Effect of AN-PEP enzyme on gastrointestinal breakdown of immunogenic gluten epitopes in healthy subjects

Celiac disease is an autoimmune disorder of the small intestine that occurs in
genetically predisposed people. Celiac disease is caused by an immune reaction
to gluten protein found in wheat, barley and rye. The immune system
cross-reacts with the small-bowel tissue, causing an inflammatory reaction,
particularly in the distal duodenum. That leads to a truncating of the villi
lining the small intestine and mal-absorption of nutrients. The only available
treatment is a lifelong gluten-free diet.
The gluten epitopes that are responsible for the immune reaction, are rich in
proline. The DSM enzyme AN-PEP (Apergillus Niger Prolyl EndoProtease)
specifically cleaves gluten epitopes by cleaving behind these proline-residues.
In vitro, AN-PEP was shown to effectively degrade gluten epitopes into
non-immunogenic fragments under gastrointestinal conditions. It remains to be
demonstrated in vivo to what extent and how fast AN-PEP can degrade gluten
epitopes in the stomach and, how much remaining epitopes enter the small
intestine. A second question is whether the caloric density of a meal can
influence the efficacy of AN-PEP to degrade gluten by delaying gastric emptying
and hence prolonging meal residence time.

Primary:
• Assess effect of AN-PEP on gluten epitope degradation in the duodenum
Secondary:
• Assess effect of AN-PEP on gluten epitope degradation in the stomach
• Assess effect of meal caloric content on the efficacy of AN-PEP on gluten
epitope degradation in the stomach and duodenum

A randomized, double-blind, placebo-controlled cross-over study in healthy
subjects (n=12 completers).

All subjects will come to the study site for 4 test days with one week in
between. At test days, a gastro-duodenal catheter will be placed in the stomach
and duodenum under x-ray control. Subsequently, subjects randomly receive, in a
cross-over fashion, one of the 4 following meals:
1. A low caloric gluten meal together with AN-PEP drink
2. A high caloric gluten meal together with AN-PEP drink
3. A low caloric gluten meal together with placebo drink
4. A high caloric gluten meal together with placebo drink
Meals (300 mL) contain fat, carbohydrate and 5.2 g of gluten powder (4 g gluten
protein).
Paracetamol is added to the meals to measure gastric emptying rate. The
dilution maker polyethylene glycol-3350 (PEG3350) will be continuously perfused
into the duodenum using the feeding catheter. PEG-3350 will be analyzed in all
intraduodenal samples, to determine the dilution of the samples with endogenous
gastrointestinal secretions.
During the 60 minutes prior to administration of the meal and during the 240
minutes thereafter fluid samples from the stomach and small intestine will be
taken through the feeding tube. Blood samples will also be collected.

None. There is a theoretical risk of creating a perforation during insertion of
a feeding tube. There is no literature on the magnitude of this risk. The risk
of placing a feeding tube in healthy volunteers is considered as nil.
Participants will experience mild discomfort during the insertion of the tube.
The placement is done under X-ray screening. The total radiation exposure is
minimal, estimated to be 0.20 mSv. This is equal to the amount of radiation
that the body is exposed to during a flight of 3 hours at 4 km altitude
(www.nrg-nl.com).
During blood sampling, a total amount of 44 ml blood will be taken per test
day. In total, 176 ml blood will be obtained from each subject during this
study. No side effects are to be expected, except from the possible occurrence
of a bruise on the site of blood sampling. This will disappear within a few
days.