Synbiotics and Fecal Microbiota Transplantation to Treat Non-Alcoholic Steatohepatitis

Study Overview
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Brief Summary
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The goal of this clinical trial is to investigate the therapeutic potential of A. soehngenii and pasteurized A. muciniphila combined with B. animalis subsp. lactis and fructo-oligosaccharides with and without conditioned vegan lyophilized fecal microbiota transplantation capsules to reduce NASH in patients with fibrotic NASH. The main questions to answer are: Can NASH be treated by altering the gut microbiota using LFMT capsules? Can NASH be treated using a syntrophic cocktail of synbiotics and will these strains strengthen the effect of FMT? What are the underlying mechanism by which the aforementioned treatments attenuate NASH? Participants will be treated with FMT-capsules or placebo, and all participants will receive a cocktail of 3 strains of probiotics and one type of prebiotic.

Detailed Description
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Main objective To investigate the therapeutic potential of A. soehngenii and pasteurized A. muciniphila combined with B. animalis subsp. lactis and fructo-oligosaccharides with and without conditioned vegan lyophilized fecal microbiota transplantation (LFMT) capsules to reduce NASH in patients with NASH and NASH-fibrosis. Secondary objective To investigate the mechanisms of A. soehngenii and pasteurized A. muciniphila combined with B. animalis subsp. lactis and FOS with and without conditioned vegan LFMT capsules in reducing NASH in patients with NASH and NASH-fibrosis. Study design: Double-blind randomized placebo-controlled intervention study. Study population: Patients between age 18-75 years with biopsy-proven NASH obtained up to 32 weeks before screening based on tandem reading of two expert liver pathologists: SAF Steatosis score ≥1, Activity ≥2, Fibrosis <4; 50% of participants should at least have NASH fibrosis stage 1, 2 or 3 according to the NASH CRN fibrosis staging system. Exclusion criteria are: Current or history of significant alcohol consumption for a period of more than 3 consecutive months within 1 year before screening (significant alcohol consumption is defined as more than 2 units/day for females and more than 3 international units/day for males, on average; 1 international unit contains ±14 grams of alcohol), liver cirrhosis or hepatocellular carcinoma, hepatitis B and/or C, auto-immune hepatitis, Wilson's disease, primary sclerosing cholangitis, primary biliary cholangitis, alpha-1-antitripsine deficiency and hemochromatosis, history of liver transplant, current placement on a liver transplant list, use of pre-, pro- or synbiotics, use of systemic antibiotics 3 month prior to randomization, use of tamoxifen, methotrexate or amiodarone, prior or planned bariatric surgery, diabetes mellitus with active GLP-1 receptor agonist treatment, bleeding disorder, International normalized ratio (INR) of prothrombin time >1.4 or platelet count <50 109/L at screening, anti-platelet/coagulant therapy use which cannot be temporarily discontinued, any major cardiovascular event within 6 months prior to screening (e.g. myocardial infarction, cerebrovascular accident), prolonged compromised immunity (e.g. recent cytotoxic chemotherapy, HIV-infection with a CD4 count < 240), active or prior history of invasive malignancy (except for curatively treated in situ carcinomas [e.g., cervix] or non-melanoma skin cancer) unless a complete remission was achieved, surgery scheduled for the trial duration period, except for minor surgical procedures in the opinion of the investigator, pregnant or nursing women, any condition which, in the investigator's opinion, might jeopardize participants' safety or compliance with the protocol, and participation in another concomitant clinical trial. A total of 48 patients will receive synbiotic treatment with A. soehngenii, pasteurized A. muciniphila, B. animalis subsp. lactis and fructo-oligosaccharides, and will be randomized 1:1 to treatment with FMT or placebo, stratified for proton-pump inhibitor use, metformin use, and histopathological fibrosis score. Intervention: Recipient participants From day -3 before the first FMT until completion of the study, all recipient participants will receive an oral daily single dose of 5 g FOS. Subsequently, participants will be randomized to lean donor FMT capsules (donors conditioned with WholeFiber) or placebo (blinded design)1. At baseline (day 0) and at week 8 (day 56) and week 16 (day 112) participants will ingest 21 LFMT or placebo capsules. The study visits will be 8 weeks apart, although a margin of -3 days to +3 days is implemented to take participants schedule and availability into account. In addition, participants will daily take 2 capsules of LFMT or placebo during the whole study period (24 weeks). The investigators have previously observed that gut microbiota composition in the recipient is affected up to 8-12 weeks after donor FMT1, so this time window ensures a stable donor gut microbiota composition during the study. During the 24-week intervention period, all participants will take daily doses of 109 A. soehngenii CH-106 cells (dosage based on previous studies including toxicology study)2,3, 1010 B. animalis subsp. lactis BLC1 (a well-studied strain marketed as a probiotic by Sacco SRL) and 3x1010 pasteurized A. muciniphila ATCC BAA-835T cells (dosage based on EFSA approval and obtained from A-Mansia Biotech). Percutaneous liver biopsies will be performed as a part of screening when participants are theoretically eligible for participation, unless a liver biopsy has been performed in the previous 36 weeks. A tandem-read by two liver pathologists blinded to any other result will determine if patients will be included in the study. At 24 weeks, another liver biopsy will be performed to examine the effect of the FMT, which will also be reviewed by two liver pathologists (again blinded to any other result). The NASH-CRN classification will be assessed on H&E slides, for steatosis, inflammation and ballooning, and with a Sirius red-stained slide for evaluation of fibrosis. RNA for RNA-sequencing will be isolated. Differential gene expression will be assessed over time (baseline and 24 weeks) and by treatment allocation (vegan donor FMT versus placebo). Feces will be collected at baseline, and after week 2, 8, 10, 16, 18, and 24, in order to investigate the changes in gut microbiome. Also, markers of gut barrier function will be assessed. Blood will be collected at baseline and at 8, 16 and 24 weeks to investigate common liver enzymes, indicators of glycemic control, lipids, and general and more NASH-specific inflammation parameters. A multiparametric MRI of liver (MRI-PDFF, MR elastography, corrected T1) and of visceral and subcutaneous fat will be performed at baseline and after 24 weeks to estimate visceral and subcutaneous adipose tissue depot volume, hepatic fat content as well as hepatic fibrosis and inflammation. Continuous glucose measurements will be performed at home using portable devices, during a consecutive period of 1 week (7 days) in the week before baseline, week 1, 9, 17 and 25. Vegan donors The fecal donor samples will be collected, lyophilized ('freeze-dried'), and encapsulated (see paragraph 6.8). Thereafter, the LFMT capsules will be stored at -80°C. Donors will be extensively screened for infectious diseases. Primary outcome To demonstrate that A. soehngenii combined with pasteurized A. muciniphila and B. animalis subsp. lactis, FOS and conditioned vegan LFMT capsules reduce NASH as defined by an improvement of liver histology in individuals with NASH and fibrosis stage 0-3, with improvement defined as reduction of steatohepatitis by ≥1 SAF-A point and no worsening of liver fibrosis, or improvement in ≥ 1 stage liver fibrosis and no worsening of steatohepatitis. Secondary outcomes To demonstrate that the combined treatment improves: non-invasive outcomes of NAFLD, i.e. multiparametric MRI of liver and surrounding subcutaneous adipose tissue (MRI-PDFF, MR elastography, corrected T1), FibroScan Elastography and Controlled Attenuation Parameters, and plasma panel Enhanced Liver Fibrosis (ELF) panel, pro-C3. change in blood markers from baseline to end of treatment, namely: liver enzymes (i.e. alanine amino transferase (ALT), aspartate amino transferase (AST), gamma glutamyl transferase (GGT), alkaline phosphatase (ALP)), inflammatory blood markers (i.e. leukocytes, monocytes, CRP, Il-1(β), Il-6, Il-11, Il-17, Il-32, TNF-α, IFN-γ, other inflammatory markers), SCFA (i.e. propionate, butyrate, acetate), lactate, ethanol, plasma lipids (i.e. LDL, HDL, triglycerides, total cholesterol), albumin, kreatinine, hemoglobin, FGF-21, adiponectin, leptin, lipopolysaccharides, estrogen, vitamin B12, folate acid,zonulin, and other metabolomic and lipodomic outcomes. microbiome read outs (composition, engraftment, strain tracking) and metabolites, fecal SCFA-composition, and fecal albumine. glycemic control, insulin resistance, body weight/BMI, waist circumference and percentage body fat MetSy criteria / % SCFA production and GLP1 release liver gene expression profile: lipogenic, inflammatory and fibrogenic pathways. liver pathology, histopathological features, immunofluorescence and assessment of pathophysiological proteins. NAFLD histology as assessed with a deep-learned algorithm scoring whole slide images of liver biopsies. Continuous glucose monitoring (4x 7 consecutive days) quality of life (general (SF36) and NAFLD/NASH-specific (CDLQ-NAFLD)).

Official Title
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Synbiotics and Fecal Microbiota Transplantation to Treat Non-Alcoholic Steatohepatitis

Conditions
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FMT, Non Alcoholic Steatohepatitis, Non-Alcoholic Fatty Liver Disease, Fecal Microbiota Transplantation, Prebiotics, Probiotics, Microbiome, Intestinal Microbiome, Gut Microbiome

Intervention / Treatment
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* Combination Product: LFMT-capsules


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: biopsy-proven NASH obtained up to 32 weeks before screening: SAF Steatosis score ≥1, Activity ≥2, Fibrosis <4; 50% of participants should at least have NASH fibrosis stage 1, 2 or 3 according to the NASH CRN fibrosis staging system based on tandem reading of two expert liver pathologists fluency in Dutch or English participants should be able to understand the information and give informed consent Exclusion Criteria: Current or history of significant alcohol consumption for a period of more than 3 consecutive months within 1 year before screening (significant alcohol consumption is defined as more than 2 international units/day for females and more than 3 international units/day for males, on average; 1 international unit contains ±14 grams of alcohol) liver cirrhosis or hepatocellular carcinoma hepatitis B and/or C auto-immune hepatitis Wilson's disease primary sclerosing cholangitis primary biliary cholangitis alpha-1-antitripsine deficiency and hemochromatosis history of liver transplant, current placement on a liver transplant list use of pre-, pro- or synbiotics use of systemic antibiotics 3 month prior to randomization use of tamoxifen, methotrexate or amiodarone prior or planned bariatric surgery active GLP-1 receptor agonist treated diabetes mellitus bleeding disorder International normalized ratio (INR) of prothrombin time >1.4 or platelet count <100 109/L at screening anti-platelet/coagulant therapy use which cannot be temporarily discontinued any major cardiovascular event within 6 months prior to screening (e.g. myocardial infarction, cerebrovascular accident) prolonged compromised immunity (e.g. recent cytotoxic chemotherapy, HIV-infection with a CD4 count < 240) active or prior history of invasive malignancy (except for curatively treated in situ carcinomas [e.g., cervix] or non-melanoma skin cancer) unless a complete remission was achieved surgery scheduled for the trial duration period, except for minor surgical procedures, in the opinion of the investigator pregnant or nursing women any condition which, in the investigator's opinion, might jeopardize participants' safety or compliance with the protocol participation in another concomitant clinical trial.

Ages Eligible for Study
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Minimum Age: 18 Years
Maximum Age: 75 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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No

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Treatment
Allocation: Randomized
Intervention Model: Parallel Assignment
Interventional Model Description: Randomized placebo-controlled trial
Masking: Quadruple


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Experimental: LFMT-capsules<br>LFMT-capsules. 3 times bulk, and further continuous administration. This arm is also treated pre- and probiotics. | Combination Product: LFMT-capsules<br>* Lyophilized fecal microbiota transplantation capsules<br>* Other names: Fructo-oligosaccharides;|
| Placebo Comparator: Placebo<br>Placebo-capsules. 3 times bulk, and further continuous administration. This arm is also treated pre- and probiotics. | Combination Product: LFMT-capsules<br>* Lyophilized fecal microbiota transplantation capsules<br>* Other names: Fructo-oligosaccharides;|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Liver histology | Alteration of liver histology in subjects with NASH and fibrosis stage 0-3, with an alteration defined as change of steatohepatitis by ≥1 SAF-A point, or a change in ≥ 1 stage liver fibrosis. | baseline and after 24 weeks | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| MRI | MRI-PDFF, MR elastography, corrected T1 | baseline and after 24 weeks | 
| Fibroscan | CAP and LSM | baseline and after 24 weeks | 
| Liver enzymes (blood) | alanine amino transferase (ALT), aspartate amino transferase (AST), gamma glutamyl transferase (GGT), alkaline phosphatase (ALP)) | baseline and after 24 weeks; 8 and 16 weeks for safety. | 
| Immunological data | leukocytes, monocytes, CRP, Il-1(β), Il-6, Il-11, Il-17, Il-32, TNF-α, IFN-γ, other inflammatory markers), | baseline and after 24 weeks | 
| Plasma lipids | plasma lipids (LDL, HDL, triglycerides, total cholesterol), | baseline and after 24 weeks | 
| Gut metabolites (in blood) | short chain fatty acids (i.e. propionate, butyrate, acetate), lactate, ethanol, | baseline and after 24 weeks | 
| Glycemic control | Continuous glucose monitoring (4x 7 consecutive days), HOMA-IR | 5 weeks during study period | 
| albumin, kreatinine, hemoglobin (blood) | albumin, kreatinine, hemoglobin (blood) | baseline, 8, 16 and 24 weeks | 
| NAFLD NASH related endocrinological and metabolic outcome parameters | FGF-21, adiponectin, leptin, lipopolysaccharides, estrogen, vitamin B12, folate acid, zonulin, and other metabolomic and lipidomic outcomes. | Baseline and 24 weeks | 
| Microbiome readouts | microbiome read outs (composition, engraftment, strain tracking) and metabolites, fecal SCFA-composition, and fecal albumine. | baseline and after 24 weeks, and 5 times in between. | 
| Body weight | Body weight measured without shoes, in kilograms | baseline and after 24 weeks | 
| Height | Height measured without shoes, in cm | baseline | 
| BMI | Body mass index, calculated from height and weight kg/m^2 (kg per meters squared) | baseline and after 24 weeks | 
| Waist circumference | Measured just cranial of the spina iliaca anterior superior, in cm | baseline and after 24 weeks | 
| Percentage body fat | Measured with a biometric device, as a percentage. | baseline and after 24 weeks | 
| Demographics and (medical) history | Sex, age, ethnicity, height, weight, comorbidities (e.g. diabetes), smoking, alcohol intake, medication use, polypharmacy (defined as chronic use of ≥ 5 different medications) | baseline and 24 weeks | 
| Quality of life with CDLQ-NASH | quality of life with NAFLD/NASH-specific (CDLQ-NAFLD)) questionnaire. | baseline and after 24 weeks | 
| Quality of life with SF36 questionnaire. | quality of life (general (SF36) questionnaire. | baseline and after 24 weeks | 
| Food diary | 5x 10 days, daily caloric intake, daily fat and sugar consumption | around the visits 0, 8, 16, 24 weeks |