adverse Event 
AR 
Adverse Reaction 
CA 
Competent Authority 
CCMO 
Central Committee on Research Involving Human Subjects; in Dutch: 
Centrale Commissie Mensgebonden Onderzoek 
DSMB 
Data Safety Monitoring Board 
EudraCT 
European drug regulatory affairs Clinical Trials  
GCP 
Good Clinical Practice 
IB 
Investigator’s Brochure 
IC 
Informed Consent 
IMP 
Investigational Medicinal Product  
IMPD 
Investigational Medicinal Product Dossier  
METc  
Medical research ethics committee (MREC); in Dutch: Medisch Ethische 
Toetsing Commissie (METc) 
(S)AE 
(Serious) Adverse Event  
SPC 
Summary of Product Characteristics (in Dutch: officiële productinfomatie 
IB1-tekst) 
Sponsor 
The sponsor is the party that commissions the organisation or 
performance of the research, for example a pharmaceutical 
company, academic hospital, scientific organisation or investigator. A 
party that provides funding for a study but does not commission it is not 
regarded as the sponsor, but referred to as a subsidising party. 
SUSAR 
Suspected Unexpected Serious Adverse Reaction 
Wbp 
Personal Data Protection Act (in Dutch: Wet Bescherming 
Persoonsgevens) 
WMO 
Medical Research Involving Human Subjects Act (in Dutch: Wet Medisch-
wetenschappelijk Onderzoek met mensen 
IBD 
Inflammatory Bowel Disease 
F. 
prausnitzii 
Faecalibacterium prausnitzii 
M. Crohn 
Morbus Crohn 
E. coli 
FISH 
Escherichia coli 
fluorescent in situ hybridization 
UMCG 
University Medical Center Groningen 
SCFAs 
Short Chain Fatty Acids 

The effect of riboflavin supplementation on Faecalibacterium prausnitzii in Crohn’s 
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SUMMARY 
 
Rationale 
Recent studies show that in patients with Inflammatory Bowel Disease (IBD) a dysbiosis 
exists in the composition of the intestinal microbiota. In particular, the potentially pathogenic 
bacterium Escherichia coli (E. coli) is often more abundant in the bowel of IBD patients, and 
the anaerobic commensal Faecalibacterium prausnitzii (F. prausnitzii) is often reduced. This 
last mentioned bacteria is known to be abundant in the intestine of healthy individuals. It is 
known to produce butyrate, which stimulates the intestinal epithelium, and to secrete anti-
inflammatory substances.  
Riboflavin – also known as vitamin B2 – is required for a wide variety of cellular processes 
and has an important role in maintaining health in humans. In a pilot intervention with healthy 
volunteers it is shown that a riboflavin supplement increases the number of F. prausnitzii and 
results in a higher production of butyrate. In Crohn’s disease patients, it is known that the 
amount of F. prausnitzii in the intestine is generally low. Furthermore, it is known that there is 
an association between the number of F. prausnitzii bacteria and the length of disease in 
remission.  
 
This study will evaluate if supplementation of the diet with riboflavin in Crohn’s disease 
patients will result in a similar increase in the amount of F. prausnitzii as in healthy 
volunteers. In this patient group, an increase in the number of F. prausnitzii bacteria in the 
bowel may result in a more favourable disease course. This will be assessed with faeces 
calprotectin and two questionnaires. Additionally we will assess if there is any modulation by 
riboflavin on the other intestinal bacteria, short chain fatty acids (SCFAs) (such as butyrate), 
and the pH of the faeces. Finally, the effect of the riboflavin on the permeability of the gut will 
be evaluated with a Chroom-EDTA test, and a number of different biomarkers of 
permeability. 
 
Hypothesis 
The hypothesis is that in Crohn’s disease patients, supplementation of the diet with riboflavin 
results in an increase in the amount of F. prausnitzii, changes in microbial composition, 
increased fatty acid production, an increase in pH and a reduction of intestinal permeability. 
These changes might result in a more favourable disease course with less exacerbations. 
 
Study design 
Prospective clinical study.  
 
Study population and sample size 
In total 84 Crohn’s disease patients will be included in this study, divided into two groups. 
Group 1 (n=42) will consist of patients with disease in remission (quiescent disease); group 2 
(n=42) will consist of patients with active disease. In this study an adaptive design will be 

The effect of riboflavin supplementation on Faecalibacterium prausnitzii in Crohn’s 
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used. First 12 patients in the disease in remission group will be analysed. The methods of 
analysis and safety aspects will be taken into account. 
 
Intervention  
Supplementation of the normal diet with 1 capsule of 100 mg riboflavin (vitamin B2) during 
three weeks. 
 
Main study parameters 
Primary Objective:  
1). To investigate the effect of a riboflavin supplement on the number of F. prausnitzii 
bacteria in the faeces of active and quiescent Crohn’s disease patients. 
 
Secondary Objectives:  
1). To evaluate the effect of a riboflavin supplement on the bacterial composition in the 
faeces of active and quiescent Crohn’s disease patients. 
2). To evaluate the effect of a riboflavin supplement on the production of short chain fatty 
acids (SCFAs) in the faeces of active and quiescent Crohn’s disease patients. 
3). To assess the effect of the riboflavin intervention on the disease severity (Harvey 
Bradshaw Index, IBD-Q and faeces Calprotectin) of active and quiescent Crohn’s disease 
patients. 
4). To evaluate the effect of the riboflavin intervention on permeability of the gut in active and 
quiescent Crohn’s disease patients (Chroom-EDTA test, and different biomarkers of 
permeability). 
5). To determine the effect of a riboflavin supplement on the faecal pH in active and quiescent 
Crohn’s disease patients. 
6). To evaluate whether riboflavin intervention leads to an increase in electrical current in the 
faeces, as a result of an increase in extracellular electron shuttling bacteria. 
 
Nature and extent of the burden and the risks associated with participation, benefit 
and group relatedness 
Participating in this study has a potential health benefit. It is known that in healthy subjects, 
the supplement riboflavin increases the amount of beneficial bacteria. In Crohn’s disease 
there is often a dysbiosis of the bacterial composition, and the beneficial bacteria are 
depleted. When a similar increase in the beneficial bacteria occurs in Crohn’s disease 
patients as seen in healthy volunteers, this may result in a more favourable disease outcome 
(staying in remission longer). Riboflavin is freely available, and commonly sold in health 
shops. There is no need for a prescription to buy this supplement, and its use is considered 
to be safe. The riboflavin supplement may give a (completely innocent) yellow discoloration 
of the urine several hours after ingestion. There is no need to discontinue the riboflavin 
supplementation. The adverse event of discoloration of urine is only temporary.  
 

The effect of riboflavin supplementation on Faecalibacterium prausnitzii in Crohn’s 
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1. INTRODUCTION AND RATIONALE 
 
Rationale 
 
Bacterial dysbiosis in IBD 
The most common types of Inflammatory Bowel disease (IBD) are M. Crohn and Ulcerative 
Colitis (UC). Although there are many differences between these diseases, both are 
characterized by an altered composition of the gut microbiome. (1)  
 
Microbiota generally play an important physiological role in the digestion of nutrients. 
However, in patients with a particular genetic predisposition it has been suggested that an 
aberrant immune response against the intestinal microbiota takes place. (2) (3) We have 
previously shown that specific bacteria are essential in developing an experimental colitis 
(model of an experimental colitis). (4) In Crohn's disease it appears that pro-inflammatory 
and anti-inflammatory microbiota are out of balance. Recently we showed that this imbalance 
is also responsible for developing a humoral immune response against the own gut bacteria 
as is demonstrated by IgG coating of bacteria. (5) From a clinical practice it has long been 
known that an elemental diet can achieve remission of disease activity. (6) The exact 
mechanism that underlies this finding is yet to be discovered. We believe that this elemental diet 
changes the bacterial composition in the intestine and restores the dysbiosis.  
 
The dysbiosis as seen in IBD is characterized by an increase in the number of potentially 
pathogenic bacterium Escherichia coli (E. coli) and a reduction of the anaerobic gram-
positive commensals in the intestine (such as bacteria belonging to the Clostridium groups IV 
en XIVa). (7) (5) (8) (9) (10) These Clostridium groups are bacteria that convert complex 
carbohydrates and sugars into fatty acids such as butyrate. One of the bacteria of the 
Clostridium group IV (and the phylum of Firmicutes) is Faecalibacterium prausnitzii (F. 
prausnitzii), a bacterium that generally forms around 10% of our gut microbiota and occurs in 
almost every human being. (11) 
 
F. prausnitzii can grow on sugars, pectin and degradation products of human mucus. From 
glucose and acetate it produces in particular butyrate, which stimulates the intestinal 
epithelium by influencing regeneration and gene expression, stimulating mucus production 
and by reducing the permeability of the gut. (12) Butyrate is also the main source of energy 
for the colonocytes in the intestine. Butyrate belongs to the group of short chain fatty acids 
(SCFAs). SCFAs are produced as a product of microbial fermentation of indigestible 
carbohydrates. (13) (14) (15) Moreover, F. prausnitzii produces soluble substances which 
have an anti-inflammatory effect on the immune system of the intestine. (7) Furthermore, in 
mice it is shown that oral administration of live F. prausnitzii or its supernatant markedly 
reduced the severity of TNBS colitis (murine model for colitis) and tended to correct the 

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dysbiosis associated with TNBS colitis models. (7) Although it is difficult to cultivate F. 
prausnitzii, due to its extreme sensitivity to oxygen, it was recently demonstrated that a 
culture medium of riboflavin and cysteine makes growth of this bacteria at low oxygen levels 
possible. (16)  
 
Riboflavin – also known as vitamin B2 – is required for a wide variety of cellular processes, 
and has an important role in maintaining health in humans. Riboflavin enables the bacterium 
F. prausnitzii to use a form of anaerobic respiration, by use of external electron acceptors 
such as cysteine and oxygen. This results in the growth of F. prausnitzii in vitro at low oxygen 
levels. This may also be the case in the intestinal wall mucosa. (16) In a pilot intervention 
with healthy volunteers it is shown that a riboflavin supplement increases the number of F. 
prausnitzii in the faeces and results in a higher production of butyrate (results yet to be 
published, see Figure 1). It is shown that in Crohn’s disease there is a depletion of vitamin 
B2 level as compared with healthy controls, and that the level of this vitamin showed a 
negative correlation with the Crohn’s disease activity index. (17) Also, in Crohn’s disease 
patients, it is known that the amount of F. prausnitzii in the intestine is generally low. There is 
an association between the number of F. prausnitzii in the bowel and the length of disease in 
remission. (see Figure 2).  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 1: A study in healthy volunteers shows an increase in the number of F. 
prausnitzii in the faeces during riboflavin supplement intake in the majority of the 
subjects.  

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The present study will evaluate if supplementation of the diet with riboflavin in Crohn’s 
disease patients will result in a similar increase in the amount of F. prausnitzii as seen in the 
healthy volunteers. Potentially an even larger effect can occur, because of the low starting 
amount. In this patient group, an increase in the number of F. prausnitzii in the bowel may 
result in a more favourable disease course. (7) A possible protective effect of F. prausnitzii in 
the development of IBD has been suggested in a number of studies. (8) (7) (11) (18) Our 
hypothesis is that supplementation of the diet with riboflavin will result in an increase in the 
amount of F. prausnitzii. We hypothesize that this will be more pronounced in the Crohn’s 
disease patients with disease in remission, than in the group with active disease. This 
because, cytokines and mediators of inflammation might counteract the beneficial effect of 
the riboflavin on F. prausnitzii. Further, we hypothesize that as a result of the increase in F. 
prausnitzii, butyrate production will increase. (8) (18) (19) (20) Butyrate is thought to play a 
beneficial role on reducing the inflammation of the intestinal mucosa; the exact 
mechanism(s) are yet to be defined. (18) We expect the pH of the faeces to increase after 
the riboflavin intervention, since, the faeces pH is shown to be lower in inflammatory 
conditions, and riboflavin might result in a reduction of the inflammation. (21) Also, the 
permeability of the gut will be evaluated with a Chroom-EDTA test, and a number of different 
biomarkers of permeability. A decrease in permeability is hypothesized as a result of reduced 
inflammation. (7) (8) Finally Khan et al demonstrated that F. prausnitzii uses riboflavin for 
electron transfer. In this study it was shown that riboflavin is required for the extracellular 
shuttling of electrons by F. prausnitzii that metabolize glucose anaerobically. We will evaluate 
whether riboflavin intervention leads to an increase in electrical current, as a result of an 
increase in extracellular electron shuttling bacteria. (16) 
 
Previous studies have analysed the effect of different nutritional supplements in Crohn’s disease 
patients. In a group of patients with inactive or mild disease it was shown that a vitamin E and C 
supplement resulted in a significant reduction in oxidative stress. (22) In an article of Ramirez-
Figure 2: F. prausnitzii 
proportions in the ileal mucosa-
associated microbiota using 
FISH at the time of surgery and 
at 6 months according to the 
endoscopic recurrence status. *, 
Significant difference, P = 0.03. 
Sokol H et al. PNAS 
2008;105:16731-16736. 
 

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Farias et al 2008 it is shown that the dietary supplement inulin is able to increase the number 
of F. prausnitzii bacteria in the bowel from 10.3% during the control period to 14.5% during 
inulin intake (increase of 41%) (23). Until now, no study has been performed that evaluated the 
effect of a vitamin B2 supplement on the intestinal microbiota.  
 
In conclusion, the present study will evaluate the effect of the vitamin supplement riboflavin on the 
intestinal bacterial composition in Crohn’s disease patients. Dysbiosis of the intestinal microbiota 
is generally seen in Crohn’s disease. Restoring this dysbiosis may lead to a more favourable 
disease course with less exacerbations.  
 
 
2. OBJECTIVES 
 
Research Goals 
 
Primary Objective:  
1). To investigate the effect of a riboflavin supplement on the number of F. prausnitzii 
bacteria in the faeces of active and quiescent Crohn’s disease patients. 
 
Secondary Objectives:  
1). To evaluate the effect of a riboflavin supplement on the bacterial composition in the 
faeces of active and quiescent Crohn’s disease patients. 
2). To evaluate the effect of a riboflavin supplement on the production of short chain fatty 
acids (SCFAs) in the faeces of active and quiescent Crohn’s disease patients. 
3). To assess the effect of the riboflavin intervention on the disease severity (Harvey 
Bradshaw Index, IBD-Q and faeces Calprotectin) of active and quiescent Crohn’s disease 
patients. 
4). To evaluate the effect of the riboflavin intervention on permeability of the gut in active and 
quiescent Crohn’s disease patients (Chroom-EDTA test, and different biomarkers of 
permeability). 
5). To determine the effect of a riboflavin supplement on the faecal pH in active and quiescent 
Crohn’s disease patients. 
6). To evaluate whether riboflavin intervention leads to an increase in electrical current in the 
faeces, as a result of an increase in extracellular electron shuttling bacteria. 
 
 
3. STUDY DESIGN 
 
Baseline measurements 
At baseline a number of parameters will be assessed: Vital signs (blood pressure, pulse, 
respirations and temperature); weight (in kilograms, without shoes) and height (in cm, without 
shoes). Also a blood sample will be taken prior to the riboflavin intervention. After the three 

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weeks of intervention this blood sample will be repeated. Laboratory analysis will take place 
of a standard set of parameters routinely determined in IBD patients. The blood sample 
consists of: ALAT, ASAT, Alkaline phosphatase, amylase, BSE, CRP, ferritin, GGT, Hb, Ht, 
creatinine, leukocyte count, MCV, trombocyte count, TYBC and iron (9.0 ml of blood (EDTA 
4.0 ml and Lithium Heparine 5.0 ml)). Additionally, both before and after the intervention two 
extra blood tubes (1 plasma and 1 serum tube) will be drawn for storage (2 x 10 ml = 20 ml 
blood). This blood will be used to determine a number of different biomarkers for 
permeability: I-FABP (plasma) and SM-22 (serum). The above means that 29 ml of blood will 
be drawn per time point. Also an urine sample will be collected. Claudin-3, a marker of 
permeability, will be measured in the urine.  
 
 
 
 
 
 
 
 
 
Baseline Wk 1 of 
riboflavin 
intervention  
Wk 2 of 
riboflavin 
intervention 
Wk 3 of 
riboflavin 
intervention 
2 wk after 
the 
intervention 
Informed consent 
 
X 
 
 
 
 
Clinical visit  
 
X 
 
 
X4 
 
HB-index 1 
 
X 
 
 
X4 
 
IBD-Q 
questionnaire 
 
X 
 
 
X4 
 
Bloodsample  
 
X 
 
 
X4 
 
Urinesample  
 
X2 
 
X3 
X4 
X 
Chroom-EDTA test 
 
X 
 
 
X4 
 
Faeces sample 
 
X2 
 
X3 
X4 
X 
Separate faeces 
sample for 
calprotectin 
 
X 
 
 
X4 
 
Table 1: Schedule of activities for participating patients: 1). HB - Index: Harvey Bradshaw Index; 2). 
Faeces and urine sample are taken at two time points prior to the riboflavin intervention. There will be at 
least six days between both time points. 3). Faeces and urine collected on day 10 after starting the 
intervention. 4). These measurements will take place on the last day of the riboflavin intervention. 

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At two time points a Chroom-EDTA permeability test will take place to assess the 
permeability of the gut. The Chroom-EDTA test is used in this hospital before in the (by the 
ethical committee approved) COLIPI clinical trial (‘’Identification of predictive parameters for 
colitis in melanoma patients treated with ipilimumab’’ (METc 2012/085)). Participants will be 
asked to come to the hospital fasted (without breakfast). Prior to drinking the Chroom-EDTA 
a pregnancy test will be performed in the urine of all female participants (dipstick test). When 
the pregnancy test is negative, participants will be asked to drink the Chroom-EDTA during 
the clinical visit. After drinking the Chroom-EDTA participants will remain fasted for an 
additional two hours. Hereafter participants will be asked to collect urine during 24 hours (see 
‘proefpersoneninformatie’ for detailed instructions). Additionally, a Harvey Bradshaw Index 
(HBI) will be taken before and after the riboflavin intervention to quantify the severity of the 
symptoms of the disease, and to evaluate if any clinical relevant improvement occurs (see 
attachment 1). The HBI is based on five items: general wellbeing (0 = very well, 1 = slightly 
below average, 2 = poor, 3 = very poor, 4 = terrible); abdominal pain (0 = none, 1 = mild, 2 = 
moderate, 3 = severe); number of liquid stools per day; abdominal mass (0 = none, 1 = 
dubious, 2 = definite, 3 = tender); and complications (arthralgia, uveitis, erythema nodosum, 
aphthous ulcers, pyoderma gangrenosum, anal fissure, new fistula and abcess 
(complications, score 1 per item)). A total score of less than 5 is generally considered to 
represent clinical remission. A score of 5 – 7 is considered as mild disease, 8 – 16 as 
moderate disease and > 16 as severe disease. (24)  
 
Riboflavin intervention 
The study is designed in such a way that the participants are their own controls (control 
period). After signing the informed consent patients are asked to collect a stool sample to 
measure the amount of Faecalibacterium prausnitzii (F. prausnitzii). After 6 days a second 
sample is requested, where after the intervention with riboflavin can commence. These two 
samples prior to the riboflavin intervention are meant to give a reflection of the amount of F. 
prausnitzii without stimulation and the mean individual degree of fluctuation (control period). 
During the riboflavin intervention period, the participant is asked to take one capsule of 
riboflavin (100 mg) per day. After ten days, and on the final day of taking the supplement 
another stool sample will be requested (see check lists). This to measure the effect of 
riboflavin on the amount of F. prausnitzii. On the second clinical visit patients will be asked to 
bring the riboflavin container. The capsules will be counted as a control to assess if the 
patient has followed the dosing instructions. The riboflavin supplement is then stopped, and 
another stool sample will be requested two weeks after stopping. Furthermore, the changes 
in bacterial composition, and bacterial products (i.e. butyrate) before and after the riboflavin 
intervention will be assessed. The stool samples will be tested on F. prausnitzii and other 
(butyrate producing) bacteria, using fluorescent in situ hybridization (FISH) with specific 
probes. On two days (day 6 and day 28, see checklist) patients will be asked to divide de 
faeces over two containers instead of one. This extra container on these days contains a 
glycerol (20%), cysteine and pbs buffer (10 ml). This is used to preserve the bacteria for 

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analysis of the electrical current and culturing experiments. The concentration of certain fatty 
acids in the stool will be measured. In this butyrate is of particular interest. In addition, a 
portion of the faeces sample will be stored, for microbiome analysis. Microbiota will be 
analysed with pyrosequencing. All findings in the area of intestinal wellbeing, frequency of 
defecation, and the consistency of the faeces will be recorded in a questionnaire on two time 
points (IBD-Q, see F1, ‘vragenlijst IBD-Q’). Also faeces will be collected for analysis of the 
faecal calprotectin. Faecal calprotectin is a neutrophilic protein in faeces, and considered to 
be a useful non-invasive tool, for differentiating IBD from non-organic disease. Also it is 
shown that repeated measurements of faecal calprotectin can predict disease course. (25) 
This measurement will be performed twice: at baseline and on the last day of the riboflavin 
intervention. For the complete schedule of activities and the different time points see Table 1 
and the checklist of the participants. On two timepoints (day 17 and day 42) all samples will 
be collected (either with car) or participants will bring the samples to the hospital themselves. 
 
 
4. STUDY POPULATION 
 
4.1 Population (base)  
The study population consists of 84 participants in total. All participants have been diagnosed 
with Crohn´s disease prior to inclusion. Two groups will be formed based on severity of 
disease. Disease activity will be primarily determined by faecal calprotectin. Participants in the 
first group (group 1) have a low calprotectin and are considered to be in remission. Participants 
in the second group (group 2) have a high calprotectin, and are considered to have (moderate) 
active disease. 42 participants will be included per group. The groups are described in detail 
below (Chapter 4.2). Of all 84 participants, the effect of the riboflavin intervention will be 
evaluated by analysing different samples at different time points (blood, urine, faeces, and a 
clinical severity disease score (HB-index) and the validated IBD-Q questionnaire).  
 
4.2 Inclusion criteria 
 
Group 1 (n=42) 
- Patients diagnosed with Crohn´s disease in remission (calprotectin < 200 µg/g) 
- Age 18-65 years 
 
Group 2 (n=42) 
- Patients diagnosed with Crohn´s disease with active disease (calprotectin > 200 µg/g) 
- Age 18-65 years 
 
Concomitant medication for Crohn’s disease is allowed in all groups.  
 

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4.3 Exclusion criteria 
A potential subject who meets any of the following criteria will be excluded from participation 
in this study: 
 
- Swallowing disorders 
- Pregnancy and lactation 
- Use of antibiotic drugs, probiotics (i.e.Yakult, Vifit, Activia etc) or specific prebiotic 
supplements in the 3 weeks prior to the riboflavin intervention (for a list of probiotic, prebiotic 
and other supplements see attachment 2) 
- Use of Methotrexate drugs 
- Colonoscopy and colon cleansing in last 3 months (26) 
- Use of a vitamin B2 supplement, or multivitamin complexes containing vitamin B (i.e. 
vitamin B-complex) in the 3 weeks prior to the riboflavin intervention 
- Severe Crohn’s disease (HBI > 12) 
 
4.4 Sample size calculation 
The primary objective in this study is to investigate whether the number of F. prausnitzii 
increases as result of a riboflavin intervention in patients with Crohn’s disease. A power 
calculation has been performed (dr. I.M. Nolte). First the mean increase in F. prausnitzii as 
seen in the healthy volunteer riboflavin pilot study was calculated. In this study bacteria were 
measured per grams of faeces. The amount of bacteria were measured with FISH. The 
mean increase was 6.14 10^8 bacteria per gram of faeces per volunteer (SD: 1.56 10^9). In 
an article of Swidsinski et al 2008 the number of F. prausnitzii bacteria in the bowel was 
measured in 82 Crohn’s disease patients using FISH. (27) The mean of F. prausnitzii was 
5.6 10^9 bacteria / ml of faeces (SD: 5.9 10^9). In this same article, it is shown that the mean 
of F. prausnitzii in healthy volunteers (n=32) is much higher: 14.9 10^9 bacteria / ml faeces 
(SD: 4.5 10^9). In the article of Swidsinski, the number of F. prausnitzii is roughly 2.5 times 
higher in the healthy group as opposed to the group with Crohn’s disease. We expect that 
the same ratio of F. prausnitzii between healthy volunteers and Crohn’s disease patients will 
be the case in our population. A similar increase as seen in the healthy volunteer group is 
hypothesized to take place after the riboflavin intervention. This results in an expected mean 
increase of F. prausnitzii of 2.45 10^8 bacteria / grams of faeces in our Crohn’s disease 
group (SD: 6.22 10^8). With a one sided test, with a power of 0.80 and an alpha of 0.05, this 
will result in a sample size of 42 patients for the group in remission. Since we want to study 
the effect in two experimental groups (one group in remission, and one group with active 
disease), 84 patients will be recruited.  
In this study an adaptive design will be used. When the first 12 patients in the disease in 
remission group are included and have completed the study procedures, the collected faeces 
samples will be analyzed. The number of F. prausnitzii with FISH will be determined and 
compared on different timepoints (primary endpoint). Also the ratio of enterobacteriaceae / F. 
prausnitzii will be determined. When the increase of the F. prausnitzii bacteria is less than 

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the increase seen in the healthy volunteer study, we will reassess the performed power 
calculation. Also safety aspects will be taken into account continuously. The HB-index and 
the reported adverse events will be noted. If there are indications to believe that the safety of 
the participants are at risk, the study will be suspended, and we will reassess continuing the 
study. 
 
 
 
 

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5. TREATMENT OF SUBJECTS 
 
5.1 Investigational product/treatment 
See chapter 7. 
 
5.2 Use of co-intervention (if applicable) 
When patients use antibiotic drugs, probiotics (i.e. Yakult, Vifit, Activia etc), specific prebiotic 
supplements or specific vitamin supplements during the 3 weeks prior to the start of the 
riboflavin intervention they cannot participate in the study (see exclusion criteria). In addition 
participants are asked to refrain from specific supplements during the study period (see 
‘proefpersoneninformatie’, attachment 8). In addition, participants are asked to refrain from 
alcohol, two days before test days. Participants are asked to refrain from smoking during the 
study period. Also participants are asked to refrain from intensive physical activity on the day 
before a test days. 
 
5.3 Escape medication (if applicable) 
Not applicable.  
 
 
 
6. INVESTIGATIONAL PRODUCT  
 
6.1 Name and description of investigational  product(s) 
Not applicable.  
 
6.2 Summary of findings from non-clinical studies 
Not applicable.  
 
6.3 Summary of findings from clinical studies 
Not applicable. 
 
6.4 Summary of known and potential risks and benefits 
Not applicable.  
 

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6.5 Description and justification of route of administration and 
dosage 
Not applicable.  
 
6.6 Dosages, dosage modifications and method of administration 
Not applicable.  
 
6.7 Preparation and labelling of Investigational Medicinal Product 
Not applicable.   
 
6.8 Drug accountability 
Not applicable. 
 
 
  
7. NON-INVESTIGATIONAL PRODUCT 
 
7.1 Name and description of non-investigational product(s) 
Riboflavin 
The non-investigational product used in this study is vitamin B2 (riboflavin) produced by DSM 
Nutritional Products. The riboflavin product (hard gel capsules) will be consumed orally. The 
daily dose will be ingested by taking one capsule per day at breakfast. Riboflavin is readily 
absorbed in the gastro-intestinal tract. Absorption is increased by intake of food.  
 
Chroom-EDTA 
Chroom-EDTA oral solution (see IMPD, versie 2, 23 Dec 2015) is a permeability marker 
which is often used in studies with human subjects. Chroom-EDTA is an extremely stable 
and inert complex with very limited toxicity.  
 
7.2 Summary of findings from non-clinical studies 
Not applicable.  
 
7.3 Summary of findings from clinical studies 
Not applicable. 
 
Riboflavin 

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In a pilot-intervention study in healthy volunteers, it is shown that a riboflavin supplement 
increases the number of F. prausnitzii, and there appeared to be a positive effect on the 
production of butyrate (results not published yet). 
 
Chroom-EDTA 
The Chroom-EDTA test is used in this hospital before in the (by the ethical committee 
approved) COLIPI clinical trial (‘’Identification of predictive parameters for colitis in melanoma 
patients treated with ipilimumab’’ (METc 2012/085)). 
 
7.4 Summary of known and potential risks and benefits 
Riboflavin 
No adverse effects associated with riboflavin consumption from food or supplements have 
been reported. In a study by Schoenen and coworkers (1994) no short-term side effects were 
reported in 49 patients treated with 400 mg/day of riboflavin taken with meals for at least 3 
months. The lack of harm resulting from high oral doses of riboflavin may be due to its limited 
solubility and limited capacity for absorption in the human gastrointestinal tract (Levy and 
Jusko, 1966; Stripp, 1965; Zempleni et al., 1996) and its rapid excretion in the urine 
(McCormick, 1994). Zempleni et al. (1996) showed that the maximal amount of riboflavin that 
was absorbed from a single oral dose was 27 mg. Similarly in a study by Stripp (1965) there 
was limited absorption of 50 to 500 mg of riboflavin with no adverse effects. 
 
Based on the studies above as well as the widespread market presence of 200 mg riboflavin 
supplements without reported adverse effects, the (US) Council for Responsible Nutrition 
(CRN) published an opinion in 2013, setting a tolerable upper intake level (UL) of 200 mg/d 
riboflavin 
 
Furthermore, the above (section 7.3) described finding of an increase in the number of F. 
prausnitzii and butyrate after riboflavin intake in healthy volunteers is promising. A similar (or 
even larger increase) in the number of F. prausnitzii (and butyrate) in the bowel of Crohn’s 
disease patients by means of a simple supplement may result in the improvement of health 
of Crohn’s disease patients. No adverse effects are known of this supplement, however 
vitamin B2 can cause a temporary and harmless bright yellow discoloration of the urine.  
 
 
Chroom-EDTA 
Chroom-EDTA oral solution is a safe diagnostic to measure the integrity of the intestines. To 
achieve better results in the research concerning the integrity of the intestines it is important 
that there are proper diagnostics. To achieve this the development of new markers is 
necessary. Without these markers it is more complicated to measure the effect of a therapy 
on the recovery of the intestines. As opposed to the labelled 51-Chroom-EDTA, the Chroom-
EDTA solution used in this study has no gamma-radiation. The risk for the humans is very 
low. No side effects of Chroom-EDTA have been reported. 

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7.5 Description and justification of route of administration and 
dosage 
Riboflavin 
Riboflavin will be taken orally (conform the intended use).The product used in this study 
contains 100 mg of riboflavin per capsule. Participants will take 1 capsule of riboflavin per 
day at breakfast. The total daily dose is 100 mg. This is a commonly used dose for riboflavin 
supplementation (sold over the counter, without prescription). Participants will be advised to 
store the riboflavin product at room temperature in a dry place and to keep the product out of 
reach of children and pets.  
 
Chroom-EDTA 
Patients will consume the Chroom-EDTA after a night of fasting (last meal 10 p.m. one day 
prior to the clinical visit). After emptying the bladder, subjects will consume a Chroom-EDTA 
drink, followed by a glass (240 ml) of water. The first dose (at baseline) will be given under 
supervision of the study nurse/investigator. After an additional two hours of fasting, 
participants are allowed to eat and drink again without restrictions. From consuming of the 
Chroom-EDTA-drink onwards for 24 hours, participants will collect urine. Urine samples will 
be collected in plastic bottles containing 10 ml 6 mol/l hydrochloric acid to prevent bacterial 
deterioration. 
 
7.6 Dosages, dosage modifications and method of administration 
Riboflavin 
No method of administration modifications will take place in the present study. 
 
Chroom-EDTA 
Chroom-EDTA solution is prepared (20 µmol/ml), using the method of Binnerts et al, in a 
food-grade environment and complete formation and stability is checked. Drinks will be filled 
with exact amounts of Chroom-EDTA, resulting in drinks containing 400 µmol Chroom-EDTA. 
The drinks will be produced in the pharmacy of A15 in accordance with GMP license (see 
attachment).  
 
7.7 Preparation and labelling of Non Investigational Medicinal 
Product 
Prior to the start of the study, DSM Nutritional Products will provide riboflavin and be 
responsible for packaging and labelling of the study supplement. The supplement will be 
packed into bottles of 23 capsules (21 days supplementation of 1 capsule per day + 2 spare 
capsules). The packages will be labelled in accordance with applicable laws and regulations. 
Labels on the study products will contain information required for usage and identification 

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purposes. The investigator will store the study product in a secure, limited access storage 
area at a cool dry place (between 15°C and 25°C), and protected from extremes of light and 
humidity. 
7.8 Drug accountability 
Riboflavin 
Participants will receive a bottle of 23 capsules at the start of the study. When possible 
products of the same batch will be used in all subjects. Subjects should return all unused 
capsules to the investigator. The investigator will then throw away the capsules. 
 
 
Chroom-EDTA 
The Chroom-EDTA oral solution will be labelled by the pharmacy A15. Chroom-EDTA will be 
stored at the pharmacy of the UMCG (contact person dr. B.G.J. Dekkers, dr. S. Hofman, 
B.H.W. Molmans and M.C. Mollema). On the day of administration the investigator will collect 
the Chroom-EDTA at the pharmacy.  
 
 
 
8. METHODS 
8.1 Study parameters/endpoints 
8.1.1 
Main study parameter/endpoint 
The main study parameter is the number of F. prausnitzii bacteria per gram of faeces. This 
will be measured with FISH and specific probes for F. prausnitzii and all bacteria will be 
counted. (28) This will be determined in the faeces samples at different time points. The 
number of bacteria from before and after the riboflavin intervention will be compared.  
8.1.2 
Secondary study parameters/endpoints  
- Before and after the riboflavin intervention, faeces samples will be analyzed for: 
- The number of butyrate producing bacteria other than F. prausnitzii per gram 
faeces, in particular Roseburia species, as measured by FISH. The numbers before 
and after the intervention will be compared.  
- The number of Enterobacteriaceae, including E. coli per gram of faeces. The 
numbers before and after the intervention will be compared.  
- The ratio of Enterobacteriaceae / Faecalibacterium is a measure of the improvement 
in the dysbiosis in intestinal microbiota.  
- The concentration of fatty acids in the stool (i.e. butyrate).  
- The level of faecal calprotectin will be measured at different time points, as marker 
of activity of Crohn’s disease. 
- Faeces pH. 

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- Microbiota analysis by pyrosequencing. 
- The Harvey Bradshaw Index, a measure of disease severity, will be taken before and after 
the intervention (see paragraph 3 and attachment 1).  
- The validated IBD-Q questionnaire will be taken before and after the intervention (see 
document F1: IBD-Q). 
- Blood and a urine sample (see paragraph 3, study design) will be taken before and after the 
intervention.  
- A Chroom-EDTA test will be performed at two time points. 
- Biomarkers of gut permeability will be measured before and after the intervention.  
- Electrical current measurements of faeces and cell culturing experiments.  
- Adverse events (AE’s), serious adverse events (SAE’s) and suspected unexpected serious 
adverse reactions (SUSAR’s). 
 
8.1.3 
Other study parameters  
Not applicable. 
 
 
8.2 Randomisation, blinding and treatment allocation 
In this current study participants will not be randomized. Also no blinding will take place. The 
study is designed in such a way that participants are their own controls.  
 
8.3 Study procedures 
Standard faeces samples (five time points: day 0, day 6, day 17, day 28 and day 42) will 
be divided into three fractions: 
- The first fraction (0.5 gram) will be fixed with 4% paraformaldehyde in Phosphate Buffered 
Saline (PBS) and used for FISH analysis with specific probes. (29)   
- The second fraction (100 µgram) will be used for the analysis of volatile fatty acids. 
- The third fraction (0.5 gram) will be stored for microbiome analysis by Next Generation 
Sequencing (microbiome analysis, by 16S RNA sequencing and MiSeq Illumina). 
- The remainder is retained for dry weight determination, pH analysis and lactate analysis. 
 
Faeces samples on glycerol, cysteine and PBS mix (two time points: day 6 en day 28): 
-These samples will be used for the electrical current measurements of faeces and cell 
culturing experiments.  
 
FISH analysis 
Fluorescent probes for F. prausnitzii (F. prau), Clostridium group XIVa (Erec), Roseburia 
(rint),  Enterobacteriaceae (EC1535) and total bacteria (EUB338) will be used to measure the 
effect of the riboflavin intervention. The hybridized bacteria will be counted with an 
epifluorescence microscope DMRA2 of Leica, automatic system, with a user modified 
version of Leica Q-win software (Leica, Wetzlar, Germany). (30)  

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Short Chain Fatty Acid analysis 
HPLC ion chromatography system (Metrohm AG, Herisau, Switzerland) will be used for fatty 
acid analysis. The concentrations of lactate, formate, butyrate, propionate and acetate will be 
measured with a conductivity detector. The 6.1005.200 Metrosep Organic Acids column 
(Metrohm AG) with 5 mm particle size and dimensions of 7.8 mm by 250 mm will be used for 
differentiating fatty acids. The mobile phase is 5mM HNO3 and acetonitrile v/v ratio of 98:2, 
respectively, with a flow rate of 0.6 ml min-1. 
 
Chroom-EDTA test 
Chroom-EDTA oral solution is a permeability marker which is often used in studies with 
human subjects. Chroom-EDTA is an extremely stable and inert complex with very limited 
toxicity. (31) Under normal physiological circumstances (i.e. healthy intestines) an amount of 
less than 2.5% of Chroom-EDTA is absorbed. When the intestine is more permeable, more 
Chroom-EDTA will enter the body passively by the intestinal paracellular route via tight 
junctions, resulting in an increased urinary Chroom-EDTA concentration. (32) In UC patients, 
Chroom-EDTA excretion reflects colonic permeability. (33) 
 
No side effects of Chroom-EDTA have been reported. (33) For the Chroom-EDTA 
permeability test, subjects will consume a Chroom-EDTA drink at baseline and on the last 
day of the riboflavin intervention. After emptying the bladder, subjects will consume a 
Chroom-EDTA drink. The first dose (at baseline) will be given under supervision of the study 
nurse/investigator. From consuming of the Chroom-EDTA-drink onwards for 24 hours, 
participants will collect urine. Urine samples will be collected in plastic bottles containing 10 
ml 6 mol/l hydrochloric acid to prevent bacterial deterioration. 
 
Chroom-EDTA solution is prepared, using the method of Binnerts et al, in a food-grade 
environment and complete formation and stability is checked. Drinks will be filled with exact 
amounts of Chroom-EDTA, resulting in drinks containing 400 µmol Chroom-EDTA. The 
drinks will be produced in the pharmacy of A15 in accordance with GMP license (see 
attachment).  
 
Faeces Calprotectin 
Calprotectin (also known as MRP-8 / MRP-14 or S100A8/A9 complex) is widely used as a 
marker for inflammatory bowel disease. Calprotectin is released when neutrophils enter the 
gut wall and are activated during the inflammatory process. (34) Calprotectin is a very stable 
compound at room temperature and can be measured within hours with a simple ELISA test. 
Literature has shown that faecal calprotectin levels correlate well with endoscopic, as well as 
histological, disease activity of patients with IBD. (35) 
 
Relevant biomarkers 

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Serum and plasma will be stored at two time points. This will allow us to look for a number 
biomarkers of permeability. Multiple biomarkers will be determined (see below).  
 
I-FABP 
Intestinal-Fatty acid binding proteins (I-FABP) is member of a class of three FABPs. I-FABP 
levels rise rapidly after enterocyte cell death. It is considered to be a promising marker of 
enterocyte injury. (36) (37) (38) I-FABP is determined in plasma. 
 
SM-22 
New promising marker of permeability (personal communication with (J.P.M. Derikx, 
MUMC)), determined in serum samples. 
 
Urine Claudin-3 
Urine Claudin-3 is a promising marker for intestinal tight junction loss. Recently it has been 
shown that in rats Claudin-3 urine levels increased rapidly in a rat hemorrhagic shock model 
(in this model intestinal barrier loss is observed rapidly after induction of hemorrhagic shock). 
This study also showed an association between claudin-3 stained biopsies and claudin-3 
urine levels in IBD patients. (36) (39) 
 
Measurement of electrical current and cell culturing experiments 
The experiments of the current measurements will take place conform the methods of Khan 
et al. A two chambered microbial fuel cell will be used. The current before and after the 
riboflavin intervention will be measured. (16) In addition a small fraction of this sample will be 
used for cell culturing experiments. 
 
8.4 Withdrawal of individual subjects 
Subjects can leave the study at any time for any reason if they wish to do so without any 
consequences. The investigator can decide to withdraw a subject from the study for urgent 
medical reasons. 
 
8.4.1 
Specific criteria for withdrawal (if applicable) 
Not applicable. 
 
8.5 Replacement of individual subjects after withdrawal 
In case of withdrawal or insufficient participation of a subject, he / she will be replaced by a 
newly recruited subject who matches the former subject. This is done in order to include the 
desired number of participants in each experimental group. 
 

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8.6 Follow-up of subjects withdrawn from treatment 
Not applicable. 
 
8.7 Premature termination of the study 
The study will be suspended if there are indications to believe that the safety of the 
participants is at risk. However it is not expected that this supplement and the procedures in 
this study may lead to adverse events. In the case of premature termination of the current 
study the institution, regulatory authorities, CCMO and the METc of the UMCG will be 
informed.  
 
 
 
 
9. SAFETY REPORTING 
9.1 Section 10 WMO event 
In accordance to section 10, subsection 1, of the WMO, the investigator will inform the 
subjects and the reviewing accredited METc if anything occurs, on the basis of which it 
appears that the disadvantages of participation may be significantly greater than was 
foreseen in the research proposal. The study will be suspended pending further review by 
the accredited METc, except insofar as suspension would jeopardise the subjects’ health. 
The investigator will take care that all subjects are kept informed.  
 
9.2 AEs, SAEs and SUSARs 
9.2.1 
Adverse events (AEs) 
Adverse events are defined as any undesirable experience occurring to a subject during the 
study, whether or not considered related to the investigational product. All adverse events 
reported spontaneously by the subject or observed by the investigator or his staff will be 
recorded. 
 
The collection of faeces and urine samples entails no risks for the subjects. The Harvey 
Bradshaw Index can be determined by taking a short questionnaire (5 items). The IBD-Q is a 
validated questionnaire used to measure the influence of the inflammatory bowel disease on 
the quality of life and the impact of the disease. 
 
No side effects of Chroom-EDTA have been reported. 
 

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Riboflavin is freely available, and commonly sold in health shops. There is no need for a 
prescription to buy this supplement, and its use is considered to be safe. The adverse event 
of discoloration of urine is only temporary.  
 
A minor risk of this study is the development of a small hematoma (‘blauwe plek’) as a result 
of the blood sampling from the inside of the elbow. All together, we believe that the risks in 
this study are minor, and acceptable for the subjects participating in this study. 
 
9.2.2 
Serious adverse events (SAEs) 
A serious adverse event is any untoward medical occurrence or effect that at any dose:  
- results in death; 
- is life threatening (at the time of the event); 
- requires hospitalisation or prolongation of existing inpatients’ hospitalisation; 
- results in persistent or significant disability or incapacity; 
- is a congenital anomaly or birth defect; 
- any other important medical event that may not result in death, be life threatening, or 
require hospitalization, may be considered a serious adverse experience when, based upon 
appropriate medical judgement, the event may jeopardize the subject or may require an 
intervention to prevent one of the outcomes listed above. 
 
The sponsor will report the SAEs through the web portal ToetsingOnline to the accredited 
METc that approved the protocol, within 15 days after the sponsor has first knowledge of the 
serious adverse events. 
 
SAEs that result in death or are life threatening should be reported expedited. The expedited 
reporting will occur not later than 7 days after the responsible investigator has first 
knowledge of the adverse event. This is for a preliminary report with another 8 days for 
completion of the report.  
9.2.3 
Suspected unexpected serious adverse reactions 
(SUSARs) 
Adverse reactions are all untoward and unintended responses to an investigational product 
related to any dose administered. 
 
Unexpected adverse reactions are SUSARs if the following three conditions are met: 
1). the event must be serious (see chapter 9.2.2); 
2). there must be a certain degree of probability that the event is a harmful and an 
undesirable reaction to the medicinal product under investigation, regardless of the 
administered dose; 

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3). the adverse reaction must be unexpected, that is to say, the nature and severity of the 
adverse reaction are not in agreement with the product information as recorded in: 
- Summary of Product Characteristics (SPC) for an authorised medicinal product; 
- Investigator’s Brochure for an unauthorised medicinal product. 
 
The sponsor will report expedited the following SUSARs through the web portal 
ToetsingOnline to the METc: 
- SUSARs that have arisen in the clinical trial that was assessed by the METc; 
- SUSARs that have arisen in other clinical trials of the same sponsor and with the same 
medicinal product, and that could have consequences for the safety of the subjects involved 
in the clinical trial that was assessed by the METc. 
 
The remaining SUSARs are recorded in an overview list (line-listing) that will be submitted 
once every half year to the METc. This line-listing provides an overview of all SUSARs from 
the study medicine, accompanied by a brief report highlighting the main points of concern.  
The expedited reporting of SUSARs through the web portal ToetsingOnline is sufficient as 
notification to the competent authority. 
 
The sponsor will report expedited all SUSARs to the competent authorities in other Member 
States, according to the requirements of the Member States.  
 
The expedited reporting will occur not later than 15 days after the sponsor has first 
knowledge of the adverse reactions. For fatal or life threatening cases the term will be 
maximal 7 days for a preliminary report with another 8 days for completion of the report.  
 
9.3 Annual safety report 
 
In addition to the expedited reporting of SUSARs, the sponsor will submit, once a year 
throughout the clinical trial, a safety report to the accredited METc, competent authority, and 
competent authorities of the concerned Member States. 
This safety report consists of: 
- a list of all suspected (unexpected or expected) serious adverse reactions, along with an 
aggregated summary table of all reported serious adverse reactions, ordered by organ 
system, per study; 
- a report concerning the safety of the subjects, consisting of a complete safety analysis and 
an evaluation of the balance between the efficacy and the harmfulness of the medicine under 
investigation. 
 

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9.4 Follow-up of adverse events 
All AEs will be followed until they have abated, or until a stable situation has been reached. 
Depending on the event, follow up may require additional tests or medical procedures as 
indicated, and/or referral to the general physician or a medical specialist. 
SAEs need to be reported till end of study within the Netherlands, as defined in the protocol. 
 
9.5 Data Safety Monitoring Board (DSMB) / Safety Committee 
Not applicable. 
 
 
 
10. STATISTICAL ANALYSIS 
 
The study population consists of two groups (an active disease group and a disease in 
remission group). The primary endpoint is number of F. prausnitzii bacteria per grams of 
faeces. The two groups will first be statistically analysed separately. A non-parametric 
statistical test will be used in which the number of F. prausnitzii bacteria prior to the intake of 
riboflavin (control period) will be compared with the number of bacteria after intake of 
riboflavin (i.e. day 28). This will be analyzed with the paired Wilcoxon test. For analysis 
between groups (active disease and disease in remission) the Mann-Whitney U test will be 
used. The MiSeq Illumina data will be analyzed with a multi-parametric test.  
 
 
10.1 
Primary study parameter(s) 
- The number of F. prausnitzii per gram of faeces will be measured with FISH and specific 
probes for F. prausnitzii, both before and after the riboflavin intervention. 
 
10.2 
Secondary study parameter(s)  
Both before and after supplementation the following parameters will be included: 
- The number of other bacteria in the faeces. Of special interest are the number of butyrate 
producing bacteria other than F. prausnitzii, in particular Roseburia species. Additionally the 
number of Enterobacteriaceae in the stool, and the ratio of Enterobacteriaceae / 
Faecalibacterium will be determined.  
- Microbiota in the faeces by pyrosequencing. 
- The concentration of short chain fatty acids (SCFAs) in the faeces samples. 
- Routine IBD blood test. 
- Faeces pH. 
- Faecal calprotecin. 
- The clinical Harvey Bradshaw Index (index of disease severity). 

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- IBD-Q questionnaire. 
- Chroom-EDTA test. 
- Electrical current measured in faeces sample. 
- Biomarkers of gut permeability (as described above). 
 
10.3 
Other study parameters 
Not applicable. 
 
10.4 
Interim analysis  
Not applicable. 
 
 
 
11. ETHICAL CONSIDERATIONS 
11.1 
Regulation statement 
This study will be conducted according to the principles of the Declaration of Helsinki 
(October 2013) and in accordance with the principles of with the Medical Research Involving 
Human Subjects Act (WMO). The study will start after approval of the medical ethical 
committee (METc) is obtained. 
 
11.2 
Recruitment and consent 
The responsible physician will first approach eligible patients with general information about 
the study. When patients are interested, and agree to receive more information from the 
physician, the physician will supply a partipant information letter ‘proefpersonen informatie’ 
for the potential participant to read at home (see document E1 and E2). If the potential 
participant is interested to participate in the study, the participant will send back a signed 
‘toestemmingsformulier’.After this, the investigator can contact the participant to make an 
appointment at the outpatient clinic. At this time more information will be given about the 
research, its goals, the duration and procedures and the possible adverse events. Also, 
additional questions of the potential participant can be answered at this time. The 
investigator will stress that participating is voluntarily, and that they can quit at any time and 
for any reason, without any consequence for their health and further treatment. Also they will 
be informed about the strict confidentially concerning the acquired medical data and further 
patient details. Hereafter the investigator will also sign the informed consent form. If needed 
the potential participant can request additional information at any time by a second opinion 
(i.e. the independent expert). Potential participants will have one week to decide whether or 
not to enrol in this study. If the potential participant does not respond within one week, a 
reminder letter will be sent. After this, the participant have one more week to respond. If 
there is no reaction after this second letter, no more attempts will be made to try and contact 

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this patient for this study Documented informed consent must be obtained from all patients 
prior to inclusion in the study. Informed consent will be asked in writing, and in the following 
manner: the patient will sign and personal date the informed consent form first and send this 
form back to the investigator. When the  investigator has signed and dated the consent form 
afterwards,  the patient will receive a copy. Finally, the general physician of each patient will 
be informed about the enrolment of the patient to the study by a standard letter. The 
informed consent procedure takes place conform Good Clinical practice (GCP). After signing 
the informed consent sheet, the study procedures will commence (see paragraph 3, study 
design). 
 
11.3 
Objection by minors or incapacitated subjects (if 
applicable) 
Not applicable. 
 
11.4 
Benefits and risks assessment, group relatedness 
Participating in this study has a potential health benefit. It is know that in healthy subjects, 
the supplement riboflavin increases the amount of beneficial bacteria. In Crohn’s disease 
there is often a dysbiosis of the bacterial composition, and the beneficial bacteria are 
depleted. When a similar increase in the beneficial bacteria occurs in Crohn’s disease 
patients as seen in healthy volunteers, this may result in a more favourable disease outcome 
(staying in remission longer). The vitamin preparation containing vitamin B2, may give a 
(completely innocent) yellow discoloration of the urine several hours after ingestion. The 
collection of faeces samples entails no risks for the subjects. 
 
11.5 
Compensation for injury 
The sponsor/investigator has a liability insurance, which is in accordance with article 7, 
subsection 6 of the WMO. 
 
 
The sponsor (also) has an insurance, which is in accordance with the legal requirements in 
the Netherlands (Article 7 WMO and the Measure regarding Compulsory Insurance for 
Clinical Research in Humans of 23rd June 2003). This insurance provides cover for damage 
to research subjects through injury or death caused by the study. 
1).  € 450.000,-- (i.e. four hundred and fifty thousand Euro) for death or injury for each 
subject who participates in the Research; 
2).  € 3.500.000,-- (i.e. three million five hundred thousand Euro) for death or injury for all 
subjects who participate in the Research; 
3).  € 5.000.000,-- (i.e. five million Euro) for the total damage incurred by the organisation for 
all damage disclosed by scientific research for the Sponsor as ‘verrichter’ in the meaning of 
said Act in each year of insurance coverage. 

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The insurance applies to the damage that becomes apparent during the study or within 4 
years after the end of the study. 
 
11.6 
Incentives 
Travel costs will be compensated: the subjects will receive a compensation of 0.19 euro/km 
when travelling with car. In addition, participants will receive a ticket for free parking at the 
UMCG on study days. The total amount of money will be paid after completing the study, and 
after the last samples have been collected. The riboflavin supplement will be handed over to 
the participant free of charge. Participating in the study will not result in costs for the 
participant.  
 
 
 
 
12. ADMINISTRATIVE ASPECTS, MONITORING AND PUBLICATION 
12.1 
Handling and storage of data and documents 
The handling of personal data complies with the Dutch ‘Personal Data Protection Act’ (Dutch: 
‘wet bescherming persoonsgegevens (wbp)’). All related documents and records will be kept 
and archived for at least 15 years. Data of participants will be handled confidentially and a 
coded identification number will be used to link the data to the specific patient. This specific 
data that can be linked to a patient will be stored separately. The coded identification number 
used in this study respectively consists of: the acronym: RISE-UP combined with the date 
and number of inclusion. For example the first participant included of the first of June 2014 
would have the code identification number: RISE-UP/01-06-2014/01. The principal 
investigator will safeguard the key to the code.  
 
The case record forms (CRF’s) will be completed and signed for correctness by the 
investigators. By signing this CRF, the medical investigator is responsible for the legibility, 
completeness and correctness of the CRF.  
 
Changes, errors and/or additions to the original CRFs shall be corrected by drawing a single 
line through the incorrect entry and writing as close to the original as possible. The correction 
must be initialled and dated by the authorized person making the changes. If necessary the 
reason for the changes must be given.  
 
12.2 
Monitoring and Quality Assurance  
Not applicable. 
 

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12.3 
Amendments  
A ‘substantial amendment’ is defined as an amendment to the terms of the METc application, 
or to the protocol or any other supporting documentation, that is likely to affect to a significant 
degree: 
- the safety or physical or mental integrity of the subjects of the trial; 
- the scientific value of the trial; 
- the conduct or management of the trial; or 
- the quality or safety of any intervention used in the trial. 
 
Non-substantial amendments will not be notified to the accredited METc and the competent 
authority, but will be recorded and filed by the sponsor. 
 
 All substantial amendments will be notified to the METc and to the competent authority.  
12.4 
Annual progress report 
The sponsor/investigator will submit a summary of the progress of the trial to the accredited 
METc once a year. Information will be provided on the date of inclusion of the first subject, 
numbers of subjects included and numbers of subjects that have completed the trial, serious 
adverse events/ serious adverse reactions, other problems, and amendments.  
 
12.5 
End of study report 
The sponsor will notify the accredited METc and the competent authority of the end of the 
study within a period of 90 days. The end of the study is defined as the collection of the last 
samples of the last patient. In case the study is ended prematurely, the sponsor will notify the 
accredited METc and the competent authority within 15 days, including the reasons for the 
premature termination. 
 
Within one year after the end of the study, the investigator/sponsor will submit a final study 
report with the results of the study, including any publications/abstracts of the study, to the 
accredited METc and the Competent Authority.  
 
12.6 
Public disclosure and publication policy 
 
The sponsor of this study are the UMCG. The goal of this study is to contribute to research, 
and to publish the results. The results of this research will be disclosed unreservedly. Both 
positive and negative trial results must be disclosed. In general, the results of this research 
will be submitted for publication to peer-reviewed scientific journals.  
 
 
 

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13. STRUCTURED RISK ANALYSIS  
 
13.1 
Potential issues of concern 
 
See section 13.2. 
 
13.2 
Synthesis 
 
All subjects included in this study have been diagnosed with Crohn’s disease. No children 
will be included in this study. Pregnancy is one of the exclusion criteria in this study.  
 
In a pilot-intervention study in healthy volunteers, it is shown that a riboflavin supplement 
increases the number of F. prausnitzii, and there appeared to be a positive effect on the 
production of butyrate (results not published yet). A similar (or even larger increase) in the 
number of F. prausnitzii (and Butyrate) in the bowel of Crohn’s disease patients by means of 
a simple supplement may have therapeutic consequences. Riboflavin is freely available, and 
commonly sold in health shops. There is no need for a prescription to buy this supplement, 
and its use is considered to be safe. The adverse event of discoloration of urine is only 
temporary. The Chroom-EDTA is safe and, no adverse effects are known. The collection of 
faeces samples entails no risks for the subjects. A minor risk of this study is the development 
of a small hematoma (‘blauwe plek’) as a result of the blood sampling from the inside of the 
elbow. All together, we believe that the risks in this study are minor, and acceptable for the 
subjects participating in this study. 

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Attachment 1. Harvey Bradshaw Index 
(See document F1: (HB index) for the Dutch version of this questionnaire). 
Date: __________ 
1. 
General well-being (yesterday) 
- 
Very well = 0 
 
 
 
q 
 
- 
Slightly below par = 1  
 
 
q 
- 
Poor = 2   
 
 
 
q 
- 
Very poor = 3 
 
 
 
q 
- 
Terrible = 4 
 
 
 
q 
 
2. 
Abdominal pain (yesterday) 
- 
None = 0   
 
 
 
q 
- 
Mild = 1 
 
 
 
 
q 
- 
Moderate = 2 
 
 
 
q 
- 
Severe = 3  
 
 
 
q 
 
3. 
Number of liquid or soft stools per day (yesterday) 
 
___________________________ 
 
4. 
Abdominal mass 
- 
None = 0   
 
 
 
q 
- 
Dubious = 1 
 
 
 
q 
- 
Definite = 2 
 
 
 
q 
- 
Definite and tender = 3 
 
 
q 
 
5. 
Complications 
 
(Check any that apply; score one per item, except for first box) 
- 
None 
 
 
 
 
q 
- 
Arthralgia  
 
 
 
q 
- 
Uveitis 
 
 
 
 
q 
- 
Erythema nodosum 
 
 
q 
- 
Aphtous ulcers 
 
 
 
q 
- 
Pyoderma gangrenosum  
 
q 
- 
Anal fissure 
 
 
 
q 
- 
New fistula  
 
 
 
q 
- 
Abcess 
 
 
 
 
q 
 
 
 
Total Harvey Bradshaw index score: ____________ 
Remission < 5 
Mild disease = 5 – 7 
Moderate disease = 8 – 16 
Severe disease > 16 

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Attachment 2. List of commercially available probiotic, 
prebiotic and other supplements that are not allowed in the 
current study 
 
 
Probiotica: 
 
1x TSglich LGG 
40+ Acidophilus Vegicaps 
Acidophilus (Hema) 
AB Kultur Drink 
ABC Dophilus 
Acidobif 
Acidophilasé 
Acidophilus (Etos) 
Acidophilus (Hema) 
Acidophilus Plus 
Acidophilus vegicaps 
Acidophilus Yoghurt met smaakjes 
Acidophilus, bifidus en bulgaricus 
Acidophilus-4 
Aciforce (Vogel) 
Activia 
Actimel (Danone) 
Aktifit 
A-Yoghurt 
Beneflora 
Bifilus 
Bio 
Biotic 
Biomild 
Bioghurt DiSt 
Colon Balans 
Colon Rein Naturel (Kruidvat) 
Colonphyta 
Cultura 
Cyclops yoghurt 
Darm conditioner 
Darm Flora Optimaal (Kruitvat) 
Darmclean 
Darmflora Plus 

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Darmo Caps 
Darmplus 
Darmflora balans (DA) 
Darm totaal (Etos) 
Ecologic 
Equiderm 
Eugalan forte 
Fidus 
Fresh'n Fruity 
Frucht Bighurt 
Fysiq 
Gefilus 
GB-Bifidus halfvolle yoghurt  
Lactobacillus acidophilus 
Lactobacillus complex (Hema) 
LGG+ 
Maxi Baby Dophilus 
Milupino Kinder Trink-Frühstück 
Mix Lactobacillus 
Multi-Billion Dophilus 
Multidophilus 
Natufood Vitaal 
Naturally acidophilus 
Nature's biotics 
Nite Cap 
Non Dairy Acidophilus plus 
Omnibionta 3 
Onaka 
Orthiflor Junior 
Orthiflor Plus 
Orthiflor 
Power Dophilus 
Power Dophilus II 
Pro'Ac 
ProBiotic 
Prolife 
ProViva 
Rela Vruchtensap 
R-LSttyoghurt 
Ser con GG 
Simfidus 
Symbalance 

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Symbio (Flor) 
Tofu Jog 
Trenev Trio  
Trifit 
Vitamel 
Vifit (Mona) 
Vitelle Lactobacillus (Trekpleister) 
Yakult 
Yolac 5 
Yosa  
VSL#3 
 
* Tevens alle zuivelproducten waarbij vermeld staat dat er Lactobacillen en/of Bifidobacteriën aan 
zijn toegevoegd. 
 
Prebiotica 
 
Fructo-oligo-sacchariden (FOS) 
Oligofructose (raftilose) 
Inuline (raftiline) (bijv Colonclean ®) 
Galactose-oligo-sacchariden (GOS) 
Lactulose 
Lactitol 
Psyllium (Vlozaad) 
Verschillende soorten vezeltabletten, bijvoorbeeld: Dagravit Vitentials ®, 
Bio-Vezel tabletten ®, Vezel complex Forte ®, Famafiber ®, etc. 
 
Overig 
 
-Vitaminesupplementen (bijvoorbeeld multivitaminen tabletten, vitamine C, vitamine E, β-caroteen, 
zink. 
-Supplementen die claimen het immuunsysteem of de gezondheid van de darmen te verbeteren.  

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