Targeting the secondary bile acid glycodeoxycholic acid as therapeutic strategy in type 2 diabetes mellitus.

Bile acids (BAs) and their receptors (e.g. TGR5, Takeda G-coupled protein 5 and
VDR, vitamin D receptor) have gained interest in development of treatment
modalities for type 2 diabetes mellitus (T2D). Postprandial hyperglycemia,
inflammation and hyperlipidemia are important risk factors for cardiovascular
disease. Preliminary data show that postprandial portal secondary BAs have high
affinity for TGR5 /VDR with important consequences for Glucagon like peptide -
1 (GLP-1) secretion and inflammation respectively. Also, BAs may promote
cholesterol elimination via the ATP binding cassette (ABC) half transporters G5
and G8 (ABCG5/G8).In this study, we want to investigate if the secondary BA
glycodeoxycholic acid (gDCA) increase GLP-1 secretion, reduce inflammation and
hyperlipidemia after a meal.

To study the effects of gDCA on postprandial GLP-1 secretion, inflammation
responses and hyperlipidemia in healthy lean male subjects and male T2D
patients. The secondary objectives are to evaluate the effect of gDCA and
ezetimibe on cholesterol elimination assessed as total faecal sterol
concentration and plasma lipid profile/composition and the effect of different
formulations on the gDCA bioavailability. The primary objective of phase 1 is
to determine safety of long-term gDCA administration in healthy volunteers.

The study design of the first pase of the study is an investigator initiated,
single center proof of concept study. The study design of phase 2 and 3 is a
randomized, placebo-controlled, cross-over, proof of concept study,

The TRADE study is divided in three phases. Each phase is described in detail
below.

Phase 1
The first phase of the study will be a pilot study where we investigate the
metabolism and safety of long-term administration of gDCA compared to long-term
administration of gDCA formulated in enteric-coated capsules (enteric-coated
gDCA). In the past no safety study is performed or published investigating the
metabolism of prolonged administration of (enteric-coated) glycine conjugated
gDCA. Therefore we will include 20 healthy male volunteers in this phase. Ten
subjects will receive 10 mg/kg/day gDCA for 30 days, the other group (N=10)
will receive 10 mg/kg/day enteric-coated gDCA for 30 days. Subjects undergo a
MMT at day 1, day 15 and day 30 in the AMC. After the last subject completed
the study, the data will be analyses in the laboratory and discussed by the
DSMB. After advise of the DSMB the project team will decide on phase 2.

Phase 2
At the second phase we include 10 T2D male patients. The 10 T2D patients will
receive in randomized order 10 mg/kg bw /day gDCA, enteric-coated gDCA and
placebo. Duration of each treatment periode is 4 weeks. Each treatment will be
followed by a wash-out period of 4 weeks.

Phase 3
In the third (final) phase we want to include 10 T2D male patients. The 10 T2D
patients will receive in randomized order 10 mg/kg bw /day gDCA, enteric-coated
gDCA and placebo. Next to that, patients will receive 20 mg ezetimibe per day
during eacch intervention period.

In addition, subjects will undergo (in phase 1) 3 mixed meal tests (MMTs). In
phase 2 and 3 patients will undergo 4 mixed meal test. Each mixed meal test
will take place before and after each intervention period. The mixed meal test
will be performed using Nutridrink compact (Nutricia, Zoetermeer, the
Netherlands), a commercial liquid meal containing a mix of essential
macronutrients. Before and after the MMT, energy expenditure (EE) is measured
by indirect calorimetry. Body composition will be measured each study visit
using whole-body air displacement plethysmography (BOD POD). 24 hour stools +
morning stool sample will be collected to investigate changes in the microbiome
and the faecal sterol concentration. We will ask participants to fill in
online or written dietary diary for 3 days prior to the MMTs to ensure the
stability and similarity of the gut microbiota during the study period. After
each MMT, appetite will be measured by the Universal Eating scale.

The burden of this study includes a screening visit and 3x (phase 1) or 4x
(phase 2 and 3) 6 hour admissions to the hospital after an overnight fast.
Next to that, administration of 10mg/ kg bw/day (enteric coated) gDCA for 30
days, placebo for 30 days (phase 2 and 3) and ezetimibe 20 mg/day 30 days
(phase 3), and 3 (phase 1) or 4 (phase 2 and 3) standardized liquid meals.
Several blood samples will be drawn via an intravenous catheter. We will
monitor the liver function tests 1 week after the start of the gDCA
administration. The total amount of blood taken will be ca 316ml (phase 1) and
433 ml (phase 2 and 3). Insertion of an IV cannula carries the risk of hematoma
or phlebitis.

gDCA is a naturally occurring bile acid metabolism and has been used in similar
doses in clinical studies. Prolonged use carries a slight risk of abdominal
discomfort and diarrhoea. In addition, prolonged treatment has been described
to cause ASAT elevation that was reversed upon discontinuing treatment. As
described above, our previous study patients and the patient with 3B-HSD have
not experienced any side effects with this dosage.

During the first phase of the TRADE study, the investigator should be aware of
the development of an increase in liver enzymes and therefore frequent control
of liver enzymes (ASAT, ALAT, AF, gGT) takes place during the study period.
Moreover, the investigator should be alert for the development of
gastro-intestinal side effects such as diarrhea after administration of gDCA.
No SAE*s are expected. We introduced a step-down procedure for gDCA dosage
which is described at E9 and in the protocol (page 25).

No clinical study in humans is performed to investigate the safety and efficacy
of prolonged dosing of gDCA or enteric-coated gDCA in humans on glucose
metabolism. Therefore we want to investigate the safety and efficacy of gDCA
and enteric-coated gDCA in the first phase (pilot) of the TRADE study in
healthy male volunteers.

The most important risks of ezetimibe in monotherapy are increased ALAT, ASAT,
CK and g-GT. The risk of developing these adverse events is considered to be
very low in our study population (0.1% - 1%). A higher risk (1-10 %) exists for
the development of fatigue and gastrointestinal symptoms such as abdominal
pain, diarrhoea and flatulence.