Synbiotics and Conditioned Fecal Microbiota Transplantation to Treat Non-Alcoholic Steatohepatitis.

The prevalence and severity of NAFLD - excessive hepatic fat accumulation
without excessive alcohol consumption - are increasing at an alarming rate,
because more and more patients are living longer with the two main factors that
drive NAFLD: obesity and T2DM. 20-30% of the global population including The
Netherlands7 has some stage of NAFLD8, and in T2DM, this is staggering 60-80%.
NAFLD is regarded as the hepatic component of the metabolic syndrome (MetSy)
and T2DM. It can silently progress from simple steatosis to NASH (prevalence
2-3%) and NASH-related fibrosis, and ultimately to cirrhosis and hepatocellular
carcinoma (fig.1)

Progression into the fibrotic stages of NAFLD is strongly associated with
liver-related and overall mortality and increased risk of atherosclerotic
cardiovascular disease, the latter most likely via induction of dyslipidemia.
Although NAFLD-NASH has a complex pathogenesis with a multiplicity of pathways,
it is thought that insulin resistance is a crucial trigger since it causes
lipolysis in peripheral adipose tissue, resulting in increased hepatopetal free
fatty acid (FFA) flux. This drives hepatic lipotoxicity, triggering NASH,
necroinflammation and activation of hepatic stellate cells, initiating
fibrogenesis.

To date, no treatment is available for progressive NAFLD stages.However, based
on insights from observational studies and animal experiments, the gut
microbiome and its metabolites are emerging as an innovative approach to treat
this challenging metabolic liver disease, Potentially detrimental alterations
in gut microbial composition have frequently been observed in NAFLD: abberant
microbiota composition, often depleted in butyrate producers, reduced
diversity, small intestinal bacterial overgrowth and signs of increased gut
permeability. This project aims to study the effects of the combination of
bacterial strains on NAFLD.

We recently published the first vegan fecal microbiota transplantation study in
patients with NASH and aberrant microbial composition. This pilot study found
anti-inflammatory effects of vegan FMT into NASH patients, with a trend towards
reduced hepatic necro-inflammation. Upon vegan FMT engraftment, the gut
microbiome of the NASH patients was enriched in Anaerobutyricum soehngenii
(also/formerly known as Eubacterium hallii) and other butyrate-producing
Lachnospiracae. Furthermore, in obese mice, A. soehngenii treatment improved
insulin sensitivity and reduced intrahepatic triglyceride content. Of note, A.
soehngenii is well known to not only convert sugars but also lactate and
acetate into butyrate, a short chain fatty acid that may protect against NASH.
Butyrate may reduce the inflammatory component of NAFLD by promoting
differentiation of regulatory T cells. It also has an anti-migratory effects on
neutrophils31, and in the gut, it protects epithelial barrier integrity, both
potentially protective effects on NASH. Moreover, since lactate is found to be
increased in T2DM; the efficient conversion of lactate into butyrate by A.
soehngenii is expected to contribute to its beneficial actions.

Another species thought to be benifical is B.animalis subsp. lactis of which
the well-studied strain BLC1 is marketed as a probiotic and well characterized.
In vitro studies have shown that this strain can form a trophic chain with A.
soehngenii to accelerate butyrate production from FOS. In addition, the BLC1
strain can in conjuction with A. soehngenii improve butyrate production in a
model of the small intestine. Moreover, B.animalis subsp lactis with FOS has
been shown to improve intestinal discomfort and promote anti-inflammatory
properties.

Pasteurized A. muciniphila is involved in host immunological homeostasis at the
gut mucosa, improvement of gut barrier function and contributes to metabolic
health. Indeed, in a study assessing the effects of pasteurized A. muciniphila
on diet-induced metabolic disorders in mice, it was not only found that
pasteurization did not diminish the beneficial effects, but it also
unexpectedly enhanced the beneficial impact of A. muciniphila on insulin
resistance and dyslipidemia. In addition, a randomized, double-blind,
placebo-controlled proof-of-concept study in overweight/obese insulin-resistant
human volunteers showed that the daily supplementation with pasteurized A.
muciniphila at a daily dose of 10^10 cells (based upon CFU count) for 12 weeks
improved several metabolic parameters such as insulin sensitivity, insulinemia,
plasma total cholesterol, as well as relevant blood markers for liver
dysfunction and inflammation while serum lipopolysaccharide (LPS) levels were
decreased, indicative of improved barrier function. A recent study in a high
fat induced fatty liver mouse model showed that administration of A.
muciniphila cells prevented fatty liver disease by regulation of the expression
of genes that regulate fat synthesis and inflammation in the liver. Since these
studies all found that pasteurized A. muciniphila cells were similar or more
efficient than the same amount of live A. muciniphila cells, we aim to use
pasteurized A. muciniphila ATCC BAA-835T cells in the present study. Of
importance, these cells have recently received EFSA approval based on the
extensive toxicological safety assessment.

Recently studies found an interesting mechanism by which A. soehngenii may
reduce NASH. In patients with MetSy, of which NAFLD is considered the hepatic
equivalent, duodenal administration of A. soehngenii strongly increased
glucagon-like peptide 1 (GLP1) secretion from the enteric endocrine L-cells,
potentially relating to the observed reduced plasma glucose fluctuations. Of
note, A. muciniphila administration induced GLP1 secretion in a mouse model.
Interestingly, clinical evidence is mounting that enhancing GLP1 may be a
plausible approach to reduce NASH. In a subset of patients from the phase 2A
LEAN trial with a GLP1-receptor agonist, it was shown that in addition to
weight loss and improved glycemic control, insulin-resistance driven
hepatopetal FFA-flux - the main contributor to lipid overload in NAFLD - was
reduced. In a recent Ph2B trial with a next generation GLP1 receptor agonist,
NASH histology improved and signs of fibrosis regression were noted.

Candidate therapeutic bacteria may be potentiated when combined with
prebiotics, i.e. non-digestible food substances, which can be fermented by
bacteria, promoting their growth. Such combinations are termed synbiotics.
Early evidence from murine studies and pilot studies with synbiotic cocktails
in NASH patients underscore the feasibility of this approach. NASH-fibrosis was
reduced by a cocktail of 4 prebiotics and 4 probiotics in a high fat-
choline-deficient diet mouse model. In addition, ALT and TNF-a were reduced in
patients with NAFLD who received a synbiotic cocktail of 7 bacterial strains
with the prebiotic fructo-oligosaccharides (FOS; oligomer of <10 fructose
molecules). In another pilot study in patients with NASH, a 12-week treatment
with a synbiotic cocktail of 5 probiotic bacteria and fructo-oligosaccharides
reduced ALT and liver stiffness on elastography. Together these reports are
supportive, yet the murine study did not decipher any mechanisms underlying the
protective effects and the human studies e.g. did not provide liver histology,
the gold standard in therapeutic development for NASH.

Furthermore, FOS is degraded to lactate and acetate by Bifidobacteria spp. such
as Bifidobacterium longum or B. animalis subsp. lactis, a well-studied strain
applied as a probiotic. The generated lactate and acetate has shown to be
effectively converted by A. soehngenii into butyrate in in vitro experiments,
including simulated ileum and colon models at ProDigest. Such a cross-feeding
mechanism can play a role in the colon ecosystem and contribute to the combined
bifidogenic/butyrogenic effect observed after addition of FOS to the diet. In
this context a recent human intervention study should be noted where
administration of B.animalis subsp. lactis with FOS was found to improve
intestinal discomfort and promote anti-inflammatory properties.

In our study, we will apply the complementary mode of actions of the individual
components in the synbiotic treatment, the established effects of their
combinations, and their expected impact on both upper intestinal tract and
colon. We hypothesize that for NASH, synbiotic combination treatment will
change the tipping point in the gut microbiome in order to reduce
steatohepatitis and liver fibrosis.

Primary objective
To investigate the therapeutic potential of A. soehngenii and pasteurized A.
muciniphila combined with B. animalis subsp. lactis and FOS with and without
conditioned vegan LFMT capsules to reduce NASH in patients with fibrotic NASH.

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 fibrotic NASH.

Double-blind randomized placebo-controlled intervention study.

Participants will either receive 3x 21 LFMT capsules on 1 day and daily 2
LFMT-capsules, or the same amount of placebo capsules.

All participants will daily ingest 10^9 A. soehngenii CH-106 cells, 10^10 B.
animalis subsp. lactis BLC1 (commercially available Sacco SRL) and 3x10^10
pasteurized A.muciniphila cells and 5 grams of FOS.

Benefits

NAFLD and NASH have an enormous and increasing prevalence: 20-30% of the gobal
population has NAFLD to some extent. NAFLD-related livercirrosis is a growing
indication for liver transplantation and the incidence of NASH-related
hepatocellular carcinoma is increasing. The disease progresses slowly and
NASH-liver fibrosis is strongly associated with atherosclerotic cardiovascular
disease and liver-related and overall mortality. Nevertheless, there is no
registered or proven treatment for progressed stages of NAFLD-NASH.
As previously stated, extensive prior research associates the gut microbiome
with NAFLD-NASH. Regardless, these studies did not entail an treatment
targeting the microbiome to reduce NAFLD-NASH, which would be a big step in the
field. Based on available literature on treatment with synbiotics in
NAFLD/NASH-patients and our pilot study on FMT in NAFLD/NASH-patients, this
trial with adequate sample size endeavours to study if modification of the gut
microbiome can attenuate NASH and liver fibrosis.
The benefits the study entails will be discussed in three sections. First, we
expect a short term benefit for participants. Our underpowered study in which
we administered FMT in NAFLD, showed a trend to reduction of necro-inflammation
(i.e. inflammation and ballooning) in histopathological examination.
Inflammation and ballooning are the most important predictors of progression of
NAFLD-NASH, and subsequent secondary complications.9 Half of the individuals
will receive FMT, and we expect that this already will attenuate NASH. All
participants will, in addition, be treated with a complementary synbiotic
combination. Multiple small clinical trials already showed a beneficial effect
of synbiotics on NAFLD-NASH, without entailing serious side effects. Therefore,
we expect an beneficial effect of the synbiotics in both study arms.
Second, participants will be able to benefit from the development of a
treatment for NASH. As stated, NASH is a slowly progressing, chronic disease,
without an effective treatment. If this study leads to a treatment of NASH, it
is likely that participants in the future will receive the investigated
treatment. Moreover, participants will assist in further understanding of the
pathophysiology of NASH and its relation to the gut microbiome, possibly
rendering further leads for the development of a therapy for NASH. Furthermore,
the current study and its subsequent insights could not only lead to a
treatment, but could also be the basis for preventive measures such as
effective and relative easily applicable lifestyle and dietary interventions
targeting the gut microbiome.
Last, the current study might benefit health on a population scale. As
mentioned, the prevalence of NAFLD and NASH is high and increasing subsequent
to the increase of its two main risk factors: obesity and T2DM. Fibrotic-NASH
is strongly associated with atherosclerotic cardiovascular events and
liver-specific and overall mortality. Considering the enormous prevalence, this
study and its possibilty to develop an effective treatment has the potential to
benefit the global population on an tremendous scale. Furthermore, in
comparison to medicinal alternatives currently investigated in the phase 2 and
3 trials for the treatment of NASH, FMT and synbiotics give a significant
reduction of adverse effects.

Risks and burdens

The study protocol is quite intense for participants. In summary, the burden
consists of the ingestion of LFMT capsules (3x on study visits 21 capsules,
daily 2 capsules) and daily ingestion of pre- en probiotica for 168 days, 2x a
liver biopsy, 2x a MRI and fibroscan, 4x 7 days of continuous glucose
measurements and 5x blood withdrawal. The risk for participants will mainly be
due to liver biopsies, with a complication rate of approximately 1 in 1000 per
procedure. Below, the risks and burdens are discussed more extensively.

FMT and synbiotics
In the literature and our center, FMT procedures have not been associated with
(serious) adverse events. Literature concerning the treatment with LFMT
capsules reports a lower number of adverse events in comparison to the infusion
of FMT via duodenal tube. The most prevalent side effects are mild,
self-resolving abdominal pain, nausea, diarrhea, and flatulence.
In regard to possible infectious transmission, fecesdonors are extensively
screened to mitigate the risk of potential infections by the FMT. Moreover,
fecesdonors are screened again after 2 months (during the storage of the
capsules) prior to administration of the LFMT capsules to the participants.
Participants with an immune deficiency are excluded from the studie. In our
center, FMT procedures have not been associated with adverse events, and donors
are extensively screened to mitigate the risk of potential infections by the
FMT procedure.

The provided synbiotics, moreover, are well-researched and deemed safe in both
toxicological studies and trials in humans, with no serious side effects
reported.

The predominant burden of the LFMT capsules and synbiotics will consist of
taking the capsules. On the days of the study visits (3x), participants will
ingests 21 capsules of FMT and 3 probiotic capsules. Daily, participants will
take 2 LFMT capsules, 3 probiotic capsules, and 5 grams of the
fructo-oligosaccharide powder. The ingestion of the bulk of 21 LFMT capsules on
the days of the study visits will be spread over the day to mitigate the
theoretical risk of clotting of the capsules in the stomach (which might
prevent normal passage to the duodenum). All capsules are designed to open in
the duodenum, therefore the content of the capsules will not reach the stomach
or a higher part of the digestive tract.

Liver biopsies
To date, liver biopsy is still the golden standard in the assessment of
progression or reduction of NASH, and there is no alternative. In therapy
development for NASH, the American FDA, for example, demands liver biopsy in
clinical trials. This study will, however, contribute to the further
development and validation of multiparametric MRI as a substitute for biopsy by
comparing the two.
An experienced interventional radiologist will perform ultrasound-guided liver
biopsy. Ultrasound-guided percutaneous liver biopsy is a safe method with very
low the risk of complications (< 1 per 1000 persons) comprising mostly bleeding
from the biopsy site. Moreover, local hemostasis after the procedure can be
observed, and patients will be screened for bleeding disorders. Liver biopsy
will be performed under local anesthesia, which can cause short discomfort.

Continuous glucose monitoring
Participants will have a continuous glucose monitoring-device (Freestyle libre)
installed for 4 times 7 days, in order to investigate the relation with GLP1.
This is usually well tolerated (as we see in diabetic patients), however the
prick of the installment can be unpleasant.

Blood withdrawal
Blood withdrawal, which happens five times, can be unpleasant, and can cause
self-limiting (sub)cutaneous hemorrhage/bruising.

Diaries and questionnaires
Last, participants have to keep a daily diary of food-intake, and have to fill
out two questionnaires on quality of life at baseline and week 24.