The Effect of Orally Applied Encapsulated Lipids in Yoghurt on Satiety and Food Intake: a Randomized Single-blind Placebo-controlled Study With Cross-over Design
Phase: NA
Conditions: Appetite Regulation
Sponsor: Maastricht University Medical Center

NL 59245.068.16  
 
Lipid encapsulation for ileal brake activation 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
RESEARCH PROPOSAL 
 
 
The effect of orally applied encapsulated lipids in yoghurt on satiety 
and food intake: a randomized single-blind placebo-controlled study 
with cross-over design 
 
 
 
 
 
 
 
 
 
 
 
 

NL 59245.068.16  
 
Lipid encapsulation for ileal brake activation 
 
 
 
 
Protocol title: The effect of orally applied encapsulated lipids in yoghurt on satiety and food 
intake: a randomized single-blind placebo-controlled study with cross-over design 
 
Protocol ID 
NL 59245.068.16 
Short title 
Lipid encapsulation for ileal brake activation 
Version 
2 
Date 
29 November 2016 
Coordinating investigator 
Prof. dr. A.A.M Masclee 
Division of Gastroenterology-Hepatology 
Dept. of Internal Medicine 
Maastricht UMC+ 
Tel: +31 (0)43-3875021 
Principal investigator 
 
Prof. dr. A.A.M Masclee 
Division of Gastroenterology-Hepatology 
Dept. of Internal Medicine 
Maastricht UMC+ 
Tel: +31 (0)43-3875021 
Sponsor 
(in 
Dutch: 
verrichter/opdrachtgever) 
Maastricht University 
Subsidising party 
Division of Gastroenterology, Maastricht UMC+ 
Independent expert (s) 
Dr. M.H.L. Christiaans 
Nephrology 
Dept. of Internal Medicine 
Maastricht UMC+ 
Tel: +31 (0)43-3875007 
Project team members 
M.N. Corstens 
Division of Food Process Engineering 
Dept. of Agrotechnology and Food Science 
Wageningen University 
 
Dr. F. Troost 
Division of Gastroenterology-Hepatology 
Dept. of Internal Medicine 
Maastricht UMC+ 

NL 59245.068.16  
 
Lipid encapsulation for ileal brake activation 
 
 
 
 
 
Dr. C.C. Berton-Carabin 
Division of Food Process Engineering 
Dept. of Agrotechnology and Food Science 
Wageningen University 
 
Prof. dr. K. Schroën 
Division of Food Process Engineering 
Dept. of Agrotechnology and Food Science 
Wageningen University 
 
Tim Klaassen 
Division of Gastroenterology-Hepatology 
Dept. of Internal Medicine 
Maastricht UMC+ 
 
Annick Alleleyn 
Division of Gastroenterology-Hepatology 
Dept. of Internal Medicine 
Maastricht UMC+ 
 
Laboratory sites 
Maastricht UMC+ 
 
 

NL 59245.068.16  
 
Lipid encapsulation for ileal brake activation 
 
 
 
 
PROTOCOL SIGNATURE SHEET 
 
Name 
Signature 
Date 
Principal Investigator 
Prof. dr. A.A.M. Masclee 
Head of Division 
Gastroenterology and Hepatology  
 
 
 
 

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TABLE OF CONTENTS 
 
SUMMARY ............................................................................................................................................................................. 8 
1. 
INTRODUCTION AND RATIONALE ................................................................................................................ 10 
2. 
OBJECTIVES ............................................................................................................................................................. 12 
3. 
STUDY DESIGN ....................................................................................................................................................... 12 
4. 
STUDY POPULATION ........................................................................................................................................... 14 
 
Population ....................................................................................................................................................... 14 
 
Inclusion criteria .......................................................................................................................................... 14 
 
Exclusion criteria ......................................................................................................................................... 14 
 
Sample size calculation ............................................................................................................................. 15 
5. 
TREATMENT OF SUBJECTS ............................................................................................................................... 16 
 
Active investigational product ............................................................................................................... 16 
 
Control product ............................................................................................................................................ 17 
6. 
INTERVENTION PRODUCT ................................................................................................................................ 17 
 
Name and description of intervention yoghurts ............................................................................ 17 
 
Summary of findings from non-clinical studies .............................................................................. 18 
 
Summary of findings from clinical studies ........................................................................................ 18 
 
Summary of known and potential risks and benefits ................................................................... 19 
 
Description and justification of route of administration ............................................................. 19 
 
Dosages, dosage modifications and method of administration ................................................ 20 
 
Preparation and labelling of Investigational Product ................................................................... 20 
 
Drug accountability ..................................................................................................................................... 20 
7. 
METHODS ................................................................................................................................................................. 21 
 
Study parameters/endpoints ................................................................................................................. 21 
7.1.1 
Main study parameter/endpoint ................................................................................................. 21 
7.1.2 
Secondary study parameters/endpoints ................................................................................. 21 
 
Randomisation, blinding and treatment allocation ....................................................................... 21 
 
Study procedures ......................................................................................................................................... 22 
 
Withdrawal of individual subjects ........................................................................................................ 24 
7.4.1 
Specific criteria for withdrawal .................................................................................................... 24 
 
Replacement of individual subjects after withdrawal .................................................................. 24 
 
Follow-up of subjects withdrawn from treatment ........................................................................ 25 
 
Premature termination of the study .................................................................................................... 25 
8. 
SAFETY REPORTING ............................................................................................................................................ 25 
 
Temporary halt for reasons of subject safety .................................................................................. 25 
 
AEs, SAEs and SUSARs ............................................................................................................................... 25 
8.2.1 
Adverse events (AEs) ....................................................................................................................... 25 
8.2.2 
Serious adverse events (SAEs) ..................................................................................................... 25 
8.2.3 
Suspected unexpected serious adverse reactions (SUSARs) ........................................... 26 
 
Annual safety report ................................................................................................................................... 26 
 
Follow-up of AEs .......................................................................................................................................... 26 
 
Data Safety Monitoring Board (DSMB) / Safety Committee ...................................................... 26 

NL 59245.068.16  
 
Lipid encapsulation for ileal brake activation 
 
 
 
 
9. 
STATISTICAL ANALYSIS ..................................................................................................................................... 26 
 
Primary study parameter(s) ................................................................................................................... 27 
 
Secondary study parameter- VAS scores for satiety and GI symptoms ................................ 27 
10. 
ETHICAL CONSIDERATIONS ........................................................................................................................ 27 
 
Regulation statement ................................................................................................................................. 27 
 
Recruitment and consent ......................................................................................................................... 28 
 
Objection by minors or incapacitated subjects ............................................................................... 28 
 
Benefits and risks assessment, group relatedness ........................................................................ 28 
 
Compensation for injury ........................................................................................................................... 28 
 
Incentives ........................................................................................................................................................ 29 
11. 
ADMINISTRATIVE ASPECTS, MONITORING AND PUBLICATION ................................................ 29 
 
Handling and storage of data and documents ................................................................................. 29 
 
Monitoring and Quality Assurance ....................................................................................................... 29 
 
Amendments .................................................................................................................................................. 29 
 
Annual progress report ............................................................................................................................. 30 
 
Temporary halt and (prematurely) end of study report ............................................................. 30 
 
Public disclosure and publication policy ........................................................................................... 30 
12. 
STRUCTURED RISK ANALYSIS .................................................................................................................... 31 
 
Potential issues of concern ...................................................................................................................... 31 
 
Synthesis.......................................................................................................................................................... 33 
13. 
REFERENCES ...................................................................................................................................................... 33 

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LIST OF ABBREVIATIONS AND RELEVANT DEFINITIONS  
 
GCP 
Good Clinical Practice 
METC  
Medical research ethics committee (MREC); in Dutch: medisch ethische 
toetsing commissie (METC) 
(S)AE 
(Serious) Adverse Event  
(S)AR 
(Serious) Adverse Reaction 
SUSAR 
Suspected Unexpected Serious Adverse Reaction 
Sponsor The sponsor is the party that commissions the organisation or performance of 
the research, for example a pharmaceutical company, academic hospital, 
scientific organisation or investigator. A party that provides funding for a 
study but does not commission it is not regarded as the sponsor, but referred 
to as a subsidising party. 
VAS 
Visual Analogue Scales 
WBP 
Personal Data Protection Act (in Dutch: Wet Bescherming Persoonsgevens) 
WMO 
Medical Research Involving Human Subjects Act (in Dutch: Wet Medisch-
wetenschappelijk Onderzoek met Mensen 
 
 

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SUMMARY 
Rationale: Sensing of lipid fractions in the small intestine can induce negative feedback 
mechanisms from different parts of the intestine to the proximal gastrointestinal (GI) tract 
including stomach, gallbladder and pancreas but also to the central nervous system. This negative 
feedback process is able to inhibit food digestion, appetite sensations and food intake, and is able 
to increase feelings of satiety and satiation. The ileal brake is considered a potent feedback 
mechanism not only during short-term intervention with ileal lipid infusion, but also as powerful 
long-term weight management strategy with orally ingested ileal lipid delivery. However, the 
major part of dietary lipids will be digested and absorbed in the proximal small intestine and is 
not likely to reach the distal ileum and induce the strong ileal brake feedback mechanism. To 
prevent orally applied lipids to be proximal digested and absorbed, we designed a food-grade 
encapsulation system that releases free fatty acids from safflower oil in the more distal small 
intestine. The current study will be an explorative study to proof the concept of ileal brake 
activation: the encapsulated lipid will be added to and mixed with yoghurt (A), and the subsequent 
satiety and food intake will be compared to an equicaloric mixture of non-encapsulated nutrients 
(control, yoghurt B) with the same amount of lipids, and the encapsulation components.  
 
 
Aim: To explore the ability of encapsulation of orally applied lipids in a yoghurt snack to modify 
ad libitum food intake and satiety, without GI symptoms. 
Hypothesis: We hypothesize that lipid encapsulates induce satiety and decrease ad libitum meal 
intake compared to control (yoghurt B), due to more distal release in the small intestine. 
Primary objective: To investigate whether encapsulation of lipids decreases ad libitum food 
intake compared to non-encapsulated lipids in a formulation with equal nutrients (control). 
Secondary objectives: 
 
- 
To investigate feelings of satiety after orally ingested lipid encapsulates 
- 
To investigate occurrence and severity of GI symptoms after orally ingested lipid 
encapsulates 
- 
To assess whether differences in food intake, satiety and GI symptoms relate to lipid 
encapsulation 
Study design: Randomized, single-blind, placebo-controlled intervention study with cross-over 
design. 
Study population: 35 human subjects (BMI 25-30 kg/m2), 18 - 65 years old. 
Intervention: Every subject receives two treatments on two different days with at least one week 
in between, following a randomized cross-over design. On the test day, the subjects will arrive in 
fasted state and receive a standardized breakfast (small, low in fat, t=0 min). Once major part of 
the breakfast has been emptied from the stomach (t=90 min), they receive a yoghurt (0.2 L) that 
contains emulsified safflower oil (6 g) either encapsulated (yoghurt A) or non-encapsulated 
(control, yoghurt B; same oil droplet size as intervention and in presence of ‘empty’ encapsulation 
matrix). Two hours after the intervention yoghurt, once the lipid will be released to the distal 
small intestine, ad libitum meal intake will be measured (t=210 min). 

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Main study parameters/endpoints: The main study parameter is the intake of ad libitum meal 
(kcal) as measured during pasta lunch, comparing intervention and control treatment. 
Nature and extent of the burden and risks associated with participation, benefit and group 
relatedness: 
The subjects (in total 35) will have to visit Maastricht University on three occasions: once to sign 
the consent, fulfil the screening and get an instruction (about half an one hour) and two times to 
attend the test days (about four hours per test day). The test days will be non-invasive: 
consumption of a breakfast, yoghurt snack, and lunch; and in between filling questionnaires on 
satiety feelings (satiety, fullness, hunger, desire to eat, desire to snack) and GI symptoms (bloating, 
discomfort, pain, nausea). These attributes will be measured using Visual Analogue Scales (VAS, 0 
to 100 mm) scores, with the most negative or lowest intensity feelings at the low end and the 
opposing terms at the high end. The subject will be asked to indicate his feeling at that moment. 
 
 

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1. INTRODUCTION AND RATIONALE 
Weight management by ileal brake activation 
 
A key strategy to control the expansion of the worldwide obesity prevalence is by large-scale 
application of dedicated dietary interventions that help weight maintenance or induce weight 
loss. A new effective dietary intervention may target to enhance natural gastrointestinal (GI) 
processes that are involved in food intake regulation, such as gastric distension, and the sensing 
of nutrients and their digestion products. Gastric distension triggers mechanosensor-mediated 
signals and suppresses the release of the stomach hormone ghrelin. The presence of 
macronutrients in the small intestine induces the release of several gut peptides that are known 
to be associated with food intake, such as cholecystokinin (CCK), peptide YY (PYY), and glucagon-
like peptide-1 (GLP-1) [1]. The ileum is believed to give the strongest signal [2], [3]. 
 
Ileal brake activation has been proven to acutely reduce food intake with direct intubation of 
any of the three macronutrients via a naso-ileal catheter (overview of literature in [3]). About 30 
years ago, ileal lipid infusion was already shown to reduce food intake [4], [5]. Moreover, ileal lipid 
infusion increased perception of satiety/fullness or decreased hunger in most studies [1], [2], [4]–
[7]. Amongst oils with different degree of fatty acid saturation, safflower oil (high in linoleic acid, 
C18:2) was found to more strongly activate the ileal brake [8]. Therefore, in the current study we 
will use safflower oil. 
The mechanism of action of inhibition of food intake by intraluminal lipids is based on sensing of 
lipid species at intestinal receptors. When activated more distally in the small intestine, 
intraluminal lipids are able to induce distal to proximal negative feedback mechanisms [9]. This 
mechanism is activated more strongly when the degradation products of lipid digestion (i.e., fatty 
acids, monoglycerides) are sensed rather than the intact lipids itself (i.e., triglycerides) (see review 
[9], paragraph 4.1.1). Hence, for effective ileal brake activation, lipase-mediated hydrolysis 
(lipolysis) of lipids is required, and in addition, proximal intestinal absorption of lipolysis 
products has to be prevented.  
 
Ileal brake activation by food 
 
To be an effective and practically applicable weight management strategy, an administration 
route is required different from direct intestinal intubation, namely orally with the regular route 
of food intake. When applied orally, however, the major part of dietary lipids will be digested and 
absorbed in the proximal small intestine and are not likely to induce the ileal brake mechanism. 
Hence, to activate the ileal brake, lipolysis needs to be controlled. Since lipolysis only occurs at the 
oil-water interface, the amount and accessibility of this interface are key in controlling it [10], [11].  
 
Many attempts have been made to control lipid digestion, especially through designing a 
protective interface structure around nano-or-micron-sized lipid droplets. Some of these 
structures were able to delay lipolysis to a certain extent, including single surfactants that provide 
steric hindrance [12], [13], more thick interfacial films produced through layer-by-layer 
adsorption of biopolymers [14]–[18], and specific Pickering-based interface structures [19], [20]. 
To truly control lipolysis, however, the structuring approach should go beyond interfacial design 
[21], and may focus on control of enzyme diffusion towards this interface by means of a larger 
structure. In vitro lipolysis has been controlled by both digestible protein gels [22], [23] and 
indigestible alginate gels [24], [25]. The latter is the approach taken in the current study. 

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Emulsion-filled Ca-alginate gel particles for ileal brake activation 
 
We have provided evidence that encapsulation of small lipid droplets into millimetre-sized Ca-
alginate gel particles is to be considered as a promising approach for ileal brake activation. The 
benefit of using alginate gel over protein gel, is the degradation of protein and resistance of 
alginate matrix against gastric proteases [23], so maintenance of the encapsulate structure. In 
addition, the pH-dependency of volume and porosity of alginate gel particles makes them very 
suitable for ileal delivery: they are known to shrink under acidic conditions, lowering the pore 
size, and hence protecting the encapsulated emulsion in the stomach; moreover, they swell at 
neutral pH, increasing the pore size and favouring lipase diffusion towards the encapsulated oil in 
the small intestine [24], [26]–[28].  
 
In humans, the transport of alginate beads in the GI system (n=10, after a fatty preload) has 
been tracked using MRI scanning, and interestingly, the beads were found to end up most in the 
ileum rather than in duodenum and jejunum [29]. This suggest an extended residence time in the 
ileum, and therefore stronger ileal brake activation. 
 
Besides, alginate may modify the ileal brake response directly as it is a water-soluble fibre, 
which can slow transit time and result in spreading of nutrients and slower absorption [30]. The 
direct effect of alginate on feelings of satiety and hunger have also been evaluated: alginate drinks 
were found to be able to modulate hunger dependent on its gelling behaviour [31] and viscosity 
[32]. On the other hand, no effect was found from compressed alginate capsules that were ingested 
30 min before meal, on gastric motor functions, satiation, appetite, nor gut hormones [33]. 
Alginate in the form of Ca-gel particles (not loaded with oil), may affect feelings of satiety (n=10, 
after a fatty preload), dependent on the gel structure, but not hunger and appetite [29]. Taking all 
together, the potential direct effect of alginate on hunger modulation may only hold for alginate 
in solution, but for sure, alginate will not oppose the beneficial effect. 
 
The fate of calcium-alginate gel particles that do contain oil has also been studied in in vivo 
studies. In rat, emulsion-filled Ca-alginate beads improved gastric integrity (in terms of 
microscopic observation) compared to free emulsion droplets or emulsion micro-clusters, and 
delayed lipid absorption [34]. These findings were confirmed in a human trial in which oil-core 
calcium-alginate-shell capsules (0.5 cm) resulted in faster gastric emptying, and delayed lipolysis 
and absorption as measured with 13C-labelled octanoic acid detected in a breath test [35], though 
these capsules would be quite large to use as a food additive. 
 
Present encapsulates 
 
We designed encapsulates of safflower oil droplets (S) (d32 ~ 25 μm) in Ca-alginate (CA) 
hydrogel particles (0.5-1.2 mm) with different pore size of the hydrogel (5-10 nm). Lipolysis was 
controlled simply by gel particle size, and pore size at neutral pH, as shown for studied in a simple 
static in vitro digestion model. As preparation for the current human trial, we added these S-CA 
particles to yoghurt-meals and studied lipolysis during dynamic in vitro digestion in a three-
compartment model of the upper GI tract (DIDGI, [36], [37]). Digestive media was sampled and 
analysed both for total extent of lipolysis and for the fraction solubilised in mixed bile micelles. To 
distinguish between i) direct inhibition of lipase by alginate [38] and ii) physical barrier to 

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encapsulated oil [24], meals containing S-CA particles were compared to control meals with non-
encapsulated lipid droplets, both in the presence and absence of empty CA particles. Lipolysis of 
non-encapsulated lipid was fast and would correspond to time scales relevant for duodenal 
delivery, whearas encapsulated lipid in CA particles showed delayed lipolysis up to four hours 
after ingestion, corresponding to ileal delivery. 
 
Present study 
The present study will be an explorative study to proof the concept of ileal brake activation using 
yoghurt that contains encapsulated safflower oil droplets in CA gel particles. The effectivity will 
be tested in a randomized, single-blind, placebo-controlled intervention study with cross-over 
design. Within each subject we will study the difference in ad libitum food intake and satiety after 
ingestion of the active yoghurt (containing encapsulated lipid) or control (equicaloric mixture of 
non-encapsulated nutrients: same amount of lipids and empty encapsulates).  
 
 
 
 
2. OBJECTIVES 
Primary objective: To investigate whether encapsulation of lipids decreases ad libitum food 
intake compared to non-encapsulated lipids in a formulation with equal nutrients (control). 
Secondary objectives: 
 
- 
To investigate feelings of satiety after orally ingested lipid encapsulates 
- 
To investigate occurrence and severity of GI symptoms after orally ingested lipid 
encapsulates 
- 
To assess whether differences in food intake, satiety and GI symptoms relate to lipid 
encapsulation  
 
 
 
 
3. STUDY DESIGN 
This intervention study will be a randomized, single-blind, placebo-controlled trial with two 
treatments in a cross-over design (see Figure 1 for a scheme of the design). The population and 
intervention are described in relation to the study design here in short, and into more details in 
the respective sections on population (section 4) and study procedures (section 7.3). 
 

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Figure 1. Overview of the study design of this explorative project on lipid encapsulation (in yoghurt) on satiety 
and food intake, t=0 defined as start of breakfast. The intervention yoghurt differs in the carrier of the lipid 
(active= encapsulated in gel particle for distal lipid release, control=non-encapsulated for fast-release)  
* VAS= Visual Analogue Scales, will be used for nine attributes per time point. 
 
Population 
Healthy subjects (male and female, any ethnic background, 18-65 years old) with overweight (BMI 
25-30 kg/m2) will be studied (inclusion criteria in section 4.2, exclusion criteria in 4.3). Thirty-
five subjects will be recruited (section 10.2 for more information). 
 
Intervention 
After inclusion, subjects will have to visit Maastricht University on three occasions: once to sign 
the consent, fulfil the screening and get an instruction (about half an one hour) and two times to 
attend the test days (about four hours per test day). Both test days will be scheduled in a period 
of a month, with at least one week in between them.  
Every subject receives both treatments on two different test days, following a randomized cross-
over design. The treatments differ in intervention yoghurt, which will be ingested in standardized 
state (relatively empty stomach, ninety minutes after a standardized breakfast that is taken after 
a 10 hour fast; detailed procedures in section 7.3). 
Test day 1
Test day 2
>7 d in between
Screening
35 healthy volunteers 
BMI 25-30 kg/m2
18 - 65 yr
Standardized 
breakfast (t=0)
Intervention
yoghurt (t=90)
(n=2 | randomized)
Ad libitum
lunch (t=210)
9:00
10:30
12:30
Test day
* VAS score:
-
Satiety
-
GI symptoms
10 h
fasting
+
+       +
Active
Control
9:30
10:00                                     10:45    11:00 11:15    11:30    11:45    12:00     12:15
*                *               *                *        *       *       *       *       *       *       *     *

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Both intervention products are low-fat yoghurts of 0.2 L that contain 6 g safflower oil (present as 
droplets with average size of 25 μm), which is encapsulated in gel particles (active, yoghurt A) or 
a non-encapsulated equicaloric mixture of nutrients (control, yoghurt B). 
 
 
 
 
4. STUDY POPULATION 
 Population  
Thirty-five healthy subjects (male and female, any ethnic background, 18-65 years old) with 
overweight (BMI 25-30 kg/m2) will be recruited from a pool of previously included subjects, and 
via advertisements.  
 
 Inclusion criteria 
In order to be eligible to participate in this study, a subject must meet all of the following 
criteria: 
 Based on medical history and previous examination, no serious gastrointestinal 
complaints can be defined. 
 Age between 18 and 65 years. A higher age comes with a higher chance of 
comorbidities. These could influence our study outcomes and therefore this age range 
was chosen. This study will include healthy adult subjects (male and female). Women 
must be taking contraceptives (only needed in women with childbearing potential). 
 BMI between 25-30 kg/m2. 
 Voluntary participation. 
 Able to participate in the study, willing to give informed consent and to comply with 
the study procedures and restrictions. 
 
 Exclusion criteria 
A potential subject who meets any of the following criteria will be excluded from participation 
in this study: 
 Milk (-protein or lactose)- allergy/intolerance. 
 History of severe cardiovascular, respiratory, urogenital, gastrointestinal/ hepatic, 
hematological/immunologic, 
HEENT 
(head, 
ears, 
eyes, 
nose, 
throat), 
dermatological/connective tissue, musculoskeletal, metabolic/nutritional, endocrine, 
neurological/psychiatric diseases, allergy, major surgery and/or laboratory 
assessments which might limit participation in or completion of the study protocol. The 
severity of the disease (major interference with the execution of the experiment or 
potential influence on the study outcomes) will be decided and documented by the 
principal investigator. 
 Administration of investigational drugs or participation in any scientific intervention 
study that may interfere with this study, to be decided by the principal investigator 
prior to the study. 

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 Major abdominal surgery interfering with gastrointestinal function (uncomplicated 
appendectomy, cholecystectomy and hysterectomy allowed, and other surgery) upon 
judgement of the principal investigator. 
 Dieting (medically prescribed, diabetic and vegetarian). 
 Pregnancy, lactation. 
 Excessive alcohol consumption (>20 alcoholic consumptions per week) 
 Intention to stop smoking. 
 Self-admitted HIV-positive state. 
 Abnormal eating behaviour. 
 Reported unexplained weight loss or gain in the month prior to screening. 
 
 Sample size calculation 
The calculations on required sample size should be based on the primary outcome: difference in 
intake of ad libitum meal. To our best knowledge, however, no data is available for this particular 
encapsulation system, so we need to base our calculation on related studies. First, food intake was 
significantly reduced with 79 kcal (SEM 41; n=15 so SD of 159 kcal) after ileal intubation (of 6 g 
lipid; food intake 435 kcal) compared to oral ingestion (of 6 g lipid, combined with saline infusion; 
food intake 514 kcal) [2]. Second, food intake reduced after active micro-encapsulate intake (655 
kcal, SD 216) compared to placebo (699 kcal, SD 238), so a reduction of 44 kcal (P=0.011) [not yet 
published randomized placebo-controlled double-blind cross-over study in 60 healthy overweight 
volunteers]. Third, intra-ileal infusion of 6 g safflower oil, the food intake (464.3 ± 90.7 kcal) was 
122 kcal (21 %) lower compared to placebo (saline infusion, 586.7 ± 70.2 kcal) [1]. On average, 
these three studies showed a difference of 82 kcal with an average SD of 156 kcal, and we used 
these values to base our calculation on. For the statistical test (p-value of 5% (α = 0.05), power 
80% (B = 0.80)) we used the formula: 
 
𝑛𝑛=
(𝑡𝑡𝑛𝑛−1,𝛼𝛼
2  + 𝑡𝑡𝑛𝑛−1,𝛽𝛽)2
𝑑𝑑2
 
 
that can be estimated by: 
𝑛𝑛≥𝜎𝜎2
𝛿𝛿2 (𝑍𝑍𝛼𝛼+ 𝑍𝑍𝛽𝛽)2 
 
with σ being the standard deviation for paired samples (156 kcal), δ being the minimal detectable 
difference in food intake (82 kcal), 𝑍𝑍α being 1.96 (for α = 0.05), 𝑍𝑍β being 0.84 (for B= 0.80). 
 
Filling the formula results in a value of n ≥ 28.3, so at least 29 subjects. However, it must be noted 
that the sample size estimation uses Z-values, derived from a normal distribution. The statistical 
test, used in this sample size, is based on a t-distribution instead of the normal distribution. Thus, 
for small samples (n < 100), the sample size n must be estimated iteratively using the appropriate 
t-values. These t-values depend on ‘degrees of freedom’ (df), which corresponds to n-1, so in our 
case for a sample size n of 29, the degree of freedom is 28 (29-1). Therefore, the following formula 
was used: 
 

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𝑛𝑛≥𝜎𝜎2
𝛿𝛿2 (𝑡𝑡𝑛𝑛−1,𝛼𝛼
2  + 𝑡𝑡𝑛𝑛−1,𝛽𝛽)2 
 
with t-value being 2.048 for a two-sided α of 0.05 with df of 17, and 0.855 for B= 0.80 with df of 
17. 
 
This leads, after a small number of iterations, to a re-estimate of n of at least 31 subjects (n ≥ 30.4). 
According to this power calculation, 31 subjects will need to complete the study. Because of 
possible dropouts, 35 subjects will be recruited for this study. 
 
 
 
 
5. TREATMENT OF SUBJECTS 
During this study, two treatments will be compared, following a randomized cross-over design. 
The treatments differ in intervention yoghurt (active or control formulation), which will be 
ingested in standardized state (relatively empty stomach, ninety minutes after a standardized 
breakfast that is taken after a 10 hour fast). The yoghurt will be provided with a glass of water or 
tea (150 mL). Two hours after ingestion of the intervention yoghurt, food intake will be measured 
in a standardized pasta lunch. Additionally, feelings of satiety and GI symptoms will be determined 
each 30 minutes before the intervention and each 15 minutes after the intervention. For more 
details on study procedure, see section 7.3. 
On each test day, subjects receive one of the two formulations described in Table 1. Both products 
are low-fat yoghurts of 0.2 L that contain 6 g safflower oil (present as droplets with average size 
of 25 μm), which is encapsulated in gel particles (active, yoghurt A) or a non-encapsulated 
equicaloric mixture of nutrients (control, yoghurt B). 
 
Table 1. Description of the composition of the interventional yoghurts, provided in randomized order. 
Formulation Name 
Composition 
Yoghurt A 
Active 
120 g yoghurt + 60 g oil-gel particles (containing 6 g oil) + 24 g water* 
Yoghurt B 
Control 
120 g yoghurt + 54 g empty gel particles + 6 g oil in 24 g water* 
* needed to produce the emulsion for the control as 20% oil emulsion. 
 
 Active investigational product 
To activate the ileal brake mechanism, 6 grams of lipid will be ingested of which at least half will 
be delivered to the ileum following different release profile dependent on encapsulation type. 
From previous research, we know that ileal infusion with 3 gram of lipid results in the activation 
of the ileal brake [6]. The lipid encapsulates (60 g total gel particles) are added to 120 g yoghurt, 
not only to allow easy consumption for the participants, but also to ensure no difference in 
appearance between control and active formulations. To have the same composition in all 
formulations, the active product is matched to the control product in terms of total water content. 
 

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 Control product 
Non-encapsulated lipid will be used as control, to be released into the stomach or proximal 
duodenum, in contrast to the active that will be delivered into the more distal small intestine. The 
control yoghurt will be equicaloric to the active yoghurt, with the same amount of lipids (and same 
droplet size), and the same encapsulation components (empty), only they are present separately 
rather than forming an oil-gel particle. In addition to being equicaloric, the two types of yoghurt 
will have the same mouth feel due to the presence of the empty encapsulation components. 
 
 
 
 
6. INTERVENTION PRODUCT  
Both intervention products are low-fat yoghurts of 204 mL in total that contain 6 g safflower oil 
(present as droplets with average size of 25 μm), which is encapsulated in gel particles (active, 
yoghurt A) or a non-encapsulated equicaloric mixture of nutrients (control, yoghurt B). The used 
low-fat yoghurt (Campina, Magere yoghurt, 0% fat) itself, to which the lipid formulations are 
added, is commercially available on the Dutch market and purchased from a local store. Besides, 
all added ingredients (whey proteins, alginate, calcium, safflower oil) are commercial available 
food ingredients, and repeatedly used in previous human trials so considered having a history of 
safe use. The intervention yoghurts will be produced in accordance with safety and quality 
regulations of the commercial manufacturer, and after preparation properly stored as described 
in section 6.7. 
 
 Name and description of intervention yoghurts 
The active intervention product will be a low-fat yoghurt (120 g, Campina, Magere yoghurt, 0% 
fat) mixed with 60 gram ‘encapsulates’ (gel particles that encapsulate safflower oil in such a way 
to deliver into the ileum). These encapsulates compose for 88 wt% of water (52.9 g per 60 g gel 
particles), and contain 2 wt% alginate (1.08 g per 60 g gel particles) and 10 wt% safflower oil (6 
g per 60 g gel particles, present as droplets with average size of 25 μm). The active intervention 
yoghurts contain 105 kcal; of which 51 kcal% comes from the lipid (see Table 2).  
 
Control product will be the same low-fat yoghurt as the active product, containing the same 
components as the 60 gram encapsulates, only with different physical structure: 54 gram empty 
gel particles (1.08 g alginate) present separately from 6 g safflower oil (same droplet size as in 
encapsulate). The control yoghurts also contain 105 kcal; of which 51 kcal% comes from the lipid 
(see Table 2).  
 
 
Table 2. Overview of the nutrient composition of the studied formulations, and caloric content. 
  
Yoghurt A 
Yoghurt B 
Name 
Active 
Control 

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Safflower oil (g) 
6.0 
6.0 
Whey protein (g) 
0.24 
0.24 
Alginate (g) 
1.08 
1.08 
Water inter-/intra-gel (g) 
76.9 
76.9 
Yoghurt (g) 
120 
120 
Total (g) 
204 
204 
Total (kcal) 
105 
105 
Lipid contribution (kcal%) 
51% 
51% 
 
 
 Summary of findings from non-clinical studies 
Lipolysis has be controlled in vitro by lipid encapsulation in both digestible protein gels [22], [23] 
and indigestible alginate gels [24], [25]. The latter is the approach taken in the current study given 
the resistance of alginate matrix against gastric proteases [23], so ensures maintenance of the 
encapsulate structure in the stomach. In addition, the pH-dependency of volume and porosity of 
alginate gel particles makes them very suitable for ileal delivery [24], [26]–[28]. 
We designed encapsulates of safflower oil droplets (S) (d32 ~ 25 μm) in Ca-alginate (CA) hydrogel 
particles (0.5-1.2 mm) with different pore size of the hydrogel (5-10 nm). Lipolysis was controlled 
simply by gel particle size, and pore size at neutral pH, as shown for studied in a simple static in 
vitro digestion model. As preparation for the current human trial, we added these S-CA particles 
to yoghurt-meals and studied lipolysis during dynamic in vitro digestion in a three-compartment 
model of the upper GI tract (DIDGI, [36], [37]). Digestive media was sampled and analysed both 
for total extent of lipolysis and for the fraction solubilised in mixed bile micelles. To distinguish 
between i) direct inhibition of lipase by alginate [38] and ii) physical barrier to encapsulated oil 
[24], meals containing S-CA particles were compared to control meals with non-encapsulated lipid 
droplets, both in the presence and absence of empty CA particles. Lipolysis of non-encapsulated 
lipid was fast and would correspond to time scales relevant for duodenal delivery, whearas 
encapsulated lipid in CA particles showed delayed lipolysis up to four hours after ingestion, 
corresponding to ileal delivery. 
 
 Summary of findings from clinical studies 
Ileal brake activation has been proven to be effective with direct intubation of all three 
macronutrients via a naso-ileal catheter (overview of literature in [3]). About 30 years ago, ileal 
lipid infusion was already shown to reduce food intake [4], [5]. Moreover, ileal lipid infusion 
increased perception of satiety/fullness or decreased hunger in most studies [1], [2], [4]–[7]. 
 
Regarding the current product, all ingredients (whey proteins, alginate, calcium, safflower oil) are 
commercially available food products with bio-base (so no synthetic additives), and have been 
repeatedly used in previous human trials (just some examples: alginate [29]–[33], calcium [29], 
[35], whey proteins even in infants [39], safflower oil [1], [6], [40]). Alginate as such has been used 
in clinical trials in multiple formulation forms, and it might reduce feelings of hunger (especially 
when present in solution) or have no effect [29]–[33].  

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Relatively large Ca-alginate gel particles (3.9-4.4 mm) have been tracked using MRI scanning in 
the human GI system (n=10) and were more visible in the ileum compared to the jejunum and 
duodenum [29]. This suggest an extended residence time in the ileum, and therefore favours ileal 
brake activation. 
One human trial has been performed on an encapsulation system containing similar components: 
oil-core calcium-alginate-shell capsules (0.5 cm), which resulted in faster gastric emptying, and 
delayed lipolysis and absorption as measured with 13C-labelled octanoic acid detected in a breath 
test [35], though these capsules would be quite large to use as a food additive. 
 
 Summary of known and potential risks and benefits 
In our previous experiments with ileal brake activation via nutrient delivery through an ileal 
catheter, high distal ileal nutrient concentrations have been reached. However, none of the 
subjects experienced any discomfort during or after direct infusion into the ileum. We do not 
anticipate on major complaints or side effects for the current study with oral application route 
and therefore less pronounced ileal nutrient concentrations. 
Besides, potential risks are expected to be minimal: First, a commercially available low-fat yoghurt 
(Campina, Magere yoghurt, 0% fat) will be used. Second, all of the added components are 
commercially available food ingredients with bio-base (so no synthetic additives)that have been 
repeatedly used in previous human trials. These include: alginate[29]–[33], calcium [29], [35], 
whey proteins [39], safflower oil [1], [6], [40]). Thus, all ingredients have a documented history of 
safe use. As mentioned previously, only for alginate we foresee a possibility for GI symptoms, for 
example abdominal bloating (3 out of 48 participants); but no significant difference (in nausea, 
fullness, bloating and pain scores) were detected compared to placebo [33]. Although alginate is 
used for multiple human applications (e.g., acid burn), it may cause discomfort and/or GI 
symptoms due to viscosity-induced delay of the GI transit. To minimize possible discomfort and 
GI symptoms, the study will be halted when pain (as burning/cramp/colic) is measured to be 
extremely unpleasant (VAS scores > 95 out of 100) in three or more subjects. 
  
 Description and justification of route of administration 
The lipids will not be administered via an ileal catheter, as the effectivity of this administration 
route has been proven already in previous studies (for an overview of studies, we refer to the 
review [3]). For an effective weight management strategy, administration should be performed 
via the normal route of food intake: oral administration. The current study will focus on the ability 
of lipid to induce the ileal brake when given orally. The lipids will be mixed with yoghurt, not only 
to allow easy consumption for the participants, but also to ensure no difference in appearance 
between control and active formulation. The lipids from the active intervention yoghurt will be 
delivered into the more distal small intestine within the two hours between ingestion and meal 
intake, and might consequently activate the ileal brake, whereas the control intervention yoghurt 
will deliver nutrients more proximally (to the stomach and duodenum). This allows investigating 
the effects of distal small intestinal delivery of undigested nutrients on food intake.  
 

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 Dosages, dosage modifications and method of administration 
To determine the dosage, we combined knowledge on effective dosage upon direct ileal fat 
perfusion with knowledge on the release profile of the current encapsulation system. Previous 
ileal brake studies have shown no dose dependency of satiety between 3 g and 9 g [6]. The current 
encapsulation system is expected to deliver at least 50% of the encapsulated lipid into the ileum, 
based on in vitro release data from static and dynamic digestion models (as summarized in section 
1 and 6.2). We chose the investigational products to contain as little lipid as possible to still be 
effective, because lipid is a highly caloric nutrient and difficult to be compensated in the 
subsequent lunch. Therefore, the intervention yoghurts contain 6 grams of encapsulated safflower 
oil to release ≥ 3 g into the ileum (≥ 50%). 
The method of administration of the active is based on an encapsulation method, using a matrix 
of alginate that is gelled by calcium divalent ions. The release of encapsulated lipid from these gel 
particles is based on pH-induced swelling at neutral pH as in the small intestine. This swelling 
increases the porosity of the gel matrix, which favours diffusion via increasing its coefficient. We 
found this diffusion coefficient under in vitro intestinal conditions to determine the release rate. 
By reverse engineering, we designed encapsulation systems that encapsulate safflower oil, and 
tuned the release profile based on gel matrix.  
 
 Preparation and labelling of Investigational Product 
Handling and labelling will be done in the food facility of the Metabolic Research Unit Maastricht 
(MRUM, Maastricht University), following relevant HACCP guidelines. All ingredients (whey 
proteins, alginate, calcium, safflower oil) are commercial available food products, so no special 
precautions have to be taken in handling of the substances (see section 12 for a structured risk 
analysis). The alginate gel particles, both empty and filled with lipid, will be produced in 
accordance with safety and quality regulations of the commercial manufacturer. After preparation 
of these particles, they will be stored not longer than four days (96 hours) in 0.5 M CaCl2 solution 
under refrigerated conditions (temperature of 4 °C) to ensure microbial safety. The ingredients 
(i.e., yoghurt, oil-gel particles, or gel particles and emulsion) will be mixed freshly (1 to 20 hours 
before consumption) and stored under refrigerated conditions as suggested by the yoghurt 
supplier (Campina, Magere yoghurt, 0% fat). A detailed description of the procedures can be found 
in a separate standard operating procedure for the active and control yoghurt. 
Labelling of the products will be according to the following code: study title (Lipid encapsulates, 
LE), subject number (01-35), yoghurt (A, B), and date. For example: LE07/A 13-02-17 means 
subject 7 of lipid encapsulates study-yoghurt A (active) produced on February 13th 2017. 
 
 Drug accountability 
Not applicable as all used substances are food grade. 
 
 
 
 

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7. METHODS 
 Study parameters/endpoints 
7.1.1 
Main study parameter/endpoint 
The main study parameter is the intake of ad libitum meal (kcal), as measured during pasta lunch 
(based on weight reduction of the plate assuming a homogenous meal). The reduction in amount 
of intake is supposed to reflect the activation of the ileal brake. The food intake will be compared 
between encapsulated lipids (active, yoghurt A) and non-encapsulated lipids in a formulation with 
equal nutrients (control, yoghurt B) within a subject. 
7.1.2 
Secondary study parameters/endpoints 
Feelings of satiety (VAS scores) 
The secondary study parameter is the effectivity to activate the ileal brake and, hence, induce 
feelings of satiety and reduce feelings of hunger, as measured on VAS scores (satiety, fullness, 
hunger, desire to eat, desire to snack). The VAS scores will be compared in time between 
intervention and control within a subject. 
 
GI symptoms (VAS scores) 
Another secondary study parameter is the occurrence and severity of gastrointestinal (GI) 
symptoms that orally ingested lipid encapsulates might give, as measured on VAS scores of GI 
symptoms (bloating, discomfort, pain, nausea). The VAS scores will be compared in time between 
intervention and control within a subject. Besides, the number of AEs and SAEs will be recorded. 
Moreover, we will assess whether differences in GI symptoms relate to encapsulate type. 
 
 Randomisation, blinding and treatment allocation 
All 35 participants receive two treatments of one day each, following a cross-over design. The 
order in which they receive the treatments will be determined through randomisation. 
Randomisation will minimize the risk of bias and enhance the validity of statistical comparisons. 
As subjects qualify for the study, they will be assigned a subject number (01 - 35, via lottery) that 
corresponds with a predefined randomization schedule. 
 
Blinding of packing and treatment will be assured for subjects. We will check at the end of the last 
test day whether the subjects were expecting to consume a control on one or more of the test days, 
and in case yes, on which day(s) they received it. Besides, on this day we will ask how they liked 
the yoghurts.  
 
The VAS questionnaires will be labelled by the researcher with study title (Lipid encapsulates, 
LE), subject number (01-35), yoghurt (A, B) and time point (t= 0, 30, 60, 90, 105, 120, 135, 150, 
165, 180, 195, 210 min). For example: LE07/ A / 120 - Subject 07, active yoghurt, VAS scores at 
120 minutes.  

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Besides, labelling of the products will be according to the following code: study title (Lipid 
encapsulates, LE), subject number (01-35), yoghurt (A, B), and date. For example: LE07/A 13-02-
17 means subject 7 of lipid encapsulates study-yoghurt A (active) produced on February 13th 
2017. 
 
 Study procedures 
Study procedure of the total study period 
Thirty-five healthy subjects (male and female, any ethnic background, 18-65 years old) with 
overweight (BMI 25-30 kg/m2) will be recruited (for more information on recruitment and 
consent see section 10.2; inclusion criteria in section 4.2, exclusion criteria in 4.3). If the volunteer 
is interested, an appointment will be made to sign the consent and complete the medical screening 
(discuss questionnaire (medical history, health status and medicine, and physical activity level 
(low, moderate or active)). If the volunteer is suitable, we will measure height and body weight as 
described below). After the subject is included, (s)he will receive an introduction lecture (of about 
half an one hour) and two test days (that consist of about four hours) will be scheduled per 
participant (with at least seven days in between the test days). Both test days will be scheduled in 
a period of a month. 
 
Height measurement for BMI calculation- Height will be measured using a standard 
stadiometer. The participant has to remove any footwear. All participants should have their height 
measured while in bare or stocking feet. The participant will be instructed to stand on the base of 
the stadiometer, and the investigator will check if the participant is standing straight with 
shoulders back and resting flat on his/her feet. The height will be recorded in centimetres (cm) to 
the nearest 0.1 cm.  
Body weight measurement- Body weight will be measured using an electronic or digital scale, 
which is regularly calibrated. Subjects are asked to remove their footwear, outer garments (e.g., 
jackets, hats, heavy sweaters, etc.), belt, heavy jewellery, and they are asked to empty their 
pockets. The participant will be asked to remain on the scale, and the investigator will read the 
screen once stabilized and record the value in kg to the nearest 0.1 kg. 
 
Procedure of the intervention study days 
The procedure of the intervention study days is summarized in Table 3. On each test day, the 
subjects will arrive at 8:50 am at Maastricht University, after a ten hour fast, so no eating and 
drinking is allowed other than ad libitum consumption of water and tea (without milk or sugar) 
after 22:00 the night before each test day.  
Upon arrival at Maastricht University (8:50 am), compliance to the previously mentioned rules 
will be checked and baseline measurements will be done (“t=0” min), including nine VAS scores 
for satiety and GI symptoms. Then, the experiment starts with the ingestion of a standardized 
breakfast (9:00 am, t=0 min). During all study days, this breakfast will be relatively light (±120 
kcal) and low in fat (≤0.5 g total lipid): 1 sandwich (tijgerbrood tarwe 1 g lipid/100g, 1 boterham: 
35g (±84 kcal)) with marmalade (0 g lipid/100g, 1 portion: 15g (±36 kcal)) and one glass of water 
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Ninety minutes after breakfast (10:30 am, t=90 min), once major part of the breakfast has been 
emptied from the stomach, subjects receive a low-fat yoghurt-like product of 0.20 L that contains 
one of the two formulations of emulsified safflower oil (6 g lipid as active or control formulation, 
for product details see section 5), following a randomized cross-over design. Each 30 minutes 
before the intervention and each 15 minutes after the intervention, the nine VAS scores for satiety 
and GI symptoms will be measured. Two hours after the intervention yoghurt (12:30, t=210 min), 
ad libitum meal intake will be measured as amount eaten from a large pasta meal (>1 kg, obtained 
from a local supermarket). After collection of these results, the test day is finished and the subject 
returns home (around 13:00 am). For an overview of the complete study design, see Figure 1. 
 
VAS scores for satiety and GI symptoms- During a test day, satiety and GI symptoms will be 
measured at 12 time points (t= 0, 30, 60, 90, 105, 120, 135, 150, 165, 180, 195, 210 min). 
Questionnaires will be used to measure satiety feelings (satiety, fullness, hunger, desire to eat, 
desire to snack) and GI symptoms (bloating, discomfort, pain, nausea). These nine attributes will 
be measured using Visual Analogue Scales (VAS, 0 to 100 mm) scores, with the most negative or 
lowest intensity feelings at the low end and the opposing terms at the high end. The subject will 
be asked to indicate his feeling at that moment. The investigator can easily quantify the rating as 
shown in previous studies (for example: METC 13-2-025; 11-3-034; 14-3-062). The scoring forms 
will be collected immediately so that they cannot be used as a reference for later scorings.  
 
 

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Table 3. Schedule of the participant prior and during the test days. 
When? 
Action/Intervention 
Measurement 
Interview 
Sign consent, complete screening (inclusion); 
and information lecture 
Screening 
questionnaire, 
height, body weight 
Test days (2x) 
 
 
8:50 am Arrival* and baseline 
VAS (S & GI, t=0 min)** 
9:00 am Standardized breakfast(t=0 min) 
 
9:30 am  
VAS (S & GI, t=30 min) 
10:00 am  
VAS (S & GI, t=60 min) 
10:30 am Intervention yoghurt(t=90 min) 
VAS (S & GI, t=90 min)** 
10:45 am  
VAS (S & GI, t=105 min) 
11:00 am  
VAS (S & GI, t=120 min) 
11:15 am  
VAS (S & GI, t=135 min) 
11:30 am  
VAS (S & GI, t=150 min) 
11:45 am  
VAS (S & GI, t=165 min) 
12:00 am  
VAS (S & GI, t=180 min) 
12:15 am  
VAS (S & GI, t=195 min) 
12:30 am Ad libitum lunch(t=210 min) 
VAS (S & GI, t=210 min)** 
13:00 am Return home 
 
VAS= Visual Analogue Scales for satiety (S) and GI symptoms (GI). * after 10 h fasting. ** VAS score 
measured just before the intervention. 
 
 Withdrawal of individual subjects 
Subjects can leave the study at any time for any reason (also without reason) if they wish to do so 
without any consequences. The responsible investigator can also withdraw a subject if continuing 
participation is in his opinion deleterious for the subject’s wellbeing. Subjects can also be 
withdrawn in case of protocol violations and non-compliance. When a subject withdraws from the 
study, a medical examination can be performed if considered necessary by the nurse 
practitioner/physician. In case of withdrawal because of AEs or SAEs haematological, blood 
chemistry and urine laboratory test or other special examinations may be performed. 
7.4.1 
Specific criteria for withdrawal 
Not applicable. 
 
 Replacement of individual subjects after withdrawal 
Thirty-five subjects will be included at the start of this explorative study. Subjects will not be 
replaced after withdrawal.  
 

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 Follow-up of subjects withdrawn from treatment 
No formal follow-up visits are planned in case a subject withdraws from the study after 
administration of a study investigational product.  
 
 Premature termination of the study 
The study will be discontinued if the treatments are intolerable for the majority of the subjects. 
 
 
 
 
8. SAFETY REPORTING 
 Temporary halt for reasons of subject safety 
In accordance to section 10, subsection 4, of the WMO, the sponsor will suspend the study if there 
is sufficient ground that continuation of the study will jeopardise subject health or safety. The 
sponsor will notify the accredited METC without undue delay of a temporary halt including the 
reason for such an action. The study will be suspended pending a further positive decision by the 
accredited METC. The investigator will take care that all subjects are kept informed.  
 
 AEs, SAEs and SUSARs 
8.2.1 
Adverse events (AEs) 
AEs are defined as any undesirable experience occurring to a subject during the study, whether 
or not considered related to the investigational product, trial procedure, or the experimental 
intervention. All AEs reported spontaneously by the subject or observed by the investigator or his 
staff will be recorded. 
8.2.2 
Serious adverse events (SAEs) 
A SAE is any untoward medical occurrence or effect that: 
- 
results in death; 
- 
is life threatening (at the time of the event); 
- 
requires hospitalisation or prolongation of existing inpatients’ hospitalisation; 
- 
results in persistent or significant disability or incapacity; 
- 
is a congenital anomaly or birth defect; or 
- 
any other important medical event that did not result in any of the outcomes listed above 
due to medical or surgical intervention but could have been based upon appropriate 
judgement by the investigator. 
An elective hospital admission will not be considered as a SAE.  
 

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The investigator will report all SAEs without undue delay after obtaining knowledge of the events, 
through the web portal ToetsingOnline to the accredited METC that approved the protocol, within 
seven days of first knowledge for SAEs that result in death or are life threatening followed by a 
period of maximum of eight days to complete the initial preliminary report. All other SAEs will be 
reported within a period of maximum 15 days after the sponsor has first knowledge of the SAE. 
8.2.3 
Suspected unexpected serious adverse reactions (SUSARs) 
Not applicable. 
 
 Annual safety report 
Not applicable. 
 
 Follow-up of AEs 
All AEs will be followed until they have abated, or until a stable situation has been reached. 
Depending on the event, follow up may require additional tests or medical procedures as 
indicated, and/or referral to the general physician or a medical specialist. 
SAEs need to be reported until end of study within the Netherlands, as defined in the protocol. 
 
 Data Safety Monitoring Board (DSMB) / Safety Committee 
Not applicable. 
 
 
 
 
9. STATISTICAL ANALYSIS 
The SPSS statistical software package Version 23.0.0.2 (IBM© SPSS Statistics, Chicago, IL, USA) will 
be used for statistical analysis. The data will be organized in this software, containing descriptive 
independent variables (ID, age, gender, BMI), fixed variables (test day (1, 2), treatment (A, B), 
time) and dependent variables (food intake and VAS scores). A descriptive analysis of the study 
population will be performed first, about the independent variables age, gender and BMI, 
including mean ± S.D. The dependent variables will be checked to meet the requirements of the 
statistical models described, including and normality, and homogeneity of variance. Continuous 
data will be reported as mean ± S.D and/or S.E.M. and statistical significance will be set at p< 0.05, 
two sided. 
 

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 Primary study parameter(s) 
The main study parameter is the intake of ad libitum meal (kcal). The difference in intake of ad 
libitum meal (kcal) will be compared within-subjects, between ingestion of the active or control 
yoghurt. 
H0: There is no significant difference in ad libitum meal intake after control and active yoghurt. 
H1: Ad libitum meal intake differs between control and active yoghurt. 
If the food intake data (2 values per subject) is normally distributed and homogeneity of variance 
holds, a paired-samples T-test will be performed to evaluate the null hypothesis that there is no 
difference in ad libitum food intake after control and active yoghurt (n=35, p ≤ 0.05). 
Missing values of the primary study parameter food intake will be left out of the analysis, because 
the paired-samples T-test compares food intake within-subjects. 
 
 
 Secondary study parameter- VAS scores for satiety and GI symptoms 
Secondary parameters include differences between treatments in feelings of satiety (as measured 
by VAS) and GI symptoms (as measured by VAS). During each of the test days, participants (n=35) 
fill in nine VAS scores (VAS1-VAS9) per time point, at 12 time points (t= 0, 30, 60, 90, 105, 120, 
135, 150, 165, 180, 195, 210 min). If the VAS data is normally distributed and homogeneity of 
variance holds, we will use a repeated measures analysis of variance (ANOVA) model that includes 
the fixed factors treatment, time, and the interaction treatment * time to evaluate the null 
hypothesis that there is no difference in VAS scores after control and active yoghurt (n=35, p ≤ 
0.05). If the results of the ANOVA indicate a significant effect of intervention yoghurt (Wilks’ 
Lambda test, p ≤ 0.05), the treatments will be compared based on pairwise comparison with a 
Tukey post hoc test. 
Missing values of the secondary study parameter will cause the intervention to be left out, due to 
the use of ANOVA. 
 
 
 
 
10. ETHICAL CONSIDERATIONS 
 Regulation statement 
The study will be conducted according to the principles of the Declaration of Helsinki (as amended 
in Tokyo, Venice, Hong Kong, Somerset West, Edinburgh, Seoul and Fortaleza. Note of clarification 
added in Washington and Tokio) and in accordance with the Medical Research Involving Human 
Subjects Act (WMO). Last updated during WMA General Assembly meeting in Fortaleza, Brazil, 
October 2013. 
 

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 Recruitment and consent 
Subjects will be recruited from a pool of previously included subjects that participated in previous 
studies, and besides, using posters at the local libraries and student frequented areas, advertising 
in local newspapers, and if needed using www.onderzoeksmachine.nl. Initial information will be 
provided verbally or via email. The written information brochure will be send by regular mail or 
email. If a volunteer is interested, an appointment will be made to sing an informed consent and 
complete the medical screening (for the procedure see section 7.3). A minimum of one week is 
provided to decide whether the volunteer would like to participate. Volunteers will not be 
contacted if they do not respond to the sent information brochure. Upon arrival for the first visit, 
the participants will have to sign an informed consent before screening can start. If participants 
withdraw from the study after the screening, these results will be destroyed. Participants can 
make at any time an appeal to the independent doctor that has been appointed to this study. 
Participants will be informed that their decision to participate is voluntary and they can withdraw 
at any time without giving a reason. At the end of the study, participants will have the opportunity 
to be informed about their individual results and the group results.  
 
 
 Objection by minors or incapacitated subjects 
Not applicable. 
 
 Benefits and risks assessment, group relatedness 
The benefits of this study can be great: it will provide knowledge about the principle of intestinal 
brake activation through dietary yoghurt that contains encapsulated lipid (effectivity compared 
to control). If found effective in this study, such a yoghurt snack could be directly implemented in 
a diet, to prevent overeating, and in that way provide a long-term weight management strategy. 
However, besides that there is no personal benefit for the subjects. 
This study is non-invasive, so we do not expect any great risks for the subjects. The oral 
consumption of a breakfast, yoghurt snack, and lunch clearly reduces the discomfort in subjects 
compared to our previous intestinal brake studies with naso-ileal catheter. Moreover, all 
consumed products are food-grade and could appear in a regular diet. To minimize discomfort 
further, the study will be halted when pain (as burning/cramp/colic) is measured to be extremely 
unpleasant (VAS scores > 95 out of 100) in three or more subjects. 
 
 Compensation for injury 
Insurance for participants will be provided in accordance with the legal requirements in the 
Netherlands (Article 7 WMO and the Measure regarding Compulsory Insurance for Clinical 
Research in Humans of 23th June 2003). Maastricht University has a standard WMO insurance a 
liability insurance for research participants.  
 

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The sponsor/investigator has a liability insurance that is in accordance with article 7 of the WMO. 
The sponsor (also) has an insurance that is in accordance with the legal requirements in the 
Netherlands (Article 7 WMO). This insurance provides cover for damage to research subjects 
through injury or death caused by the study. 
1. € 650.000,-- (i.e., six hundred and fifty thousand Euro) for each subject who participates 
in the Research; 
2. € 5.000.000,-- (i.e., five million Euro) for all subjects who participate in the Research;  
3. € 7.500.000,-- (i.e., seven million five hundred thousand Euro) for the total damage 
incurred by the organisation for all damage disclosed by scientific research for the 
Sponsor as ‘verrichter’ in the meaning of said Act in each year of insurance coverage. 
The insurance applies to the damage that becomes apparent during the study or within four years 
after the end of the study. 
 
 Incentives 
Once completed the full study, the subjects will receive 75 euro (including travel expenses). In 
case a subject stops participation during the study, he or she will be reimbursed pro rato. 
 
 
 
11. ADMINISTRATIVE ASPECTS, MONITORING AND PUBLICATION 
 Handling and storage of data and documents 
Data are handled confidentially and anonymously in accordance to WBP 
(in 
Dutch: 
Wet 
Bescherming Persoonsgevens; Personal Data Protection Act). To guarantee the privacy of the 
participants, subject numbers will be used in such a way that no personal information about the 
participants will be available (allocated via lottery). All questionnaires and data will be coded 
(using study title (Lipid encapsulates, LE), and subject number (01-35), yoghurt (A, B) and time 
point (t= 0, 30, 60, 90, 105, 120, 135, 150, 165, 180, 195, 210 min). For example: LE07/ A / 120- 
Subject 07, active yoghurt, VAS scores at 120 minutes. The researcher keeps the key of the code 
in a locked cabinet. Next to the research team, the data is assessable for the METC, IGZ and 
monitors. No human material will be taken during this study. All primary documents and data 
shall be kept for 15 years after the end of the experimental phase of the study for possible 
inspection. All future studies will always be sent to the METC for approval.  
 
 Monitoring and Quality Assurance  
The study will be monitored by the Clinical Trial Centre Maastricht (CTCM). 
 
 Amendments  
Amendments are changes made to the research after a favourable opinion by the accredited METC 
has been given. All amendments will be notified to the METC that gave a favourable opinion.  

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A ‘substantial amendment’ is defined as an amendment to the terms of the METC application, or 
to the protocol or any other supporting documentation, that is likely to affect to a significant 
degree: 
- the safety or physical or mental integrity of the subjects of the trial; 
- the scientific value of the trial; 
- the conduct or management of the trial; or 
- the quality or safety of any intervention used in the trial. 
All substantial amendments will be notified to the METC and to the competent authority. 
 
Non-substantial amendments will not be notified to the accredited METC and the competent 
authority, but will be recorded and filed by the sponsor. 
 
 Annual progress report 
The current study is expected to take substantially shorter than a year. In case of considerable 
delay, the sponsor/investigator will submit a summary of the progress of the trial to the 
accredited METC once a year. Information will be provided on the date of inclusion of the first 
subject, numbers of subjects included and numbers of subjects that have completed the trial, SAE/ 
SARs, other problems, and amendments.  
 
 Temporary halt and (prematurely) end of study report 
The investigator/sponsor will notify the accredited METC of the end of the study within a period 
of eight weeks. The end of the study is defined as the last patient’s last visit. The sponsor will notify 
the METC immediately of a temporary halt of the study, including the reason of such an action. In 
case the study is ended prematurely, the sponsor will notify the accredited METC within 15 days, 
including the reasons for the premature termination. 
The investigator/sponsor will send the publication on the results of the study to the accredited 
METC after peer-reviewed by a scientific journal; if not accepted within one year after the end of 
the study, the manuscript will be send as submitted to the journal within one year after the end of 
the study to the accredited METC. 
 
 Public disclosure and publication policy 
All trial results, both positive and negative, will be disclosed in agreement with the CCMO 
statement on publication policy. Based on the results of this study, at least one publication will be 
submitted for publication to a peer-reviewed scientific journal. The authorship of the article shall 
be determined in appropriate consultations based on a considerable contribution to the set-up 
and execution of the study and an active participation in publication. None of the parties 
concerned has the right of veto considering publication. 
 

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12. STRUCTURED RISK ANALYSIS  
 Potential issues of concern 
a. Level of knowledge about mechanism of action 
Ileal brake activation has been proven to be effective with direct intubation of all three 
macronutrients via a naso-ileal catheter (overview of literature in [3]). About 30 years ago, ileal 
lipid infusion was already shown to reduce food intake [4], [5]. Moreover, ileal lipid infusion 
increased perception of satiety/fullness or decreased hunger in most studies [1], [2], [4]–[7]. The 
mechanism of action is based on sensed lipid species at intestinal receptors, which induce distal 
to proximal negative feedback mechanisms [9]. This mechanism is induced more strongly when 
the degradation products of lipid digestion (fatty acids, monoglycerides) are sensed rather than 
by the intact lipids itself (triglyceride molecules) (clear overview of literature in [9], paragraph 
4.1.1). Hence, for efficient ileal brake activation, lipase-mediated hydrolysis of lipids is required, 
and in addition, proximal absorption of degradation products has to be prevented. 
To be an effective weight management strategy, a different administration route is required than 
direct intubation, namely orally with the normal route of food intake. When applied orally, 
however, the major part of dietary lipids will be digested and absorbed in the proximal small 
intestine and are not likely to induce the strong ileal brake mechanism. Hence, to activate the ileal 
brake, the digestion of lipid needs to be controlled. To truly control lipid digestion, the structuring 
approach should go beyond interfacial design [21], and may focus on control of enzyme diffusion 
towards its substrate by means of a larger structure (more details on this approach can be found 
in section 1). 
 
b. Previous exposure of human beings with the test product(s) and/or products with a similar 
biological mechanism 
Humans have not been exposed to the current test products. However, humans have been exposed 
regularly to the separate components present in the current products: low-fat yoghurt, safflower 
oil, whey protein, calcium, alginate. First of all, the low-fat yoghurt (Campina, Magere yoghurt, 0% 
fat) is a product from a local supermarket that is included in the diet of major part of the Dutch 
population. Safflower oil is a vegetable oil that can be part of a normal diet. Moreover, the exact 
same safflower oil has been intubated at different locations in the small intestine in previous ileal 
brake studies by the current principal investigator without GI symptoms. The biopolymers whey 
protein and alginate can be used in food preparations, and are safe to use in vivo. No GI symptoms 
were observed in the previous study by the current principal investigator, where whey protein, 
carbohydrates and alginate were present (but no lipid), even at higher concentrations [Alleleyn 
and co-workers, in press]. 
 
c. Can the primary or secondary mechanism be induced in animals and/or in ex-vivo human cell 
material? 

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The primary mechanism, the ileal brake, has been induced multiple times in vivo in clinical trials, 
as described in 12.1a). Moreover, the transposition of the ileum to the jejunum in rat resulted in 
higher GLP-1 and PYY levels, and weight loss [41]. In an ex vivo pig study increased GLP-1 and PYY 
release was found after ileal exposure to nutrients [42]. These results all contribute to the 
suggestion that satiety hormones are involved in the ileal brake mechanisms. 
 
d. Selectivity of the mechanism to target tissue in animals and/or human beings 
The satiety hormone receptors (GLP-1r, PYYr) are expressed in the distal gut regions [43]. And 
after stimulation of ex vivo ileal tissue, satiety hormone release was the highest in the most distal 
part of the small intestine [42]. In older animal studies it has been shown that signals arising in 
the lower duodenum and upper jejunum have no effect on the short-term control of food intake 
whereas signals from the more distal jejunum and ileum significantly inhibited total daily food 
intake [44]. Furthermore a study of Maljaars et al. (2011) showed that ileal fat infusions had the 
most pronounced effect on food intake and satiety compared to oral, duodenal and jejunal fat 
ingestion [2]. The data all contribute to the selectivity of the mechanism to the target tissue: the 
ileum.  
 
e. Analysis of potential effect 
To our best knowledge, no data is available for this particular encapsulation system about food 
intake and satiety. Our encapsulation system, however, had been extensively studied in simulated 
digestive conditions, including both static and dynamic in vitro models. Based on these models, 
the behaviour of the encapsulation system and rates of lipolysis were being estimated. Based on 
previous studies using the same oil, it is known that already at 3 g intra-ileal infusion, satiety can 
be induced compared to control (orally taken lipid) [6]. Intra-ileal infusion of 6 g safflower oil, the 
food intake (464.3±90.7 kcal) was 21 % lower compared to placebo (saline infusion, 586.7 ±70.2 
kcal) [1]. Besides, food intake was significantly reduced with 79 kcal (SEM 41; n=15 so SD of 159 
kcal) after ileal intubation (of 6 g lipid; food intake 435 kcal) compared to oral ingestion (of 6 g 
lipid, combined with saline infusion; food intake 514 kcal) [2]. In another study on encapsulation, 
food intake reduced after active micro-encapsulate intake (655 kcal, SD 216) compared to placebo 
(699 kcal, SD 238), so a reduction of 44 kcal (P=0.011) [not yet published randomized placebo-
controlled double-blind cross-over study in 60 healthy overweight volunteers]. On average, these 
three studies showed a difference of 82 kcal with an average SD of 156 kcal. 
 
f. Pharmacokinetic considerations 
Not applicable. 
 
g. Study population 
The study population includes healthy overweighed subjects (BMI 25-30 kg/m2), in the age of 18-
65 years.  
 
h. Interaction with other products 
Not applicable. 
 

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i. Predictability of effect 
This study is non-invasive, with the primary outcome variables being food intake and the 
secondary study parameters measured using a questionnaire, so no biomarkers will be used. The 
questionnaires will include VAS scores for satiety (satiety, fullness, hunger, desire to eat, desire to 
snack) and GI symptoms (bloating, discomfort, pain, nausea). To minimize possible discomfort, 
the study will be halted when pain (as burning/cramp/colic) is measured to be extremely 
unpleasant (VAS scores > 95 out of 100) in three or more subjects. 
 
 
j. Can effects be managed? 
Not applicable. 
 
 Synthesis 
The overweight of the subjects (BMI 25-30 kg/m2) might bring a higher risk compared to persons 
with normal weight. To reduce this risk, potential subjects are medically screened and only 
included when relatively healthy.  
The meals and yoghurts are bought from a local supermarket and only used before the expiry date. 
In addition, all added ingredients are food-grade according to EU regulation: safflower oil, calcium, 
alginate and whey protein. New batches will be ordered for this study and opened only in rooms 
that are suitable for human consumption. Handling and labelling will be done in the food facility 
of the Metabolic Research Unit Maastricht (MRUM, Maastricht University), following relevant 
HACCP guidelines. 
This study is non-invasive, so we do not expect any great risks for the subjects. The oral 
consumption of a breakfast, yoghurt snack, and lunch clearly reduces the discomfort in subjects 
compared to our previous intestinal brake studies where naso-ileal feeding catheters were 
introduced. To minimize discomfort further, the study will be halted when pain (as 
burning/cramp/colic) is measured to be extremely unpleasant (VAS scores > 95 out of 100) in 
three or more subjects. 
 
 
 
 
 
 
 
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