A phase IV, randomized, double-blind, placebo-controlled, parallel-group trial to assess the effect of 12-week treatment with the glucagon-like peptide-1 receptor agonist (GLP-1RA) liraglutide or dipeptidyl peptidase-4 inhibitor (DPP-4i) sitagliptin on the cardiovascular, renal and gastrointestinal system in insulin-naïve patients with type 2 diabetes (T2DM).

Currently published data on the effects and side effects of the new
blood-glucose lowering incretin-based agents (glucagon like peptide-1 receptor
agonists (GLP-1RA) and dipeptidyl peptidase-4 inhibitors (DPP-4i)) are
primarily derived from large-scaled registration studies in patients with type
2 diabetes mellitus (T2DM). By definition these international, multi-center,
randomized-controlled trials (RCTs) focus on glycemic control as a primary
outcome. Already in these phase III trials, but increasingly so during the
post-marketing period, incidental reports have suggested a possible association
between the use of these agents and changes in cardiovascular parameters
(increased heart rate), acute renal failure and pancreatitis. Indeed, GLP-1
receptors are present in most organ systems of the human body and consequently,
pharmacological interventions enhancing GLP-1 activity may influence the
functioning of these organs, in addition to their favorable effects on the
endocrine pancreas. Currently, for most of the registered incretin-based
compounds, large-scaled long-term outcome trials are underway, as required by
the recently issued Federal Drug Administration (FDA) and European Medicines
Agency (EMA) guidance. These trials may hopefully provide more clarity with
respect to the possible causal relationship between the use of these drugs and
fore-mentioned adverse events and the forthcoming clinical implications.
However, the results of these trials may not become available before 2016.
Conversely, to date, studies in humans detailing the effects on these organ
systems, biological processes and underlying mechanisms, which could explain
these associations, are lacking. Therefore, as part of the EU-FP7 program, the
EMA has endorsed a project to study the pharmaco-epidemiological aspects of
these potential side effects and the underlying mechanisms within the SAFEGUARD
consortium. The present study is part of the SAFEGUARD Work Package 6,
mechanistic studies (WP-6 leader: prof. M. Diamant).

Overarching Aim: to detail the (mechanisms underlying the) actions of GLP-1RA
and DPP-4i on the cardiovascular, renal and gastrointestinal system patients
with T2DM.

For the sake of clarity, we divide the study objectives into 3 parts:

Primary objectives:
Cardiovascular part: to assess the acute effects of GLP-1RA and the long-term
effects of both GLP-1RA and DPP-4i on resting heart rate variability.
Renal part: to assess the acute effects of GLP-1RA and the long-term effects of
both GLP-1RA and DPP-4i on glomerular filtration rate.
Gastrointestinal part: to assess the long*term effect of GLP*1RA and DPP*4i on
fecal elastase-1.

Secondary objectives:
Cardiovascular part: to measure the acute effects of GLP-1RA and the long-term
effects of both GLP-1RA and DPP-4i on blood pressure, heart rate, hemodynamic
variables, autonomic nervous system (ANS) function, microvascular function,
arterial stiffness, plasma lipid spectrum, glycemic variables (all of the
fore-mentioned variables will be assessed in the fasting and postprandial
state), body anthropometrics and body fat content.
Renal part: to measure the acute effects of GLP-1RA and the long-term effects
of both GLP-1RA and DPP-4i on renal plasma flow, renal tubular function and
renal damage parameters.
Gastrointestinal part: to measure the acute effects of GLP-1RA on exocrine
pancreatic function, and to measure the long*term effect of GLP*1RA and DPP*4i
on different aspects of pancreatic exocrine function/structure, plasma
pancreatic enzymes, gallbladder motility, liver enzymes, hepatic function,
hepatic steatosis and gastric emptying speed.

Exploratory objectives:
To explore the long-term effects of GLP-1RA and DPP-4i on various relevant
biomarkers.


For the pilot study preceding the trial, we have added the following objective:
To assess the changes following infusion of L-NMMA and the combination of
infusion of exenatide and L-NMMA in GFR and ERPF in healthy male subjects.
Also, we will validate 'contrast-enhanced ultrasound' which can potentially be
used for measuring renal perfusion non-invasively.

A double-blind, randomized, placebo-controlled, 3-armed parallel-group trial to
assess the effect of 12 weeks of treatment with the GLP-1RA liraglutide, the
DPP-4i sitagliptin or matching placebos on the function of the cardiovascular,
renal and gastrointestinal system in 60 T2DM patients. On the first day of the
(consecutive) baseline examination days preceding the long-term intervention
study, a sub study will be performed to assess the acute effects of GLP-1RA on
the cardiovascular and renal systems. This will be a double-blind, randomized,
placebo-controlled acute intervention with infusion of either the GLP-1RA
exenatide or placebo. In a subset of 12 patients an acute MRI intervention will
be performed during the baseline testing days of the main study (double blind,
randomized, placebo-controlled, cross-over study with infusion of the GLP-1RA
exenatide and placebo).

Cardiovascular parameters will be assessed using an oscillometric blood
pressure device (Dinamap®; systolic, diastolic, mean blood pressure, heart
rate), a continuous beat-to-beat blood pressure and electrocardiographic
hemodynamic monitor (NexFin®; finger blood pressure (systolic, diastolic and
mean), heart rate, stroke volume, cardiac output/-index/-contractility,
systemic vascular resistance, rest heart rate variability, autonomic nervous
system function/baroreceptor reflex sensitivity), capillary videomicroscopy and
Laser Doppler (microvascular function) and applanation tonometry (SphygmoCor®;
arterial stiffness). Blood will be sampled for the determination of plasma
lipid spectrum (triglycerides, total-, HDL- and LDL-cholesterol), glycemic
variables (glucose, HbA1c) and cardiovascular biomarkers (hsCRP, NT-proBNP).
Body anthropometrics (body weight, height, body-mass index (BMI) and waist
circumference) and bio-impedance analysis (body fat content) will be performed.
Renal parameters will be measured by the gold-standard inulin- and
para-aminohippurate (PAH) clearance method, to quantify glomerular filtration
rate (GFR) and effective renal plasma flow (ERPF), respectively. Fractional
excretion of sodium and urea will be measured as marker of tubular function and
urinary albumin/creatinine-ratio, NGAL, KIM-1 as markers of renal damage.
Gastrointestinal parameters include exocrine pancreatic function assessments
using validated laboratory methods for pancreatic exocrine enzyme (activity),
such as fecal elastase-1, plasma lipase, amylase; as well as the 13C-mixed
triglycerides breath test and secretin-enhanced Magnetic Resonance
CholangioPancreatography (sMRCP). MRI and 1H-MRS will be used to measure
pancreatic and hepatic structure and hepatic fat content, respectively.
Abdominal ultrasound will be used to assess gallbladder emptying speed.
Finally, liver enzymes (AST, ALT, GGT, ALP) and liver function (plasma albumin)
will be measured in plasma samples and acetaminophen absorption kinetics will
be measured to estimate gastric emptying speed.

Preceding this study, a pilot study will be performed in order to
operationalize 4 methods (the inulin/PAH clearance, 13C-mixed triglycerides
breath test, sMRCP and gallbladder ultrasoud). Healthy volunteers participating
in this pilot study will undergo the same testing days as the main study,
including the acute infusion study. Also, 'contrast enhanced ultrasound' will
be performed during the renal test days to validate this technique. An extra
testing day, which will not be performed in the main study, will be added to
the pilot study to investigate the interaction between exenatide and L-NMMA on
renal hemodynamics (GFR and ERPF). The long term intervention will not be used
in the pilot study.

Subjects will be randomized to either of the following arms:

Treatment arm 1: liraglutide 1.8mg s.c. once daily + sitagliptin placebo
Treatment arm 2: sitagliptin 100mg oral once daily + liraglutide placebo
Treatment arm 3: liraglutide placebo + sitagliptin placebo

Moreover, during the first baseline visit, an infusion with exenatide or
placebo will be given.

Participants in the Acute MRI sub study (12 participants of the main study)
will receive 2 additional MRI scans with placebo and exenatide.

Participants in the pilot-study will not be randomized for the long-term
intervention, but will undergo the acute intervention with exenatide. Also, on
an additional testing day both exenatide and L-NMMA will be administered.

We are well aware of the possible demand that may be imposed on the
participants in this study. Participants of the pilot study will have to visit
the clinical research unit for a total of 5 times (spread over 2 weeks),
participants in the main study will travel 9 times, spread over 17 weeks
(participants in the Acute MRI protocol: 11x). The duration of the visits
ranges between 30 minutes and 10 hours. A total amount of 492.5 mL of blood
will be drawn in the main study, and 404.5 mL in the pilot study. Some of the
tests procedures can be perceived as demanding (i.e. the renal protocol,
MRI-scans and fecal collection). However, we have gained ample experience with
similarly demanding mechanistic drug intervention studies in T2DM patients. We
have built in different ways to alleviate the burden for participants,
including clear, repeated communication, frequent contacting and intensified
(diabetes) care. During test-days, we provide meals and allow participants to
read or watch TV/DVDs when possible.
The study examinations/tests are considered to be safe. No radiation or
invasive procedures (besides intravenous peripheral catheters) are involved.
During the study tests, some *diagnostic agents* need to be administered.
Inulin, para-aminohippuric acid and stable-isotope labeled 13C-MTG are inert
and have no side effects. For acetaminophen side-effects are very rare.
Secretin can cause transient nausea and abdominal pain.
All medication used in this study has a marketing authorisation and is
considered to be safe. Exenatide and liraglutide are known to cause mild and
transient nausea, and sometimes vomiting. Sitagliptin can cause
nasopharyngitis. As these agents have been used in previous studies at the VU
University Medical Center there is ample experience. As in all drug
intervention trials, in this study, we will closely monitor patients for
adverse drug and study events during the follow-up visits and by telephone
consultations (one, two and six weeks after the start of the study medication).
Moreover, the research physicians are available for questions at all times.