(Patho)Physiological aspects of the bile salt-FXR-FGF19-axis: potential consequences in Crohn's disease.

After a meal, gallbladder contraction evacuates bile salts into the intestine,
with subsequent bile salt transport to the ileum and by active transport,
reabsorption into the enterohepatic circulation. In the ileal enterocyte,
reabsorbed bile salts activate the bile salt nuclear receptor FXR (Farnesoid X
Receptor) with the result that: 1. toxic intracellular bile salt concentrations
in the ileal enterocyte and in the liver cell are prevented by regulation of
expression of various FXR target genes involved in intracellular bile salt
transport and bile salt neosynthesis 2. *ileal brake* is activated through
enhanced expression of the FXR target gene fibroblast growth factor (FGF) 19,
which functions, after its secretion by the ileal cell, as a hormone inducing
transition of post-prandial into fasting state, including gallbladder
dilatation 3. adequate intestinal barrier function and antibacterial defense
(both known to be disturbed in inflammatory bowel disease) are maintained,
through regulation of expression of various pivotal FXR target genes. In vitro
studies suggest that an anti-inflammatory effect is generated through NF*B
inhibition. In patients with Crohn*s disease, absorption of bile salts in the
ileum into the enterohepatic circulation is thought to be impaired, either
through active ileal inflammation or through faster passage of intestinal
contents through small and large intestinal tract. We hypothesize that in
patients with Crohn's disease this, or constitutively decreased ileal FXR
expression (for example due to polymorphisms in the FXR gene), may lead to
impaired activation of intestinal FXR and FXR target genes involved in
antibacterial defense.

To study potential abnormal functioning of the bile salt-FXR-FGF19 axis in
patients with clinically quiescent Crohn*s colitis.

Patients with Crohn*s colitis with an indication for surveillance colonoscopy
and disease controls who need to undergo colonoscopy to exclude significant
disease who meet the in- and exclusion criteria will be asked for their
interest to participate in this study by their treating physician. If patients
are interested, patients will receive oral and written information about the
study from the investigator and within 1-2 weeks they will be contacted by the
investigator who will answer remaining questions and who will check the disease
activity. If patients consent to participate in the study, they will be asked
to visit the endoscopy department 7 days before the scheduled colonoscopy.
During this visit, informed consent will be signed by both the patient and the
investigator. When the signed informed consent is obtained, disease activity
index scoring, fasting gallbladder volume determination by ultrasound and
collection of 3 ml blood from a peripheral venous cannula for determination of
plasma FGF19 levels will be performed. Thereafter, the FXR ligand
chenodeoxycholic acid (CDCA) will be ingested (15 mg/kg), followed by
ultrasonographic determination of gallbladder volume and collection of 3 mL
blood every hour during 6 hours. The next six days CDCA (15 mg/kg) is ingested
at bedtime. Stools will be collected during 24 hours at the day before the
colonoscopy. On the day of the clinically indicated colonoscopy patients are
fasted because of colonoscopy, except that in the early morning CDCA (15 mg/kg)
is ingested. Upon arrival at the outpatient clinic fasting gallbladder volume
is assessed by ultrasound and 13 ml of blood is withdrawn ( 3 ml for FGF19
analysis and 10 ml for SNP analysis). Thereafter, patients will receive a bowel
preparation during four hours. During colonoscopy 6 biopies in the ileum and
cecum are taken. These will be immediately placed in liquid nitrogen and stored
at -80°C for determination at a later stage of mRNA expression levels of FXR,
Angiogenin 1, FGF19, iNOS, CAR12 and other FXR target genes of potential
relevance at a later stage. The schematic outline of the study is depicted in
figures 1 and 2.

Chenodeoxycholic acid (15 mg/kg) will be administered daily for a period of
eight days. The risk associated with chenodeoxycholic acid is considered to be
very low. There is extensive experience with chenodeoxycholic acid from
dissolution therapy of cholesterol gallstones during decades and with the
exception of sligthly increased stool frequency, and minor increases of liver
biochemistry, no relevant side effects have been noted.

- Patients will be contacted by telephone once during this study. This contact
will last a maximum of 10 minutes.
- Patients will bring an additional visit to the UMC Utrecht. During this 7
hours lasting visit, 3 ml of blood will be withdrawn from an indwelling
catheter seven times and gallbladder volume will be determined by
ultrasonography seven times.
- Patients will collect their stools during 24 hours the day prior to the
colonoscopy.
- In this study chenodeoxycholic acid (CDCA) is used. No relevant side effects
have been noted during decades of experience with CDCA, except slightly softer
stools and, exceptionally, mild increases of serum transaminases. These effects
have been shown to be reversible after cessation of CDCA ingestion.
- 3 ml of blood will be withdrawn at eight moments (two punctures; 7 times
during the first visit and one time at the day of colonoscopy) for FGF19
analysis. In addition to the 3 ml blood for FGF19 analysis, 10 ml will be
withdrawn for SNP analysis at the day of the colonoscopy. Thus, a total of 34
ml blood will be withdrawn using only two punctures. After venepuncture a
hematoma may develop.
- Gallbladder volumes will be assessed eight times (7 times during the first
visit and one time at the day of the colonoscopy) by ultrasonography.
Ultrasonographic examinations are non-invasive, safe procedures that will cause
little harm to the patient. If patients do not consent to these hourly
measuremnents, only two ultrasonographic examinationswill be will be performed.
- The colonoscopy is performed for clinical indications. These endoscopical
examinations are routinely performed and considered as safe procedures. Taking
additional biopsies of ileum and cecum for mRNA expression analysis will extend
the duration of the colonoscopy with a few minutes.