The effect of an encapsulated nutrient mixture on ileal brake activation: A double-blind randomized study to investigate the effects on body weight, food intake and satiety

Obesity and weight management
Worldwide the incidence of overweight and obesity is rapidly increasing with
negative impact on health and health care costs1. The efficacy of the currently
available treatments is relatively limited on the long term2. So far, surgical
intervention has been proven to be the only strategy to overcome severe obesity
in the long-term3. Furthermore, in a recent meta-analysis it was concluded that
dietary supplements and exercise alone does not contribute to improved weight
loss after caloric restriction diets4. Therefore, it is important to develop
new effective strategies that induce weight loss or help weight maintenance
after weight loss. A promising mechanism for weight management is to activate
the so called *ileal brake*.

Ileal brake
The ileal brake refers to an intestinal feedback mechanism that is triggered by
nutrients at a specific location in the small intestine, resulting not only in
modulation of gastrointestinal secretions and motility but also of food intake
and hunger5. The ileal brake can be activated by ileal infusion of undigested
lipid6,7, and recently it was found that ileal infusion of sucrose and casein
suppresses food intake to the same extend as equicaloric amount of lipids8. In
addition to the reduction of food intake, an increase in feelings of satiety
and decrease in hunger was found.
Since ileal brake activation seems to be a promising target for weight
management strategies alternative nutrient delivery techniques have to be used.
Several studies investigated the effects of nutrient infusion in all regions of
the small intestine9. However all studies used intestinal feeding catheters in
order to infuse nutrients directly into the desired part of the small
intestine. In order to investigate the effect of long-term ileal brake
activation in overweight individuals, a different approach for nutrient
delivery needs to be chosen. An alternative for distal small intestinal
nutrient delivery via feeding catheters is encapsulation of nutrients.
Encapsulation can be used to deliver nutrients to a specific intestinal
location. By encapsulation, nutrients are covered/surrounded with an edible
coating. This coating is defined as a thin layer of edible material applied to
the surface of the nutrients. This provides a barrier against digestion in the
stomach and the proximal parts of the small intestine. Comparable approaches
have already been used for site-specific drug delivery. Two examples of systems
able to deliver drugs to the ileo-colonic region are the ColoPulse system
described by Schellekens et al. (2010)10 and the ileo-colonic delivery system
described by Varum et al. (2013)11. Both methods are invented to accelerate
drug release in the ileo-colonic region and use specific Eudragit® polymer
coatings. Adjusting these polymer coatings results in a more proximal (ileum)
delivery. However these techniques were developed for drug delivery. To
activate the ileal brake it is preferred to use a completely food grade
application. Another food grade approach to deliver nutrients to the distal
small intestine is micro-encapsulation. This micro-encapsulation technique will
be used in the present study to deliver a mixture of nutrients consisting
mostly sucrose (60%) , casein (30%) in a shell of whey protein (10%) to the
distal small intestine.
The delivery method patented by AnaBio Technologies Ltd.
(http://www.AnaBio.ie/) comprises an active component, encapsulated within a
protein matrix of preferably whey protein. The mechanism by which their
technique delivers the active to the preferred intestinal location is based on
pH, surface porosity of the microspheres, and reaction to specific enzymes.
These three parameters are used to design the micro-encapsulation beads to
deliver the active ingredient to the preferred intestinal location (e.g.
duodenum, jejunum or ileum). Furthermore the size of the micro-beads is
important for intestinal transit time. AnaBio uses their data collected from
human and animal studies to relate the size of the micro-beads to intestinal
transit time, dose volume and physiological characteristics. The micro-bead
size (micron range) can be chosen based on the population group and the
required transit time, an average used size is 150 micron. The advantage of
these small particles is that the particles can be mixed with a food product.
The ratio active : encapsulated protein matrix is 95 : 5%. This means that if
sucrose will be encapsulated 95% of the micro-bead is pure sucrose and 5% is
whey protein. We know from our previous research8 that infusion with 13 grams
of sucrose is able to activate the ileal brake. For the present study we will
use a mixture of encapsulated sucrose microbeats (60%) and encapsulated casein
microbeats (30%) in order to simulate the composition of a small mixed
carbohydrate and protein rich meal.

Efficacy small intestinal delivery
AnaBio provided data (which cannot be shared due to IP applications) of four in
vivo studies of which 3 were human studies and 1 pig study. In these studies
the efficacy of their delivery method has been investigated. It can be
concluded from these studies that the whey protein micro-encapsulation
technique is a specific intestinal delivery method for specific to either the
ileum (2 human studies and 1 pig study) or the jejunum (1 human study).



Present study
In the present study the effect of 6 weeks ileal brake activation on body
weight, BMI and waist circumference will be investigated. It is hypothesized
that 6 weeks ileal brake activation by delivery of an encapsulated nutrient
mixture to the distal small intestine results in reduced body weight. This will
be studied by daily ingestion of an encapsulated nutrient mixture (e.g. 60%
sucrose and 30% casein encapsulated in 10% whey protein) either delivered to
the distal small intestine (active) or stomach (placebo) before lunch and
dinner for 6 weeks in a randomized, double blind parallel placebo controlled
trial. This will be studied in the second phase of the study (phase 2). Pre
intervention, study day 1 and 2 (SD1 and SD2), middle (SD4) and post (SD5)
intervention period the efficacy of the to decrease food intake (>68 kcal) will
be evaluated.
It is unknown whether the ileal brake will be activated by the encapsulated
nutrient mixture delivered to the distal small intestine (active) to the same
magnitude as ileal brake activation by ileal sucrose and casein infusion.
Therefore, we will study the ileal brake activation efficacy of the
encapsulated nutrient mixture ingestion on ad libitum food intake. This will be
done in the first phase of the study (phase 1). Pre-intervention the efficacy
will be tested in a randomized placebo controlled cross over design. Within
every subject the difference in ad libitum food intake after active or placebo
ingestion will be studied. If the active encapsulated nutrient mixture does not
result in a reduction of food intake the intervention will not start.

First part of study (Phase 1)
Aim: To evaluate the efficacy of ileal brake activation, by ingestion of an
encapsulated nutrient mixture delivered to the stomach (placebo) or distal
small intestine (active) analysed by the amount of food consumed during a
subsequent ad libitum meal.
Hypothesis: We hypothesize that delivery of encapsulated nutrient mixture to
the distal small intestine (active) results in a reduction in food intake
compared to encapsulated nutrient mixture delivery to the stomach (placebo).
Primary objective: To investigate whether delivery of an encapsulated nutrient
mixture to the distal small intestine (active) decreases the amount of food
consumed during a subsequent ad libitum meal compared to delivery of an
encapsulated nutrient mixture to the stomach (placebo).
Secondary objective: To investigate whether intake of encapsulated nutrient
mixture delivered to the distal small intestine (active) increases satiation
compared to delivery of the encapsulated nutrient mixture to the stomach
(placebo), as analysed by VAS-scores.
Tertiary objective: To analyse whether the appearance of glucose and insulin is
delayed in plasma after encapsulated nutrient delivery to the distal small
intestine (active) compared to encapsulated nutrient delivery to the stomach
(placebo).

Main study (Phase 2)
Aim: To investigate the effect of six weeks ileal brake activation by nutrient
delivery to the distal small intestine (active) on body weight. This will be
studied by daily ingestion of an encapsulated nutrient mixture which will be
delivered to the distal small intestine (active) or stomach (placebo) before
lunch and dinner for a period of 42 ± 2 days (6 weeks).
Hypothesis: We hypothesize that 6 weeks ileal brake activation by nutrient
delivery to the distal small intestine (active) at lunch and dinner results in
body weight reduction.
Primary objective: The difference in body weight before and after 6 weeks ileal
brake activation by nutrient delivery to the distal small intestine (active;
group 1) compared to nutrient mixture delivery to the stomach (placebo; group
2).
Secondary objective: To investigate whether intake of encapsulated nutrient
mixture delivered in the distal small intestine (active) decreases the amount
of food consumed during a subsequent ad libitum meal compared to encapsulated
nutrient mixture delivered in the stomach (placebo) in time, analysed pre-,
middle and post intervention.
Tertiary objective: To investigate whether intake of encapsulated nutrient
mixture (active) increases satiation analysed by VAS-scores compared to
placebo pre, middle and post intervention.
Other objective(s): To evaluate effects of 6 weeks encapsulated nutrient
delivery to the distal small intestine (active) on glucose and insulin plasma
concentrations compared to nutrient delivery to the stomach (placebo). This
will be analysed by monitor blood glucose and insulin responses in time pre-
and post intervention.

First part of study (Phase 1)

Study design: Randomized placebo controlled double blind cross-over study.
Go/no go: After phase 1 has been completed, a go/no go decision will be taken,
before the start of phase 2. In case of a significant decrease (>68 kcal) in ad
libitum food intake after ingestion of the active (phase 1) we will continue
with phase 2. If ad libitum food intake is not decreased (>68 kcal) after
ingestion of the active in phase 1, phase 2 will not be conducted.

Main study (Phase 2)

Study design: Randomized placebo controlled double blind parallel intervention
trial.
Main study parameters/endpoints: Difference in body weight and BMI before and
after the intervention between the placebo group and the intervention group.

Ingestion of micro encapsulated casein and sucrose (ileal delivery)

Blood sampling: on each test day (test day 1-4) a flexible intravenous cannula
(Biovalve 1,0mm) is inserted into an antecubital vein in the fore-arm for blood
sampling. Per time point 8mL of blood is drawn, totalling 72mL per test day
(with a total of 288mL for the 4 test days). After collection (immediately
after collection DPP-IV inhibitor will be added to the tube), K2EDTA tubes will
be centrifuged at 2500 rpm for 10 min at 4°C. The supernatant will be collected
and this will be centrifuged again at 4000 rpm for 10 min at 4°C. Plasma will
be collected in 1-mL aliquots and stored at -80°C until analysis. During blood
sampling, the volunteers will remain seated in a comfortable chair, with an
adjustable back. No side effects are expected when sampling blood in this
manner.

VAS scores for satiety and GI symptoms Scores for satiety feelings (e.g.,
satiety, fullness, hunger, prospective feeding, desire to eat, desire to snack)
and gastrointestinal symptoms (burning, bloating, belching, cramps, colics,
warm sensation, sensation of abdominal fullness, nausea and pain) will be
measured using Visual Analogue Scales (VAS, 0 to 100 mm) anchored at the low
end with the most negative or lowest intensity feelings (e.g., extremely
unpleasant, not at all), and with opposing terms at the high end (e.g.,
extremely pleasant, very high, extreme). Volunteers will be asked to indicate
on a line which place on the scale best reflects their feeling at that moment.
The scoring forms will be collected immediately so that they cannot be used as
a reference for later scorings.

Ingestion of micro encapsulated nutrients: We don't expect any problems with
ingestion of micro encapsulate since it is very easy to swallow and easy to mix
with other food products (yoghurts, drinks etc).