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Effect of dietary intervention on intestinal microbiota in patients with NAFLD. VERSION 2, 2010.11.30

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1. COPAGE

CLINICAL STUDY PROTOCOL CONFIDENTIAL

Study Title: Effect of dietary intervention on intestinal microbiota in patients with non-alcoholic fatty liver disease

Protocol Number: 10-231

Name of the Sponsor: Geneva University Hospitals

Sponsor representative: Prof. Antoine Hadengue

Version: 2.0 dated 30 Novembre 2010


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2. STUDY SUMMARY SHEET

Title of study: Effect of dietary intervention on intestinal microbiota in patients with non-alcoholic fatty liver disease (NAFLD). Protocol No:10-xxx

Sponsor’s representative: Antoine Hadengue, Gastroenterology and Hepatology, (HUG) Principal Investigator: Claude Pichard, Nutrition, (HUG) Sub-investigators:

Jacques Schrenzel, Infectious diseases (HUG) Alain Golay, Chronic diseases education (HUG) Sylvain Terraz, Radiology, (HUG) Laura Rubbia-Brandt, Clinical pathology, (HUG) Vladimir Lazarevic Infectious diseases (HUG)

Study centre: University Hospital of Geneva, 4 rue Gabrielle Perret-Gentil, 1211 Genève 14, Switzerland

Study Hypothesis: In patients with NAFLD/NASH, changes in liver lipid composition and function tests following a short dietary intervention are associated with changes in gut microbiota

Study period: - Study duration for the participant: 7-10 weeks (1-4 weeks screening period, 3 weeks of intervention + 3 weeks of follow-up) - Expected study completion date: 30.04.2012

Study type: Single arm before-after study

Number of patients: 20 subjects with obesity and NAFLD / NASH Main criteria for inclusion: Obesity defined as BMI>30 Abnormal liver function tests defined as ALT > 1.5 times the upper limit of normal NAFLD present at liver biopsy Age > 18 years, < 60 years Main exclusion criteria: Inability or unwillingness to give consent Parenteral nutrition or other ongoing dietary intervention Bulimia Other known cause of chronic liver disease, including hepatitis B or C, iron overload, Use of substances known to alter intestinal permeability, including alcohol and NSAIDs

Intervention: The phase 2 Eurodiet® program will be used as the basis for standardized hypo-caloric diet during a 3-weeks intervention period. Commercially available products to reach 1000 kcal/day. These products will be offered free of charge.


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Primary Objective:

To assess the impact of dietary intervention on the relative abundance of fecal Bacteroidetes (expressed as the bacteroidetes to firmicutes ratio) in patients with obesity, abnormal liver function tests and NAFLD

Secondary Objectives: 1. To compare fecal microbiota from patients with NAFLD or NASH at baseline 2. To assess fecal microbiota changes in patients with NAFLD or NASH after dietary intervention 3. To measure liver fat content at baseline and after dietary intervention 4. To assess changes in liver function tests and ultrasensitive CRP, cytokines and serum LPS in relation to

changes in microbiota and liver lipid composition 5. To measure orocecal transit time, an index of intestinal pullulation, at baseline and after dietary

intervention in patients with NAFLD or NASH 6. To measure intestinal permeability at baseline and after dietary intervention in patients with NAFLD or

NASH 7. To assess body composition changes in relation to changes in microbiota and liver lipid composition

Statistical methods: Baseline and end-of-treatment changes for both bacterial genders and subspecies will be compared using paired-sample Wilcoxon signed-rank test. ANOVA and paired t-test for comparison of other changes within groups. Pearson or Spearman tests will be used to assess correlations between changes in microbiota and changes in liver fat content, liver function tests, CRP, cytokines and intestinal pullulation and permeability.

Sample size: 20 patients with NAFLD/NASH will be studied before and after dietary intervention.

Assessment of end-points: Fecal microbiota will be analysed with 454-Flex metagenomics

Ultrasensitive CRP and serum LPS changes as compared with baseline Serum cytokines as measured with ELISA Liver fat content and composition will be measured using MR spectroscopy Small intestinal overgrowth and intestinal permeability will be assessed using standard 13C breath tests and polyethyleneglycol absorption test Body mass composition changes will be assessed using bioelectrical impedance analysis

Safety : All adverse events will be recorded throughout the study, in compliance with GCP ICH E6 and national regulations.


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Contractual signatories

I, the undersigned, have read the foregoing protocol and the “Participant information and consent form” document attached to the protocol and agrees to conduct the study in compliance with such documents, GCP and the applicable regulatory requirements. NAME DATE SIGNATURE

SPONSOR’s REPRESENTATIVE: A. Hadengue

PRINCIPAL INVESTIGATOR: C. Pichard


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3. TABLE OF CONTENTS

1. COPAGE ...........................................................................................................................1

2. STUDY SUMMARY SHEET ...................................................................................................2

3. TABLE OF CONTENTS .........................................................................................................5

4. GLOSSARY ........................................................................................................................7

5. ADMINISTRATIVE STRUCTURE OF THE STUDY .....................................................................9

6. BACKGROUND INFORMATION ......................................................................................... 11

6.1. Non-alcoholic fatty liver disease and its management ............................................................... 11

6.2. Human gut microbiota ............................................................................................................ 11

6.3. Microbiota changes in obesity ................................................................................................. 11

6.4. Microbiota, intestinal permeability and endotoxemia in NAFLD/NASH ........................................ 12

6.5. Hypothesis of the present study............................................................................................... 12

6.6. Description of the study population ......................................................................................... 12

6.7. Description of the intervention ................................................................................................ 13

6.8. Relevance of the results expected from this study ..................................................................... 13

7. STUDY OBJECTIVES AND PURPOSE ................................................................................... 14

7.1. Primary Objective ................................................................................................................... 14

7.2. Secondary Objectives .............................................................................................................. 14

8. STUDY DESIGN ............................................................................................................... 14

8.1. Primary endpoint .................................................................................................................... 14

8.2. Secondary end-points ............................................................................................................. 14

8.3. Study characteristics ............................................................................................................... 14

8.4. Study plan .............................................................................................................................. 15

8.5. Discontinuation of the study .................................................................................................... 15

9. DATA COLLECTED IN THE CASE REPORT FORMS (CRF) ........................................................ 16

10. SELECTION AND WITHDRAWAL OF PARTICIPANTS ......................................................... 18

10.1. Inclusion criteria ..................................................................................................................... 18

10.2. Exclusion criteria .................................................................................................................... 18

10.3. Withdrawal criteria ................................................................................................................. 18

10.4 Procedure for replacement of withdrawn patients .................................................................... 19

11. TREATMENT OF PARTICIPANTS .................................................................................... 19

11.1. Treatment administered.......................................................................................................... 19


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11.2. Treatment management ......................................................................................................... 19

11.3. Concomitant treatments and prohibited medications ................................................................ 19

11.4. Treatment compliance ............................................................................................................ 20

12. ASSESSMENT OF END-POINTS ...................................................................................... 20

12.1. Microbiota composition .......................................................................................................... 20

12.2. Serum LPS concentration ......................................................................................................... 21

12.3. Anthropometric measurements and body composition. ............................................................ 21

12.4. Cytokines ELISA ...................................................................................................................... 21

12.5. Liver fat content. MRI ............................................................................................................. 22

12.6. Intestinal permeability. ........................................................................................................... 22

12.7. Orocecal transit time. Breath test ............................................................................................ 22

13. ASSESSMENT OF SAFETY .............................................................................................. 22

13.1. Safety measurements ............................................................................................................. 22

13.2. Adverse Events ....................................................................................................................... 22

14. STATISTICS ................................................................................................................. 24

14.1. Determination of sample size .................................................................................................. 24

14.2. Statistical comparisons for metagenomic studies. ..................................................................... 24

14.3. Other statistical Analyses ........................................................................................................ 25

15. QUALITY CONTROL AND QUALITY ASSURANCE .............................................................. 25

15.1. Monitoring ............................................................................................................................. 25

15.2. Audit and Inspection ............................................................................................................... 25

16. ETHICS ....................................................................................................................... 26

16.1. Ethics Committee ................................................................................................................... 26

16.2. Patient Informed Consent ....................................................................................................... 26

17. DATA HANDLING AND RECORD KEEPING ...................................................................... 26

17.1. Study Data ............................................................................................................................. 26

17.2. Data Management .................................................................................................................. 27

17.3. Archiving ................................................................................................................................ 27

17.4. Handling of Biological Samples................................................................................................. 27

18. INSURANCE ................................................................................................................ 27

19. OWNERSHIP OF THE RESULTS – PUBLICATION POLICY .................................................... 28

20. REFERENCES ............................................................................................................... 29

21. BUDGET ..................................................................................................................... 31


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4. GLOSSARY ADR : Adverse Drug Reaction AE : Adverse Event ALAT : Alanine amino-transferase AP : Alkaline phosphatase ASAT : Aspartate aminotransferase AUC : Area Under the Curve BF Body Fat BMI : Body Mass Index BP : Blood Pressure bpm : beats per minute (heart rate unit) CI : Confidence Interval cm : Centimetre Cmax : maximum concentration Cmin : minimum concentration C/P : Cholesterol/ Phospholipid molar ratio CRF : Case Report Form CRP : C-Reactive Protein e.g. : Exempli gratia (for example) FFM Fat Free Mass g : gram GCP : Good Clinical Practice GGT : Gamma-Glutamyl Transferase GI : Gastrointestinal GLP : Good Laboratory Practice GMP : Good Manufacturing Practice h : hour Hb : Haemoglobin Hc Hematocrit HBV : Hepatitis B virus HCV : Hepatitis C virus HIV : Human Immunodeficiency Virus HUG : Hôpitaux Universitaires de Genève (University Hospitals of Geneva) ICH : International Conference on Harmonisation i.e. : Id est (that is) IEC : Independent Ethics Committee INR : International normalized ratio IQR : Inter-quartile range IRB : Institutional Review Board IU : International Unit IV : IntraVenous (route) kg : kilogram L Lipopolysaccharide LFTs Liver Function Tests LPS Litre

mol : micromole

mg : milligram min : minute mL : Millilitre mmHg Millimetre of mercury mm : Millimetre mmol : millimole MRI Magnetic Resonance Imaging msec : millisecond NA : Not Applicable


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NAFLD : Non-alcoholic fatty liver disease NAS NAFLD Activity Score NASH : Non-alcoholic steatohepatitis ng : nanogram PD : PharmacoDynamics PK : PharmacoKinetics po : per os (orally) PT : Prothrombin Time QC : Quality Control RBC : Red Blood Cells RCT Randomized Controlled Trial SAE : Serious Adverse Event SBP : Systolic Blood Pressure sec : second TG : Triglycerides WBC : White Blood Cells WHO : World Health Organization


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5. ADMINISTRATIVE STRUCTURE OF THE STUDY

Sponsor’s representative:

Prof A. Hadengue Service de Gastroentérologie et Hépatologie Hôpitaux Universitaires de Genève Rue Gabrielle Perret-Gentil 4 1211 Genève 14 Tel : +41 22 372 93 40 Fax : +41 22 372 93 66

Principal investigator: Prof C. Pichard Service d’endocrinologie, diabétologie et nutrition Hôpitaux Universitaires de Genève Rue Gabrielle Perret-Gentil 4 1211 Genève 14 Tel : +41 22 372 93 45

Sub-investigator:

Prof. J. Schrenzel Genomic Research Lab (CEBUG) Service des maladies infectieuses Hôpitaux Universitaires de Genève Rue Gabrielle Perret-Gentil 4 1211 Genève 14 Tel : +41 22 372 73 08

Sub-investigator:

Dr Sylvain Terraz Service de radiodiagnostic Hôpitaux Universitaires de Genève Rue Gabrielle Perret-Gentil 4 1211 Genève 14 Tel : +41 22 372 70 35

Sub-investigator:

Prof A. Golay Service de thérapie des maladies chroniques Hôpitaux Universitaires de Genève Rue Gabrielle Perret-Gentil 4 1211 Genève 14 Tel : +41 22 372 97 04

Sub-investigator:

Prof L. Rubbia-Brandt Service de pathologie clinique Hôpitaux Universitaires de Genève Rue Gabrielle Perret-Gentil 4 1211 Genève 14 Tel : +41 22 372 49 03


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Sub-investigator:

Dr. V. Lazarevic Genomic Research Lab (CEBUG) Service des maladies infectieuses Hôpitaux Universitaires de Genève Rue Gabrielle Perret-Gentil 4 1211 Genève 14 Tel : +41 (0)22 372 93 38


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6. BACKGROUND INFORMATION

6.1. Non-alcoholic fatty liver disease and its management

Non-alcoholic fatty liver disease (NAFLD) ranges from non-alcoholic steatosis alone, through to the necroinflammatory disorder of non-alcoholic steatohepatitis (NASH) and cirrhosis [1]. NAFLD is considered as the hepatic manifestation of the metabolic syndrome. Because of the increasing prevalence of obesity, it has become one of the most widespread forms of chronic liver disease throughout the world. In the western world, the prevalence of NAFLD ranges from 16% in lean to 76% in obese individuals [1]. Both NAFLD and NASH are associated with significant liver, cardiovascular and overall mortality and morbidity [2, 3]. Indeed, it is now foreseen that non-alcoholic chronic liver disease is about to become the leading cause of cirrhosis and hepatocellular cancer in the industrialized countries [4]. In patients with NAFLD/NASH, abnormal lipid metabolism, genetic factors and insulin resistance [5] contribute to hepatic lipid storage [1]. Insulin resistance, in turn, correlates with lipotoxicity, oxidative stress and production of proinflammatory mediators, all associated with inflammation-induced hepatocellular injury [1]. Yet, the means by which oxidative stress and cytokines instigate and maintain this pathological state are still poorly understood. Once NAFLD/NASH has been established and staged, lifestyle intervention, mainly represented by diet and exercise, is the first line of therapy. A recent meta-analysis of all available Randomized Controlled Trials (RCTs) in NAFLD/NASH [6] shows that weight loss, while being safe, dose-dependently improves histological disease activity and associated cardiometabolic risk factors in NASH. A 2-3% body weight loss improves steatosis and associate metabolic parameters, together with transaminases. More important weight losses were required to ameliorate necroinflammation and overall disease activity. No “specific pharmacological treatment outside clinical trials can be recommended »[6].

6.2. Human gut microbiota

The human gut is home to a diverse and complex collection of microorganisms, referred to as the “gut microbiota”. The latter, mostly contained in the colon, counts around 1013 to 1014 bacterias, outnumbering by ten-fold the number of human body cells and representing more than a hundred times the human genome. Intestinal microbiota has a significant metabolic activity that contributes to the extraction of energy from “non-digestible” foods, xenobiotic detoxication, production of vitamins and other functions. Gut microbiota is known to have a plastic composition, influenced by modifying factors such as diet, alcohol, antibiotic or NSAID use [7]. These factors can reduce bacterial populations thought to be “beneficial”, such as Bifidobacteria and Lactobacilli, and promote overgrowth of “noxious” strains, a situation referred to as dysbiotic. Finally, upon exposure to fat foods, gut microbiota may trigger lipid storage signals to the host [8, 9].

6.3. Microbiota changes in obesity

Germ free mice gain less total body fat than conventionally-raised mice, i.e. with a conventional gut microbiota. When conventionalized (i.e. given a “normal” intestinal microbiota taken from the distal gut of a conventionally-raised mouse), these germ free mice increased total body fat by 60% within two weeks in spite of a significant reduction in food intake. In genetically obese mice, compared to normal mice, 16S rRNA gene sequence analysis revealed that the abundance of Bacteroidetes was reduced by 50% while Firmicutes increased proportionally [10]. Weight reduction paralleled correction of these changes [10]. Microbiota is also a known modulator of a number of lipid species in the serum, adipose tissue, and liver, with its greatest effect on triglyceride and phosphatidylcholine species [9].


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The human gut of obese persons is characterized by reduced bacterial diversity at the phylum-level together with altered representation of bacterial genes and metabolic pathways [11]. The ratio of Bacteroidetes to Firmicutes is dramatically decreased in comparison to that of lean controls. Both altered diversity and Bacteroidetes to Firmicutes are progressively reversed after diet-induced weight loss [12].

6.4. Microbiota, intestinal permeability and endotoxemia in NAFLD/NASH

Changes in microbiota are thought to induce inflammation in the liver via an increase in intestinal permeability [11, 13-15], which has been reported both in alcoholic liver disease and NAFLD [15]. Increased permeability can be produced by a fatty diet, alcohol, NSAIDs, certain bacterial strains or other agents. Endotoxins, among other bacterial products, are lipopolysaccharides (LPS) that originate from the cell wall of Gram-negative bacteria, and may reach intestinal or liver antigen-presenting cells. This may trigger a necro-inflammatory process that encompasses altered production of cytokines such as TNF-α, and oxidative stress [16] .

In patients with NAFLD, small intestinal bacterial overgrowth is more frequent than in controls and is associated with high levels of plasma endotoxin and TNF-α [14, 17, 18]. Dietary changes, such as high fructose intake may cause both steatosis and increased endotoxinemia. It has therefore been hypothesized that changes in intestinal microbial composition in response to a high-fat diet may account for liver lipid storage, increased permeability and elevated endotoxin plasmatic levels. However, qualitative or quantitative changes in gut microbiota, as well as changes associated with therapy, have not been explored systematically in patients with NAFLD.

6.5. Hypothesis of the present study

We hypothesize that, following a dietary intervention in patients with NAFLD, changes in gut microbiota are associated with changes in endotoxemia, liver function, inflammation and liver lipid composition.

6.6. Description of the study population

This study will include patients with obesity, as defined by BMI>30, abnormal liver function tests (LFTs) as defined by ALT > 1.5 times the upper limit of normal, and NAFLD on liver biopsy.

Although not all patients with NAFLD/NASH have abnormal LFTs, high ALT are associated with excess mortality in these patients (see section 6.1). Standard clinical management in these patients includes:

- Exclusion of other frequent causes of liver disease, such as excess alcohol intake, hepatitis B and C, and iron overload.

- Evaluation of liver fibrosis and inflammation. This usually includes a liver biopsy, allowing precise diagnosis (i.e. is this NAFLD and only NAFLD?), grading (i.e. is this NASH or only steatosis ?) and staging (i.e. scoring fibrosis) of the disease [19]. Although fibrosis itself can be estimated by non-

invasive means (such as fibroscan ), these methods are either unaccessible in patients with obesity or insufficiently validated. Thus, liver biopsy results usually guide treatment strategies, clinical monitoring, and the need to screen for hepatocellular carcinoma [20, 21]. Liver biopsy is usually scored using an unweighted score for steatosis (0–3), lobular inflammation (0–3) and ballooning (0–2), called the NAFLD activity score (NAS). It was found that the majority of biopsy samples with a total NAS score ≥5 correlated with the diagnosis of definite steatohepatitis [19].

- The first line of treatment in NAFLD/NASH is lifestyle intervention, mainly diet and exercise [6, 22, 23]. While regular physical activity per se reduces liver fat, independently of its weight-losing effects, physical exercise alone is seldom attained and sustained. In addition, initial weight reduction is an appropriate goal, primary to adding additional strain to the muscles and joints . Thus, the first objective of lifestyle intervention in these patients is often represented by a 3-4 Kg weight reduction through dietary intervention.


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6.7. Description of the intervention

Standard management of patients with NAFLD/NASH associated with obesity includes exercise, dietary counseling, psychological support and weight loss. In patients with NAFLD, small changes in body weight (i.e. 3-4 kg), induced by diet, are known to be associated with rapid improvement in LFTs. However, the desired weight reduction should be gradual (<1.6 kg/week), because faster weight loss has exacerbated liver injury [24]. It is often observed that initial weight reduction, by reducing muscular and osteo-articular strain may help many patients to sustain exercise.

In this study, we project to start with a more drastic diet than usually proposed in this population. A 1000 calories controlled diet will be offered free of charge for 3 weeks, in order to study the effects of a standardized diet in intestinal microbiota and its correlates.

Patients will consume a standardized breakfast, 2 snacks and lunch or dinner (e.g. 4 Eurodiet® products per day) and 1 meal (lunch or dinner) will be prepared by the patient according to the dietician’s recommendations (based on proteins and vegetables).

Patients will be followed on a weekly basis by a dietician specialized in obesity management. A 3-day diet diary will be filled-in on weekly basis and during the 3 weeks of the intervention period. The composition and preparation of the 1 meal per day (non-Eurodiet® product, supposed to be prepared by the patient) will be discussed regularly in order to maintain the daily caloric intake ~1000 kcal

Table 1 Composition of hypocaloric diet prescribed to study participants

6.8. Relevance of the results expected from this study

In animals, indigenous gut microbial community, including the bateroidetes / firmicutes balance can be significantly reshaped [7] by simple interventions, which encompass oral transplantation and dietary changes.

In patients with NAFLD/NASH, identifying changes in intestinal microbiota at baseline and in response to diet, could be a first step to delineate pre-disease biomarkers with predictive power [25], thus favoring preventive health management of populations. In addition, once specific changes in microbiota have been identified, re-engineering of the imbalanced gut microbiomes with specially designed foods/diets and/or metabolically active commensal bacteria may become accessible.

Nb of products/day

Energy (kcal)

Proteins (g)

Lipids (g)

Carbo-hydrates

(g)

Sugar (g)

Fructose (g)

Polyols (g)

Starch (g)

Fibres (g)

4 1059 125.5 42.3 38.0 20.1 3.1 4.6 1.4 24.8


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7. STUDY OBJECTIVES AND PURPOSE

7.1. Primary Objective

To assess the impact of dietary intervention on the relative abundance of fecal Bacteroidetes (as expressed by the bacteroidetes to firmicutes ratio) in patients with obesity, abnormal liver function tests and NAFLD.

7.2. Secondary Objectives

1. To compare fecal microbiota patients with NAFLD and with or without NASH at baseline 2. To compare fecal microbiota from patients with NAFLD and with or without NASH after dietary

intervention 3. To measure liver fat content at baseline and after dietary intervention using non-invasive MRI 4. To assess changes in liver function tests, ultrasensitive CRP and serum cytokines ((TNF-α, IL-6, IL-

10, PAI-1, MCP-1, LPS) and expression of hepatic metabolic parameters (PGC-1α, PPAR-γ, CPT-1, MTP-1), in relation to changes in microbiota and liver fat content

5. To measure oro-caecal transit time, an index of small intestinal pullulation at baseline and after dietary intervention in patients with NAFLD/NASH

6. To measure intestinal permeability changes in relation to changes in gut microbiota 7. To assess body composition changes, as assessed by bioelectrical impedance analysis, in relation

to changes in microbiota and liver lipid composition.

8. STUDY DESIGN

8.1. Primary endpoint

Fecal microbiota will be evaluated as the ratio of Bacteroidetes to Firmicutes within the fecal flora.

8.2. Secondary end-points

1. The relative abundance of fecal microbiota individual species will be assessed at baseline in patients with NAFLD only and patients with NASH.

2. Variations in species abundance will be assessed from baseline to week 3 of dietary intervention.

3. Liver fat content will be measured after dietary intervention using non-invasive MRI and compared to that obtained at screening (to be validated against reference liver biopsy and considered to reflect baseline).

4. Changes from inclusion visit to end of treatment visit in transaminases, serum CRP and serum LPS will be assessed.

5. Intestinal bacterial overgrowth will be assessed at baseline and after 3 weeks of intervention, using orocecal transit time, a marker of bacterial activity measured by non-invasive breath-test.

6. Intestinal permeability will be measured at baseline and after dietary intervention.

7. Anthropometric and body composition measurements will be performed at baseline and after 3 weeks.

8.3. Study characteristics

This will be a single arm before-after study.


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8.4. Study plan

The study will be conducted in accordance with the ethical principles stated in the Declaration of Helsinki 1964, as revised in Seoul, 2008, and in compliance with the protocol, Good Clinical Practices (GCP), the LPth (Loi sur les produits thérapeutiques 812.21) and the OClin (Ordonnance sur les essais cliniques des produits thérapeutiques 812.214.2).

The expected duration of the study is one year. Patients will be recruited from Jan 2011 to March 2011. For individual participants, the duration of the study will be of approximately 10 weeks depending on the duration of the screening period. This will include:

8.4.1. Screening

During the screening period, the clinical and laboratory exams conducted in routine clinical care will take place. Standard of care in patients with obesity and abnormal liver function tests usually includes a physical examination, liver ultrasound and a liver biopsy (only if ALT > 1.5 times the upper limit of normal), and laboratory analyses of blood samples (hepatitis B and hepatitis C markers, transferrin saturation and ferritin, haematology and chemistry). The results of these routine tests will be used to assess the patient’s eligibility.

Patients information documents will be provided during the screening period, to ensure patients have enough time to read the documents and prepare questions.

8.4.2. Inclusion / Baseline visit

If the results of the screening analyses indicate that the patient is potentially eligible to take part in the study, an inclusion visit (baseline visit) will be conducted. During this baseline visit, informed consent will be sought. If the patient agrees to take part in the study, all tests listed on table 1 will be conducted, including MRI to assess liver fat content and bioelectric impedance to assess body composition.

Dietary intervention will start 0 to 5 days after the baseline/inclusion visit, i.e. after completion of all tests.

8.4.3. Dietary intervention (3 visits)

During these visits, patient compliance will be assessed and tolerance to the standardized diet will be checked (see 12. Assessment of safety). The “end of treatment” visit will be conducted 3 weeks after diet initiation.

8.4.4. Follow-up

Three weeks after the end of the hypo-caloric diet, a follow-up visit will take place and again safety parameters will be checked.

8.5. Discontinuation of the study

Study discontinuation could result from:

- decision of the sponsor in case of new safety information is published that raises concern for participants

- decision of the sponsor for safety reason concerning all participants

In case of premature termination of the study, the Ethics Committee will be informed.


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9. DATA COLLECTED IN THE CASE REPORT FORMS (CRF)

Source documents are “original documents, data and records (e.g. hospital records, clinical and office charts, laboratory notes, memoranda, subject’s diaries or checklist, pharmacy dispensing records, recorded data from automated instruments, microfiches, photographic negatives, microfilms or magnetic media, X-rays, subject files and records kept at the pharmacy, at the laboratory and at medico-technical departments involved in clinical trials) “ *ICH E6 1.52+

Source data are “all information in original records and certified copies of original records of clinical findings, observations, or other activities in a clinical trial necessary for the reconstruction and evaluation of the trial. Source data are contained in source documents” *ICH E6 1.51+

The following data will be recorded directly on the case report form (CRF):

- Compliance to diet, as reported by the patient to the dietetician

- Body weight

- Blood Pressure (BP), Pulse Rate (PR)

- Adverse Events

- Concomitant medications started after baseline

- Treatment compliance

The following data will be transcribed from source documents to the CRF:

- Personal details and demographic characteristics (age, sex, BMI, smoking and drinking habits)

- Medical history

- Results of clinical examination

- Concomitant medications at baseline

- Results of laboratory investigations

- Results of liver biopsy

- Results of bioelectric impedance

- Results of breath test

N.B. Results of metagenomic studies will not be transcribed on the CRF, but kept separately as an anonymous database.

The investigator will allow the monitors, the representatives of the Ethics Committee and of the Regulatory Authorities to have direct access to source data / documents and CRF/study documents.


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Table 2: Investigational schedule table

Assessment / Procedure Screening (day -28 to -1)

Inclusion visit (day -5 to 0 of diet)

Treatment 1 (day 7)

Treatment 2 (day 14)

End of diet (day 21)

Follow-up visit (day 42)

CLINICAL EVALUATION Informed consent

x

Medical History x

Physical examination x x x x x x

Concomitant medications x x x x x x

Liver Biopsy x

DIET RECALL x x x x x

Compliance x x x

Adverse Events x x x x

STANDARD LAB Complete blood count (CBC)

x x x

Thyroid Stimulating Hormone (TSH) x

PT and INR x x x

ASAT, ALAT, GGT, bilirubin x x x x

Albumin x

Creatinine x x x

Plasma cholesterol and triglycerides x x

Urine alcohol x x x

US-CRP x x x

Hbs Ag and HCV Ab x

Transferrin sat. (< 50%) x

Ferritin (< 500 ng/ml) x

SPECIFIC TESTS Serum cytokines

x x

Body composition (Bioelectric impedance) x x x

Breath-test pullulation x x

Intestinal permeability x x

MRI x x

Stool collection x x x


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10. SELECTION AND WITHDRAWAL OF PARTICIPANTS

10.1. Inclusion criteria

Patients need to meet the following criteria will be eligible to participate in the study:

- Age > 18 years, < 60 years

- BMI > 30

- ALT > 1.5 times the upper limit of normal

- NAFLD present at liver biopsy. The NAFLD activity score (NAS) score will be used [19]. It includes semi-quantitative assessment of steatosis (0–3), lobular inflammation (0–3) and ballooning (0–2). NAFLD will be defined as a steatosis score > 2. NASH will be defined as a NAS score > 5 [19]

10.2. Exclusion criteria

The following criteria must not be present in patients selected for the study:

Inability or unwillingness to give written consent

Known diagnosis or treatment for bulimia

Alcohol Consumption > 14 units per week (2 units per day). In addition, active alcohol consumption will be ruled out by urine analysis

Presence of HBsAg, OR anti-HCV antibodies

Blood Ferritin level > 500 ng/ml OR Transferrin saturation > 50%

Moderate to severe renal failure as defined by creatinine clearance < 60 ml/min

Hypothyroidism as defined by TSH > 6 mU/L

Any previous surgery interfering with intestinal absorption, such as gastric by-pass

Any known gastro-intestinal disease interfering with intestinal absorption (i.e. celiac disease)

Any medication with Orlistat, Colestipol, anticoagulants, Alfa-amylase, Alfa-glucosidase inhibitors known to alter intestinal digestion / absorption

Use of antimicrobial agent in the 4 weeks preceding inclusion

Parenteral nutrition or other ongoing dietary intervention

Diarrhoea (defined as more than 2 stool per day) within 7 days before enrolment

Use of substances known to alter intestinal permeability, such as corticoids, NSAIDs

Diagnosis of cancer upon inclusion in the study OR any other severe condition interfering with the normal conduct of the study

Known pregnancy

Active participation in another clinical study

10.3. Withdrawal criteria

Treatment may be interrupted and participants may be withdrawn from the study in case of:

- withdrawal of consent


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- occurrence of a SUSAR (see section 13. Safety assessments for a definition)

- weight loss superior to 1.6 kg per week

- any other medical concern that may significantly interfere with the completion of the dietary intervention or with microbiota analysis (for ex. medical need to administer antibiotics).

- pregnancy revealed during dietary intervention

- non-compliance that may become obvious to the patient himself or to the dietetician.

When the investigator has no news of the participant, he/she must make every effort to contact him/her, to establish the reason for the discontinuation of treatment, and to suggest the participant comes to an end-of-study visit. If all these attempts to contact the participant fail, the investigator can then declare the participant “lost to follow-up”. The investigator should document all these attempts in the corresponding medical file.

In the case of withdrawal from the study due to an adverse event, the investigator must make every effort to collect the information relating to the outcome of the event. This information is recorded in that part of the CRF which concerns adverse events. If the investigator cannot collect the information from a visit, he must collect it from the doctor ensuring the follow-up of the participant.

10.4 Procedure for replacement of withdrawn patients

In case a patient is withdrawn because of any of the above criteria, he/she will be replaced by a newly recruited patient meeting the inclusion/exclusion criteria listed above. This is meant to meet the number of 20 patients who achieve completion of the study and can be analysed

11. TREATMENT OF PARTICIPANTS

11.1. Treatment administered

Food intake standardization is needed to reduce the impact of food products on the observed qualitative and quantitative variability of the microflora. The easiest way of obtaining a significantly reduced food intake is to prescribe a commercially available diet either as a powder or a liquid. The Eurodiet® program will be used as the basis for standardized hypo-caloric diet during the 3-weeks intervention period. Its composition is described in table 1.

11.2. Treatment management

Professional dieteticians will support the subjects on a weekly basis throughout the 3 weeks. The Eurodiet products will be distributed during these weekly visits. At each visit, compliance will be checked as described under section 11.4. Participants interrupting their diet will be asked to bring unused products back to the hospital.

11.3. Concomitant treatments and prohibited medications

Details of all previous and concomitant treatments will be recorded by the investigator in the CRF.

The following medications will be avoided, unless clinically indicated during the 3 week dietary intervention:

- drugs known to interfere with intestinal permeability or absorption, including NSAIDs, orlistat, and colestipol, Alfa-amylase and alfa-glucosidase inhibitors.

- anti-vitamin K agents, since low-fat diet may alter vitamin K status.


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- antimicrobial agents

In case of clinical indication, these substances will be administered as prescribed by the clinician in charge, and the patient will be withdrawn from the study (see paragraph 10.3).

11.4. Treatment compliance

Food intake control is needed as obese patients are known to have a limited capacity to control their food intake (compliance). Evaluation of the compliance to the prescribed food items will be assessed by

- Counting the number of packages eaten per week. For each intervention visit, patients will be asked to return all food packages (eaten and not eaten)

- Measuring body weight changes (kg)

- Measuring fat-free mass and fat mass changes assessed by bioelectrical impedance analysis (BIA) (kg)

- Asking patients to complete a weekly questionnaire (“3 day-food recall” including 2 week days and 1 weekend day) throughout the 6 weeks of the study.

12. ASSESSMENT OF END-POINTS

12.1. Microbiota composition

12.1.1. Stool sampling, DNA extraction and amplification

Stools will be collected in sterile plastic 50 mL containers and frozen at -20°C until processing. DNA will be extracted from 100 mg feces using the FastDNA Spin Kit for Soil (MP Biomedicals) according to the manufacturer’s instructions. The bead-beating step will be carried out in a FastPrep Instrument (Bio 101) as described by Andersson et al. [26]. Extracted DNA will be stored at -20°C until usage.

PCR amplification and high-throughput sequencing. PCR primers will be chosen from the conserved areas flanking the V1-3 region so as to match most known 16S rRNA sequences of intestinal bacteria [27]. Composite PCR primers will also include the 454 Life Science adaptors, a 8-base sample-specific sequence tag that will allow distinguishing different PCR products when they are sequenced in parallel during a single run (i.e. for multiplexing). The V1‐3 amplicons will be generated in PCR reactions using the PrimeStar HS Premix (Takara), 2 μL of extracted template DNA and 0.5 μM of each forward and reverse primer. PCRs will be run using 30 cycles of 98°C for 10 seconds, 60°C for 15 seconds, and 72°C for 1 min. Purified samples will be pooled and sequenced using the Roche GS FLX instrument which produces more than 400’000 reads of over 200 bases. Pyrosequencing will be performed as previously described [Margulies, M. et al. Nature 437, 376–380 (2005)]. Briefly, samples will be nebulized to 200 nucleotide fragments, ligated to adaptors, fixed to beads, suspended in a PCR reaction mixture-in-oil emulsion, amplified, and sequenced using a GS Flex (Roche). The Newbler de novo shotgun sequence assembler (Roche) will be used to assemble sequences based on flowgram signal space. This process includes overlap generation, contig layout, and consensus generation.

12.1.2. Data Analysis

Sequences containing uncalled bases, incorrect primer sequences or runs of ≥10 identical nucleotides will be removed. After trimming primer sequences, reads shorter than 200 nt will also be discarded. We will then create a multiple alignment of the sequences using MUSCLE [28] using the following parameters: –maxiters 2 and -diags. Sequences corresponding to E. coli 16S rDNA positions 300-514


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will be extracted from the multiple alignments and, after removing duplicates, realigned using MUSCLE to calculate distances. Bacterial diversity will be assessed using RDP pyrosequencing tools Aligner, Complete Linkage, Rarefaction and Shannon Index and Chao1 Estimator [29, 30]. Taxonomic composition and abundance of the fecal bacterial community will be analyzed by the MG-RAST [31] and the RDP classifier [29]. In order to measure phylotype richness in the fecal communities and for easier comparisons with other metagenomic studies, Chao1 richness estimation and rarefaction curves will be calculated at 95%, 97% identity that arbitrarily correspond to the genus and species level. The Shannon index, the evenness index and the Good’s coverage index will be used to evaluate community diversity, evenness and sampling completeness.

12.1.3. Validation of the metagenomic approach

Validation of the metagenomic approach. Stool microbial composition changes detected by sequencing will be validated by using quantitative PCR (qPCR). Taxon-specific non-16S rRNA targets will be used to validate the data obtained by pyrosequencing.

12.2. Serum LPS concentration

Serum (2 mL) will be immediately frozen at -20°C and stored at -80°C until usage. LPS serum concentration will be measured by the group of Prof Patrice Cani (UC Louvain, Belgium) using the Endosafe-MCS (Charles River laboratories, Lyon, France) based on the Limulus amaebocyte Lysate (LAL) kinetic chromogenic methodology that measures colour intensity directly related to the endotoxin concentration in a sample. Serum will be diluted 1/10 with endotoxin free buffer to minimize interferences in the reaction (inhibition or enhancement) and heated 15min at 70°C. Each sample will be diluted with endotoxin-free LAL reagent water (Charles River Laboratories) and treated in duplicate; two spikes for each sample will be included in the determination. All samples will be validated for the recovery and the Coefficient Variation determination. The lower limit of detection will be assessed (it is typically around 0.01EU/mL

12.3. Anthropometric measurements and body composition

Body height will be measured to the nearest 0.5 cm and body weight to the nearest 0.1 kg on a balance beam scale. Subjects will be indoor clothing and no shoes.

Fat-free mass (FFM) and body fat (BF) will be assessed by BIA as previously described [32]. Whole-body resistance will be measured with four surface electrodes placed on the right wrist and ankle. Briefly, an electrical current of 50 kHz and 0.8 mA will be produced by a generator (Nutriguard, Data Input, Germany) and applied to the skin by using adhesive electrodes (3M Red Dot T, 3M Health Care, Borken, Germany) with the subject lying supine [32] . The skin will be cleaned with 70% alcohol before application of the contact electrodes.

FFM prediction equations

FFM will be determined with the Geneva formula Single prediction equation for bioelectrical impedance analysis in adults aged 20–94 years [32] as follows :

FFM (kg) = - 4.104 + (0.518 x height2 (cm)/resistance (Ω)) + (0.231 x weight (kg)) + (0.130 x reactance (Ω)) + (4.229 x sex *1 for men and 0 for women+).

BF (kg)= body weight (kg) - FFM (kg).Percentiles of FFM and BF will be determined according to references values as previously published [32].

12.4. Cytokines ELISA

We will use the BioRad BioPlex® (Luminex® technology) to analyze serum cytokines as previously described [33].


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12.5. Liver fat content

Liver fat content will be measured by MRI at baseline and at 3 weeks using standard MRI software available in our hospital. Importantly, MRI results will be validated against liver biopsy at baseline, allowing non-invasive measurement of liver fat at 3 weeks

12.6. Intestinal permeability.

Polyethylene glycol (PEG) 3350 will be used as the permeability probe. PEG 400 will serve as an internal control for possible changes in intestinal motility and absorption. The two components of PEG will administered orally; urine will be collected for the following 8 hours, and the concentration of each PEG molecule will be measured using high-performance liquid chromatography (Waters HPLC system). The ratio of urinary excretion of each molecule will be determined. The differential urinary excretion of the two PEG molecules (the PEG 3350/PEG 400 ratio), which corrects for possible changes in intestinal motility and absorption, will provide the "intestinal permeability index".

12.7. Orocecal transit time (Breath test)

Orocecal transit time will be measured by Lactose (13C) ureide Breath test as described earlier [34].

13. ASSESSMENT OF SAFETY

13.1. Safety measurements

Safety measurements performed during the study are indicated in Table 2. During both treatment and follow-up the following will be monitored:

- weight

- BP

In addition, at the end of the diet we will measure

- ASAT and ALAT

- Bilirubin

- PT and/or INR

13.2. Adverse Events

13.2.1. Definitions

According to the International Conference on Harmonization definitions:

A Serious Adverse Event (SAE) is “any untoward medical occurrence that at any dose: 1. results in death or 2. is life-threatening* or 3. requires in-patient hospitalisation or prolongation of existing hospitalisation or 4. results in persistent or significant disability/incapacity or 5. is a congenital abnormality/birth event or 6. is a medically important event.” A medically important event is defined as a medical event that may not be immediately life-threatening or result in death or hospitalisation but, based upon appropriate medical and scientific judgement, may jeopardise the patient/subject or may require intervention (e.g., medical, surgical) to prevent one of the other serious outcomes listed in the definition above. Examples of serious events include angioedema.

An Adverse Event (AE) is “any untoward medical occurrence in a patient or clinical investigation subject administered a pharmaceutical product and which does not necessarily


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have a causal relationship with the treatment” An AE can therefore be any unfavourable and unintended sign (including an abnormal laboratory finding), symptom, or disease temporally associated with the use of a medicinal product, whether or not related to the medicinal product. An example would be an unrelated pulmonary infection contracted during the follow-up period.

Concerning Adverse Drug Reactions (ADR), the ICH states that “all noxious and unintended responses to a medicinal product related to any dose should be considered as an adverse reaction”. An unexpected Adverse Drug Reaction is “an adverse reaction, the nature and severity of which is not consistent with the applicable product information (e.g. Investigator’s Brochure)”. In the present study, expected ADR include diarrhoea or other gastro-intestinal symptoms.

A Serious, Unexpected, Suspected Adverse Reaction (SUSAR) is an adverse reaction which is both serious and unexpected. It includes all serious adverse events that may be related to tocofersolan and that are not described under the Background section of the study protocol.

* the term "life threatening" in the definition of "serious" refers to an event in which the patient was at risk of death at the time of the event; it does not refer to an event which hypothetically might have caused death if it were more severe.

Note: The term “severe” is often used to describe the intensity (severity) of a specific event (as in mild moderate or severe myocardial infarction); the event itself, however, may be of relatively minor medical significance (such as a severe headache). This is not the same as serious, which is based on patient/event outcome or action criteria usually associated with events that pose a threat to patient’s life or functioning. Seriousness (not severity) serves as a guide for defining regulatory reporting obligations

13.2.2. Recording of Adverse Events

AEs, both serious and non-serious, will be recorded in the CRF by the investigator, whether or not they are related to the treatment.

The investigator must document as an AE:

- any unfavourable and unintended sign, including an abnormal finding from an additional examination (lab tests, X-rays, ECG, …) deemed clinically relevant by the investigator,

- any symptom or intercurring disease,

- any worsening during the study of a symptom or a disease already present when the participant entered the study (for example worsening of cirrhosis)

and which:

- is detected during a study visit or at an additional examination

- occurred since the previous study visit and is notified by the participant

Grading of AEs will be based on the Common Terminology Criteria for Adverse Events (CTCAE) v.4. For each AE, the relationship to dietary intervention will also be evaluated by the investigator and recorded in the CRF.

13.2.3. Notifying Serious Adverse Events

The investigator should inform the sponsor-investigator immediately (within 24 hours) in case a SAE occurs. All SAEs are to be reported annually to the Ethics Committee by the investigator.

In case of a SUSAR, the investigator must notify the Ethics Committee within 15 days of the event. If the SUSAR leads to death, the event must be notified as soon as possible and within 7 days.


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13.2.4. Follow-up of Adverse Events

The investigator must ensure that follow-up of the participant is appropriate to the nature of the event, and that it continues until resolution. If the AE has not resolved at the participant's final visit in the study, the participant must be followed up suitably and any information on the outcome of the event will be noted in the CRF.

If the follow-up of the participant is not done by the investigator him/herself (e.g. in the case of a surgical intervention), the investigator must establish and maintain contact with the physician and/or department in charge of follow-up of the participant, in order to have additional information and report it on the corresponding forms appended to the CRF. The investigator is responsible for ensuring that the patients attend all follow-up visits. If needed, the investigator should visit the patient in his/her home to conduct the follow-up visits and to establish the reason for non-attendance.

14. STATISTICS

14.1. Determination of sample size

No previous study on changes in fecal microbiota and its changes after diet has been performed in patients with NAFLD/NASH. We therefore decided to calculate the sample size based on changes in liver function tests known to occur in patients with NAFLD/NASH subjected to diet and exercise. This offers the advantage that a clinically significant outcome is chosen for sample size calculation. It entails that the study is only powered to evidence changes in microbiota which could be correlated to clinically significant changes in liver function.

Previous studies on dietary interventions in patients with NAFLD/NASH showed that moderate weight loss is attainable and associated with significant improvements in serum aminotransferases, insulin resistance, adipocytokines as well as liver steatosis. According to these studies, a baseline standard deviation of 0.5 in serum aspartate aminotransferase (ALAT) values can be expected. Diet, together with a 2-3 kg decrease in body weight can be associated with a 33% decrease in ALAT. For the study of a continuous response in ALT from matched pairs of study subjects, with a standard deviation of 0.5 and a true difference in the mean response of matched pairs of 0.3, we will need to study 20 pairs of subjects to be able to reject the null hypothesis,with a power of 80%. The Type I error probability associated with this test of this null hypothesis is 0.05.

14.2. Statistical comparisons for metagenomic studies.

Samples from different individuals will be compared in terms of the phylogeny of their microbial communities using UniFrac, where larger values are assigned to changes in more distant taxa [35]. Based on the presence and absence of operational taxonomic units (unweighted UniFrac), samples will be hierarchically clustered. Hierarchical clustering and principal coordinates analysis will be carried out using UniFrac, as previously described [35]. Distances between samples will be subject to principal component analysis including abundance data (weighted Unifrac). The taxonomic composition will be assigned using the RDP Classifier [29] with a 50% confidence cut-off. We will compare fecal bacterial communities to determine a “core microbiome” in terms of the shared species within NASH/NAFLD patients and healthy controls. Effects of the dietary intervention on the intrapersonal flora diversity will be assessed in UniFrac, as described above, by comparing the two time point samples. Average changes in relative Bacteroidetes content will be provided to compare groups.


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14.3. Other statistical Analyses

All primary analyses will be conducted on per-protocol basis, i.e. patients that have not been compliant to the diet will not be included in these analyses.

We will compare baseline and end-of-treatment changes for both bacterial genders and subspecies using paired-sample Wilcoxon signed-rank test. This will also be analysed according to the presence of NASH or NAFLD only. We will present a multivariate analysis explaining the delta change of bacterial genders, taking into account the baseline covariates, such as age, BMI, and others. We will also compare changes in both microbiota and liver fat content between patients that change weight in response to diet and those who don’t.

Correlation studies will be performed using Pearson or Spearman tests including the following: - Changes from inclusion visit to end of treatment visit in transaminases, serum CRP and

serum LPS will be assessed and correlated to changes in fecal microbiota (primary end-point) and in liver lipids (end-point 4).

- Changes from inclusion visit to end of treatment visit in intestinal permeability, orocecal transit time, and body composition will be assessed and correlated to changes in fecal microbiota (primary and secondary end-points) and in liver lipids (end-point 4).

All analyses will be done using SPSS software.

15. QUALITY CONTROL AND QUALITY ASSURANCE

15.1. Monitoring

Prior to study start, a clinical monitor will visit the site and facilities where the study will take place in order to ensure compliance with the protocol and GCP requirements. A training session will be organized during which the study protocol and the study procedures will be discussed with all the study staff.

Study monitoring will be carried out at regular intervals, depending on the recruitment rate, and arranged between the investigator and monitor. The monitor will:

inspect the site, the facilities and the material used for the study,

consult all of the documents relevant to the study,

check that the CRF have been filled out correctly,

directly access source documents for comparison of data therein with the data in the case report forms,

verify that the study is carried out in compliance with the protocol, GCPs and Swiss regulatory requirements.

15.2. Audit and Inspection

The investigator must allow the representatives of the Regulatory Authorities, members of the Ethics Committee and persons responsible for the audit:

to inspect the site, facilities and material used for the study,

to meet all members of his/her team involved in the study,

to have direct access to study data and source documents,

to consult all of the documents relevant to the study.


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16. ETHICS

16.1. Ethics Committee

The study protocol, the participant information and consent form, and the list of investigators document will be submitted to the Internal Medicine Department Ethics Committee by the Principal Investigator. The study will not start before written approval by the Ethics Committee has been obtained, the local regulatory requirements have been complied with, and the signature of the clinical study protocol of each contractual party involved has been obtained.

16.2. Patient Informed Consent

The investigator or a person designated by him/her is to collect written consent from each patient before, or at the latest on, the day of the baseline visit. Prior to this, the investigator or his/her delegate must inform each patient of the objectives, benefits, risks and requirements imposed by the study, as well as the nature of the diet intervention.

The patient will be provided with an information and consent form in clear, simple language. He/she must be allowed ample time to inquire about details of the study and to decide whether or not to participate in the study.

Two original information and consent forms must be completed, dated and signed personally by the patient and by the person responsible for collecting the informed consent. The patient will be given one signed original information and consent form. The second original will be kept by the investigator.

17. DATA HANDLING AND RECORD KEEPING

17.1. Study Data

The investigator will be responsible for reporting the patient’s personal details and ID number in a subject identification list. To ensure confidentiality, this list will be kept in a separate, locked cupboard together with the signed informed consent forms and the investigator should be the only one to have access to it. The investigator must agree to provide access on site to the auditor, representatives from the Ethics Committee and/or the Regulatory Authorities.

A Case Report Form (CRF) will be designed to record all the data described by the protocol and collected by the investigator. A CRF will be completed for each participant. The CRF, together with all trial related forms and Standard Operating Procedures (SOP) will be produced by the Service de Gastroentérologie before being distributed to all investigators.

The investigator, or the designated person from his/her team, agrees to complete the CRF sheets, at each participant visit, and all other documents provided by the sponsor (e.g. documents relating to the treatment management and biological samples shipping). The investigator will ensure accuracy, completeness, legibility and timeliness of the data reported in the CRF and in all required documents. All corrections and alterations of data on the CRF or source document must be made by the investigator or by the designated person from his/her team and must be dated and signed.

All laboratory data will be recorded in CRF. The reference ranges for the laboratory should be available as an aide mémoire so that values outside the normal ranges can be evaluated and commented upon by the investigator.


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17.2. Data Management

All data will be entered and stored in a database and data validation will be carried out by the study monitor.

As a result of data validation, data may require some changes. A data clarification form will then be issued and sent to the investigator for confirmation or correction and signature. In the case of obvious errors changes will not be subject to the investigator’s approval. A record of these data changes will be provided to the investigator when the study is completed.

17.3. Archiving

The investigator will keep all information relevant to the study for at least 10 years after the end of the study. As required by Swiss regulations, the investigator must arrange for the retention of the subject identification codes for a sufficient period of time to permit any medical follow-up which may be warranted, including follow-up for delayed toxic reactions. It must be possible to identify each trial subject by name against subject and product container identification codes, treatment assignment, and the CRF. Subject files and other supporting data must be kept for at least 10 years after the end of the study.

The sponsor must make appropriate arrangements for the retention of all other essential documentation pertaining to the clinical trial in a form which can be retrieved for future reference. The protocol, documentation, approvals and all other essential documents related to the trial, including certificates that satisfactory audit and inspection procedures have been carried out, must be retained by the sponsor. Data on adverse events must always be included. All data and documents should be made available if requested by relevant authorities.

17.4. Handling of Biological Samples

The samples collected in view of future analyses will be anonymized and labelled with the study title, date of collection and patient’s ID number. These samples will be kept in a locked freezer in the Service of Gastroenterology to which only the investigator and his team will have access.

Part of the stool and serum samples will be kept in case of:

- A result needs to be replicated due to technical problems

- A result needs to be validated by further metagenomic analyses

- Additional sub-species analyses need to be conducted

- For future research

Aliquots may be shipped to INRA, France for additional analyses of gut microbiota. These samples will be kept for a maximum of 10 years before being destroyed.

18. INSURANCE

Clinical trials insurance will provided by the University Hospitals of Geneva to cover all pecuniary consequences of public liability that may incur under the terms of common law on account of damage which may result from the clinical trial.


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19. OWNERSHIP OF THE RESULTS – PUBLICATION POLICY

Geneva University Hospitals, acting as the study sponsor, assumes full responsibility relating to this function and retains exclusive property rights over the results of the study, which it may use as it deems fit.

After obtaining approval by Ethics Committees, the study will be registered on the International Clinical Trials Registry Platform (www.clinicaltrial.gov). This will ensure that results comply with the International Committee of Medical Journals Editors requirements.

Any draft publication and/or communication shall be submitted to the sponsor’s representative at least 30 days before the forecasted date of communication and/or submission for a publication. The sponsor’s representative shall make comments on the draft within 15 days, for a publication, and 7 days, for an abstract, of receipt of the project. The investigator, who submitted the draft, shall take the sponsor's comments into due consideration. In any case, should the investigator who submitted the draft decide not to modify the project according to the sponsor's comments, it shall provide the sponsor with the grounds of its decision in writing

Name of the signatories of the publication of the core study:

Jacques Schrenzel and Alain Golay will share first authorship, Jean-Paul Vallée, Patrick Linder, Laurence Genton, Laura Rubbia-Brandt, Vladimir Lazarevic, Sophie Cunningham. Claude Pichard and Antoine Hadengue will share last authorship.

It is expected that additional publications may derive from the core study. It is understood that first and last signature of these additional papers will remain in the hands of respective co-investigators. For example

- Jean-Paul Vallée will be in charge of the validation of MRI techniques

- Claude Pichard will be in charge of subanalysis of metabolic effects of the diet etc.


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21. BUDGET

Assessment / Procedure Cost per

procedure

Baseline

measurements

3 weeks

measurements

Total Cost

Year 1 Year 2

STUDY NURSE (50%) 60’000 x

LIQUID MEALS 500 25’000 x

STANDARD LAB 500 30 20 25’000 x

SPECIFIC TESTS

Cytokines 600 20 20 24’000 x

Body composition (BIA) 50 30 20 2’500 x

Indirect calorimetry 450 30 13’500 x

Orocecal Breath-test 180 30 20 7’200 x

Intestinal permeability 180 20 20 7’200 x

MRI 800 30 20 40’000 x

ENDOTOXINEMIA 160 20 20 6’400 x

METAGENOMICS 5800 20 20 232’000 x

DATA ANALYSIS 5’000 x

CLINICAL MONITORING (30%) 35’000 x

TOTAL 482’800 215’400 267’400

Financed by:

- FLAGS foundation

- Radiology Dpt

- Nutrition Unit

92’000

40’000

25’000

40’000

25’000

92’000

To be financed by CRC 325’800 150’400 175’400

In addition the Centre de Recherche Clinique (CRC) will be solicited for protocol submission to the Ethics Committee, Clinical and Data monitoring and statistical analysis.

clinical study protocol - hug · clinical study protocol confidential study title: effect of...

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