Treatment Effect of Colesevelam for Bile Acid Diarrhoea - a Randomised Placebo-controlled Trial
Phase: PHASE4
Conditions: Bile Acid Malabsorption, Chronic Diarrhea
Sponsor: Lars Kristian Munck

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SINBAD 
Treatment effect of colesevelam for bile acid diarrhoea  
– a randomised placebo-controlled trial. 
 
Danish Region Zealand scientific ethics committee: SJ-641  
EudraCT number: 2016-001452-22 
 
 
 
 
Primary research group 
 
Sponsor 
Lars Kristian Munck, MD, DMSci 
Dept. of Internal Medicine 
Zealand University Hospital, Køge 
4600 Køge, Denmark 
 
Coordinating Investigator 
Christian Borup, MD 
Dept. of Internal Medicine 
Zealand University Hospital, Køge 
4600 Køge, Denmark 
 
Protocol co-authors and supervisors 
Jüri Johannes Rumessen, MD, DMSci 
Dept. of Internal Medicine 
Zealand University Hospital, Køge 
4600 Køge, Denmark 
 
Signe Wildt, MD, PhD 
Dept. of Internal Medicine 
Zealand University Hospital, Køge 
4600 Køge, Denmark 
 
 
Pierre Nordine Bouchelouche, MD, 
Dept. of Clinical Biochemistry 
Zealand University Hospital, Køge 
4600 Køge, Denmark 
 
Jesper Graff, MD, DMSci, PhD 
Dept. of Clin. Phys. and Nuclear Medicine  
Hvidovre University Hospital,  
2650 Hvidovre, Denmark 

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TABLE of CONTENTS 
Primary research group........................................................................................................................ 1 
Study locations ..................................................................................................................................... 4 
Monitoring ........................................................................................................................................... 5 
Abbreviations ....................................................................................................................................... 5 
Aims ...................................................................................................................................................... 6 
Summary of expected improvements from the study ........................................................................ 6 
Background .......................................................................................................................................... 6 
Bile acid diarrhoea ....................................................................................................................... 6 
Present diagnosis of bile acid diarrhoea ...................................................................................... 7 
Biochemical markers of bile acid diarrhoea ................................................................................ 7 
Biochemical tests in our population – choice of C4 cutoff .......................................................... 8 
Plasma bile acid species ............................................................................................................... 9 
Faecal bile acids in diagnosis of bile acid diarrhoea .................................................................... 9 
Treatment of Bile Acid Diarrhoea ................................................................................................ 9 
Design ................................................................................................................................................. 10 
Study subjects ............................................................................................................................ 10 
Inclusion criteria ......................................................................................................................... 10 
Exclusion criteria ........................................................................................................................ 10 
Withdrawal criteria ............................................................................................................................ 11 
Endpoints ........................................................................................................................................... 11 
Primary endpoint ....................................................................................................................... 11 
Secondary endpoints ................................................................................................................. 12 
Further statistical analysis plan for ancillary endpoints .................................................................... 13 
Diagnostic validation endpoints ................................................................................................. 13 
Elucidation of further aspects of treatment effect .................................................................... 14 
Endpoints for Patient Reported Outcomes ............................................................................... 15 
Power calculation ............................................................................................................................... 15 
Methods ............................................................................................................................................. 16 
Bristol stool form scale .............................................................................................................. 16 
Response criteria in chronic watery diarrhoea – Hjortswangs criteria ..................................... 16 
Patient-reported outcomes ....................................................................................................... 16 
Blinding............................................................................................................................................... 17 
Emergency unblinding ............................................................................................................... 18 

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Randomisation ................................................................................................................................... 18 
Biochemistry ...................................................................................................................................... 18 
Biobank .............................................................................................................................................. 19 
Study medication ............................................................................................................................... 19 
Colesevelam ............................................................................................................................... 19 
Placebo ....................................................................................................................................... 20 
Study plan........................................................................................................................................... 21 
Pre-screening – Written invitation............................................................................................. 21 
Study visit 1 – Inclusion and start of baseline registration ........................................................ 21 
Study visit 2 – Assessment of eligibility by stool diary results ................................................... 23 
Telephone consultation: Start of intervention .......................................................................... 24 
Telephone consultation: Start of endpoint period – compliance and AEs ................................ 25 
Study visit 3 – Treatment end and repeated questionnaires (Q-2) ........................................... 25 
End of the clinical study phase................................................................................................... 25 
Six-month follow-up ................................................................................................................... 26 
Adverse events ................................................................................................................................... 26 
Schedule ............................................................................................................................................. 26 
Rights .................................................................................................................................................. 26 
Planned publications .......................................................................................................................... 27 
Ethics .................................................................................................................................................. 27 
Recruiting ........................................................................................................................................... 27 
Information and consent ........................................................................................................... 28 
Results ........................................................................................................................................ 28 
Risks and nuisances .................................................................................................................... 28 
Data security and access .................................................................................................................... 28 
Data from the participants’ medical chart ................................................................................. 29 
Primary data responsibility ........................................................................................................ 29 
Investigators with data access for analysis ................................................................................ 29 
Economy ............................................................................................................................................. 29 
Compensation and insurance in case of injury caused by the study ......................................... 29 
References.......................................................................................................................................... 30 
Appendix 1: Study overview chart ..................................................................................................... 34 

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Study locations 
Zealand University Hospital 
Principal Investigator: Christian Borup, MD 
Department of Internal Medicine, Section of Gastroenterology 
Lykkebækvej 1, DK-4600 Køge 
 
Investigator Bente Sonne, MD, DMSci 
Department of Clinical Physiology and Nuclear Medicine 
Smedelundsgade 60, DK-4300 Holbæk 
 
Hvidovre University Hospital: 
 
Gastro-Unit, Medical Division 
DK-2650 Hvidovre, Kettegårds Allé 30, DK-2650 Hvidovre 
Principal Investigator: Christian Borup, MD  
Investigators: Camilla Nøjgaard, MD, PhD, and Hans B. Timm, MD. 
 
Investigator Jesper Graff, MD, DMSci, PhD 
Department of Clinical Physiology and Nuclear Medicine 
DK-2650 Hvidovre, Kettegårds Allé 30, DK-2650 Hvidovre 
 
Aarhus University Hospital: 
 
Principal Investigator: Søren Peter German Jørgensen, MD, PhD 
Hepato-gastroenterological Department V  
Skejby Hospital, Palle Juul-Jensens Blvd., 8200 Aarhus N 
 
Investigator Tine Nygaard Gregersen, MD, PhD 
Department of Nuclear Medicine and PET 
Skejby Hospital, Palle Juul-Jensens Blvd., 8200 Aarhus N 
 
Aalborg University Hospital: 
 
Principal Investigator: Lars Vinter-Jensen, MD, DMSci          Investigator Anna Zaremba, MD, PhD  
Department of Medical Gastroenterology 
Aalborg University Hospital, Mølleparkvej 4, DK-9000 Aalborg 
 
Investigator Trine Borup Andersen, MD, PhD 
Department of Nuclear Medicine 
Aalborg University Hospital Syd, Hobrovej 18-22, DK-9000 Aalborg 
 
 

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Cooperating Departments 
Investigator: Dominique Rainteau, Ass. Professor, PhD 
Dep. de Chimie, Sorbonne Universités  
UPMC Univ Paris 06, Paris, France INSERM-ERL 1157, CHU Saint-Antoine 27, Paris, France 
 
Investigator: Morten Dahl, MD, DMSci, PhD 
Dept. of Clinical Biochemistry, Zealand University Hospital Køge 
Lykkebækvej 1, DK-4600 Køge 
 
The Hospital Pharmacy of the Capital Region 
Marielundvej 25, 2730, DK-Herlev, Denmark 
 
Svend Høime Hansen, PhD 
Department of Clinical Biochemistry, 3-01-1, Rigshospitalet, Copenhagen University Hospital 
Blegdamsvej 9, DK 2100 Copenhagen Ø. 
 
All investigators listed above have had opportunity to add to the scientific content of this protocol. 
Monitoring 
The GCP-Unit at Copenhagen University Hospitals 
Bispebjerg Hospital, Bygning 51, 3.sal  
Bispebjerg Bakke 23  
2400 København NV, Danmark, Tlf.: +45 3863 5620 
E-mail: gcp-enheden.bispebjerg-frederiksberg-hospitaler@regionh.dk 
 
The GCP-Unit at Aalborg and Aarhus University Hospitals 
Aarhus Universitet 
Olof Palmes Allé 15 
8200 Aarhus N, Tlf: +45 7841 3950, E-mail: anjor@clin.au.dk  
 
Abbreviations 
BAD: Bile Acid Diarrhoea, BSF: Bristol Stool Form scale, C4: 7α-hydroxy-4-cholesten-3-one, CDCA: 
chenodeoxycholic acid, DMA: Danish Medicines Agency, ELISA: enzyme-linked immunosorbent 
assay, FGF19: fibroblast growth factor 19, GSRS: Gastrointestinal Symptoms Rating Scale, HPLC: 
high performance liquid chromatography, HRQoL: Heath Related Quality of Life, IBS-D: Diarrhoea-
predominant irritable bowel syndrome, LC-MS/MS: liquid chromatography-tandem mass 
spectrometry, ROC: Receiver Operating Characteristics, SeHCAT: 75Selenium conjugated Tauro-
homocholic acid retention test, SF36v2: Short Form 26 version 2, SHS: Short health Scale. 

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Aims 
1. To determine the efficacy and safety of colesevelam for treating bile acid diarrhoea (BAD). 
2. To correlate both the current scintigraphic 75-Selenium conjugated Tauro-homocholic acid 
retention test (SeHCAT) and the biochemical marker of BAD 7α-hydroxy-4-cholesten-3-one 
(C4) with colesevelam treatment response, and to validate the cut-off values determined in 
the VABAD trial (1). 
 
Summary of expected improvements from the study 
The present diagnosis of BAD rests on the radionucleotide based SeHCAT test that has limited 
accessibility. Patients with BAD therefore often endure an extensive diagnostic workup including 
endoscopies before referral for SeHCAT. Consequently, the patients are often misdiagnosed and 
suffer impaired quality of life although treatment for BAD exists. A biochemical test will allow 
timely screening of patients with chronic diarrhoea in analogy with tests for celiac disease, 
increase awareness of BAD, reduce diagnostic delay, and the exposure to unnecessary invasive 
examinations. Demonstrating the treatment effect of colesevelam in a randomised placebo-
controlled trial and correlating each biochemical test result with the subsequent treatment effect 
will improve both the diagnostic strategy and the treatment of BAD considerably. This crucial link 
between test and expected treatment effect is currently missing and providing this will help 
doctors treat and advise their patients better. 
 
Background 
Bile acid diarrhoea 
Bile acid diarrhoea (BAD) is a common cause of chronic watery diarrhoea affecting an estimated 
1% of the general population (2). BAD is detected in 20–30% of patients with diarrhoea 
predominant irritable bowel syndrome (IBS-D) (3), up to 40% of patients with microscopic colitis 
(4), and in patients with Crohn’s disease without inflammatory activity (3, 5-9). 
Bile acids are synthesised in the liver, excreted in the bile and facilitate the lipid absorption in the 
small intestine as micelles. Bile acids that are not thus absorbed are normally reabsorbed in the 
terminal ileum by the ileal bile acid transporter and returned to the liver via the portal vein. The 
bile acid pool recirculates approximately 20 times per day and 95–97% is reabsorbed at each 
passage of the small intestine (5, 7). Non-absorbed bile acids enter the large bowel and give active 
secretion when present in higher concentrations causing watery diarrhoea. 
Bile acid diarrhoea is historically classified as: 
Type 1 
secondary to disease in the terminal ileum and resection of the terminal ileum. 

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Type 2 
idiopathic or primary BAD 
Type 3 
secondary to other diseases such as microscopic colitis and cholecystectomy.  
New insight into the regulation of bile acid synthesis and its enterohepatic circulation has 
demonstrated that patients with primary BAD have normal reuptake of bile acids, and 
overproduction of new bile acids that surpass the ileal re-uptake capacity (5, 7, 8, 10). This has 
provided new possibilities for both diagnosis and treatment of BAD. 
Present diagnosis of bile acid diarrhoea 
The present diagnostic test for BAD is the SeHCAT retention test that originally was introduced by 
Thaysen (11). The 75Selenium decays by γ-emission with a half-life of 120 days and when bound to 
Taurine-conjugated homocholic acid (HCAT) it recirculates in the enterohepatic circulation. The 
ratio between γ-emission measured seven days apart is the test result and reflects the loss to the 
large intestine. Retention values representing severe (SeHCAT retention 0–5%), moderate (5–10%) 
and mild BAD (10–15%) have never been validated in placebo-controlled trials (12), but usually 
moderate and severely reduced retention is associated with diarrhoea and with treatment effect 
(12). The SeHCAT test is not available in the US and many other countries and 75Selenium is 
manufactured in one facility only. Thus, diagnosis of BAD is often delayed and misdiagnosis is 
common as reflected by the high proportion of BAD among IBS-D patients (3). In lack of better 
options, clinicians may rely on the subjective treatment effect of a bile acid sequestrant reported 
by the patient. This approach has not been validated (13) and has several diagnostic pitfalls 
making the interpretation difficult (13, 14). Bile acids can be measured in the stools but it is 
cumbersome and used only for research purposes (15). 
 
Biochemical markers of bile acid diarrhoea 
New knowledge has emerged on the physiology and regulation of enterohepatic bile acid 
circulation (16) and of bile acid synthesis (16, 17). 7α-hydroxy-4-cholesten-3-one (C4) is the key 
intermediary molecule in bile acid synthesis and a marker for the de novo synthesis of bile acids. 
High levels of C4 in serum are associated with BAD (18-21) and compared with SeHCAT, C4 has a 
sensitivity of 90% and specificity of 79% for detecting BAD (19, 21). However, C4 requires analysis 
by high-performance liquid chromatography (HPLC) with tandem mass spectrometry and has thus 
primarily found clinical use at centres with a special interest in BAD and no access to SeHCAT (15). 
Upon evaluation of their clinical use of C4, Brydon et al chose a 30 ng/mL cut-off. This had 
correlation to observational treatment response, but unfortunately not to the SeHCAT test (22). 
Several factors may influence C4 of which diurnal variation and food intake are best described 
(23). Plasma concentration of C4 increases acutely 4–6 hours after intake of alcohol (24). C4 is 
decreased in cholestasis and may be marginally decreased in hepatic cirrhosis (25). Statins and 
fibrates decrease the amount of cholesterols available for bile acid synthesis. Thus, atorvastatin 
lowers a medically or surgically induced elevated C4 (26). 

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The hormone FGF19 is released from enterocytes in the terminal ileum into the portal circulation 
in response to bile acid absorption through stimulation of the farnesoid X receptor (17). FGF19 
inhibits hepatic bile acid synthesis and relaxes the gallbladder. It also has an insulin-like effect 
increasing liver gluconeogenesis, decreasing gluconeogenesis, but unlike insulin decreases 
lipogenesis and increases protein synthesis in the liver (27) – all physiological anti-diabetic effects.  
Walters et al. showed that the pathogenesis of primary BAD is an impaired negative hepatic 
feedback by FGF19 leading to overproduction of bile acids (28, 29). FGF19 correlates inversely with 
C4 in healthy volunteers (18)  and fasting values of FGF19 correlate with SeHCAT (10). 
Unfortunately, fasting FGF19 alone varies considerably both within and between individuals. A 
single low fasting value of FGF19 <145pg/mL has 58% sensitivity and 84% specificity for moderate 
BAD (SeHCAT ≤10%)  (10), which in a clinical context is insufficient. 
 
Biochemical tests in our population – choice of C4 cutoff  
 
We studied FGF19 after stimulation with a mixed meal (30) and then 
with combinations of the meal plus chenodeoxycholic acid (31) and 
pilot results were promising. Based on these preliminary results, we 
did a prospective validation study (VABAD) of 71 consecutive 
diarrhoea patients referred for SeHCAT. We found that neither 
stimulated nor fasting FGF19 were better than C4. The fasting FGF19 
receiver operating characteristics (ROC) analysis versus SeHCAT ≤ 
10% showed an area under the curve (AUC) of 0.73 and for C4 it was 
0.83. Thus, C4 is the better biochemical test. Of the 71 VABAD 
patients, 59 fulfilled the Hjortswang criteria for diarrhoea (described 
below) that is a prerequisite in SINBAD. Table 1 shows ROC results for this subpopulation. The C4 
cut-off value of 46 ng/mL had optimal combined diagnostic characteristics with a diagnostic 
accuracy of 74%. 13 (22%) of the 59 patients had C4 > 46 ng/mL. The cut-off > 58 had higher PPV, 
and 11 of 59 (19%) had C4 > 58ng/mL. We chose the 46 ng/mL cutoff for the primary endpoint to 
keep false negative test results at a minimum.  
TABLE 1 
Results from the 
VABAD study 
SeHCAT<=10% 
N=25 
Median (IQR) 
SeHCAT>10% 
N=34 
Median (IQR) 
ROC analysis, AUC 
(95% CI) 
Cutoff for 
positive test 
Sens./spec. 
(%) 
PPV/NPV 
(%) 
Fasting C4 
47 (21–71)** 
11 (9–23) 
0.82 (0.71–0.93)** 
C4 > 25 
72 /82 
64 / 80 
 
C4 >  46  
52 / 91 
72 / 74 
C4 > 58 
44 / 94 
77 / 70 
Fasting FGF19 
75 (57–146)* 
115 (8 –220) 
0.69 (0.54–0.83)* 
FGF19 ≤ 85 
60 / 77 
65 / 72 
 
 
 
 
 
 
 
FGF19 in pg/mL and C4 in ng/mL. The statistical significance of the ROC curves was tested nonparametric versus true area=0.50. 
*p=.02; **p=.00003 

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Plasma bile acid species 
In the VABAD study, patients with bile acid diarrhoea had fewer secondary bile acids in plasma, 
less lithocolic acid and less sulfate-conjugated bile acid species (1). The diagnostic utility of these 
possible biomarkers is yet unclear. However, the VABAD study was not powered to make 
conclusions in this subject. Therefore, we will assess this in further exploratory analyses. 
Faecal bile acids in diagnosis of bile acid diarrhoea 
Measurements of faecal bile acids are used for diagnosis of BAD in the USA (15). Excretion of total 
bile acids in 48-hours on a set diet has primarily been used. However, this procedure is 
cumbersome. Increased faecal primary bile acids > 5% and > 10% possible are alternatives (32, 33). 
Further, it is expected that patients with bile acid diarrhoea in adjunct to increased primary bile 
acids in faeces will have less secondary bile acid species. Recently, a combination of primary bile 
acids >10% in a random single stool sample combined with elevated C4 was shown to be a good 
diagnostic test (34). Further, primary bile acids correlate well with SeHCAT; levels > 15% had 83% 
PPV for SeHCAT <10% (35). It is plausible that a combination of C4 with elevated primary fecal bile 
acids could correlate better to the SeHCAT scintigraphy and to treatment response than C4 alone. 
However, there only are few reports, and therefore we will assess this in exploratory analyses.  
Treatment of Bile Acid Diarrhoea 
Based on observational data, sequestrants such as cholestyramine, colesevelam, and colestipol 
seem effective treatments of BAD (12). They bind to anions like bile acids and these are then 
excreted with the faeces lowering cholesterol and alleviating symptoms of bile acid diarrhoea. All 
are licensed for treating hypercholesterolaemia, but cholestyramine has the additional indication 
for BAD. Cholestyramine is a well-established treatment for BAD, but there are no placebo-
controlled trials with cholestyramine for BAD since formulating a placebo for cholestyramine has 
been impossible. One recent study compared cholestyramine with hydroxypropyl cellulose as an 
intended placebo in 26 participants with functional diarrhoea (36). Unfortunately, hydroxypropyl 
cellulose had an active effect with a per protocol (PP) response rate of 38% compared with 64% 
for cholestyramine (p=0.22). A 23% dropout rate for cholestyramine reflects the side effects of 
cholestyramine and the intention to treat (ITT) cholestyramine response rate dropped to 58%.  
Colesevelam is used off-label for treating BAD often as second-line therapy due to its higher price, 
however many BAD patients who do not tolerate cholestyramine benefit from colesevelam, and 
because of this placebo-controlled studies have been warranted (13, 37). A placebo-controlled 
trial of colesevelam for suspected BAD in patients with Crohn's disease and diarrhoea despite 
inflammatory remission (i.e. suspected BAD), found diarrhoea ITT remission rates of 67% (10/15) 
for colesevelam and 27% (3/11) for placebo (p=0.057). Unfortunately, the extreme inclusion and 
exclusion criteria caused slow recruitment and the study was terminated prematurely (38). 
A recent open, non-controlled trial with obeticholic acid, a potent FXR agonist that stimulates 
FGF19 synthesis, demonstrated that obeticholic acid reduced bile acid synthesis and had a clinical 
effect in patients with primary BAD and some cases of type 2 BAD (39). This demonstrates the 
effect of a new pharmacological class of treatment for BAD. 

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Design 
Randomised, placebo-controlled, parallel groups, double-blinded multicenter, phase IV trial. 
Null hypothesis:  
Colesevelam and placebo have the same effect on BAD.  
Intervention:  
Colesevelam (tablets of 625mg) or identical placebo capsules. One to three capsules taken twice 
daily. Start dose is two capsules twice daily. The dose is titrated by a study nurse who is 
independent of the investigators. 
Parallel arms of 12 days blinded treatment, of which the first five days is run-in and the last seven 
days measure endpoints. 
Study subjects 
Inclusion criteria  
 Patients referred to Clinical Physiological/Nuclear Medicine departments for SeHCAT at 
Holbæk, Hvidovre, Aarhus, and Aalborg University Hospitals 
 Suspected BAD 
 ≥ 18 and < 80 years of age. 
 Women of fertile age must use safe contraception during the treatment part of the study  
o spiral or hormonal contraception, ie. birth control pill, hormonal implant, 
transdermal patch, vaginal ring, or contraceptive depot injection. 
o Sexual abstinence from heterosexual intercourse. May be accepted as safe 
contraception by the investigator in a case by case assessment a 
o Vasectomised partner 
 Ability to give informed consent after written and oral information in the Danish language 
Note a) sexual abstinence from heterosexual intercourse is deemed a highly effective method of 
anticonception by the Clinical Trials Facilitation Group. It is accepted in this trial only because the 
duration is short – three weeks.  
 
 
Exclusion criteria  
 
 Inflammatory bowel disease, including microscopic colitis  
 Acute suspected or proven viral gastroenteritis within the recent 4 weeks 
 Acute non-viral gastroenteritis within the recent 8 weeks 
 Investigator-assessed debilitating chronic disease e.g. WHO performance score 3–5 

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 Prior treatment with colesevelam 
 Treatment with laxatives or anti-diarrhoeal drugs during the study 
o Except for stable dose the last four weeks of psyllium husk and opioids for pain 
 Pregnancy  
 Breastfeeding women 
 Crucial medication that cannot be separated appropriately from colesevelam  
o i.e. taken one hour before or 4 hours after colesevelam 
 Oral anticoagulation, both warfarin, and new oral anticoagulation  
 Treatment with cyclosporine within two months 
 Bowel obstruction (subileus or ileus) 
 Biliary obstruction 
 Short bowel syndrome 
 Bowel ostomy 
 Allergy to colesevelam or its constituents  
 Allergy to placebo constituents (excluding lactose)  
 Investigator-assessed high risk of non-compliance 
 If on statin/fibrate medication, unwilling to pause medication between study visits 1 and 2 
Withdrawal criteria 
Upon inclusion (i.e Visit 1) we screen for:  
  1) Biliary obstruction – plasma total bilirubin must be < 2 x upper normal limit 
  2) Pregnancy – plasma/urine HCG must be negative in all women of childbearing potential 
The screening result must be available before randomisation (i.e. at Visit 2). 
Endpoints 
Primary endpoint  
Placebo-controlled ITT diarrhoea remission rate defined by the Hjortswang criteria for colesevelam 
in patients with BAD defined by C4 > 46 ng/mL. 
 
Handling of missing data: drop-outs are set as treatment failures in the ITT analysis if less than five 
of the last seven treatment days that comprise the endpoint assessment period are complete. If 
five or more of these days are complete, a mean of these days will be used for calculating the 
Hjortswang response criterium.  
 
Statistical analysis: data will be longitudinally correlated (baseline, run-in, endpoint assessment 
period for each patient) and nested within study sites. Thus, data will not be independent and 
cannot correctly be assessed with simple statistical tests. Therefore, we will fit an appropriate 
generalized linear mixed-effects model with an unstructured covariance pattern of the chance of 
response with colesevelam versus placebo. In addition, baseline adjustments for the severity of 

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diarrhoea (mean per day sum of Bristol type 6 and 7 stools) and the severity of bile acid 
malabsorption (visit 2 C4 value) will be made to avoid skewed randomisation of these prognostic 
covariates by chance (40). This baseline adjustment is clinically relevant as, in the future, diarrhoea 
severity and C4 should be available for physicians before starting therapy.  
The model fit will be assessed by inspection of scaled residual plots and QQ-plots. 
 
Sensitivity analyses: The robustness of the statistical modelling of the primary endpoint will be 
addressed in sensitivity analyses of different approaches.  
Different handling of missing values: 1) any missing diary day of the last seven treatment days and 
the patient treatment is set to failure; 2) excluding patients who drop out due to large pill size (the 
DB caps used for trial blinding are significantly larger than the colesevelam tablet), 3) using 
multiple imputations for missing values.  
Strict Hjortswang’s response criteria: no watery stools (Bristol stool type 6 or 7) during the last 
seven treatment days.  
Different C4 cut-off used to define bile acid diarrhoea:  C4 > 25 ng/mL; C4 > 58 ng/mL.  
If possible, given that the study is not powered for stratification, we will assess remission rates in 
stratification intervals defined by C4 0–=25 ng/mL, >25–46 ng/mL.  
Different statistical modelling and assessment: 1) added adjustment for patient sex and age, 2) 
model without baseline adjustment, 3) model without random effect of study site, 4) a simple 
Fisher’s exact test of a 2x2 table (response versus treatment allocation) reporting remission rates. 
Secondary endpoints  
1. PP analysis for the primary endpoint 
Statistical analysis: this is an efficacy (de jure) estimand for colesevelam. Modelling as for the 
primary endpoint but with imputation of missing data as if all patients had adhered (41, 42). This 
includes all patients with bile acid diarrhoea and complies with a per protocol principle. 
 
2. Placebo-controlled diarrhoea ITT remission rate for colesevelam defined by the Hjortswang 
criteria in patients with BAD defined by SeHCAT ≤ 10% 
Statistical analysis: as the primary endpoint but with baseline adjustment for SeHCAT, not C4. 
Sensitivity analyses: as for the primary endpoint. Instead of differing C4 cutoffs, the SeHCAT 
cutoffs ≤ 5%, and ≤ 15% will be assessed. 
 
3. Placebo-controlled diarrhoea PP remission rate for colesevelam defined by the Hjortswang 
criteria in patients with BAD defined by SeHCAT ≤ 10% 
Statistical analysis: this is an efficacy (de jure) estimand for colesevelam. Modelling as for the 
primary endpoint with baseline adjustment for SeHCAT and with imputation of missing data as if 
all patients had adhered. 
 

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4. Placebo-controlled effect of colesevelam in all treated patients  
Statistical analysis: as for the primary endpoint but instead of the binary Hjortswang response 
criteria as outcome, continuous variables will be modelled with suiting baseline adjustment: 
a. the absolute number of stools (mean per day over 6 or 7 days) 
b. the total number of Bristol 6 and 7 stools (mean per day over 6 or 7 days) 
 
5. The de facto effectiveness of colesevelam on health related quality of life assessed as the sum 
of Short Health Scale scores.  
Statistical analysis: modelling with adjustment for baseline SHS score 
 
The secondary endpoints are tested with correction for multiple statistical testingand significant 
results are reported as such.  
The secondary endpoints will also be tested at the 0.05 significance level and reported as 
hypothesis-generating results acknowledging the risk of type 1 error. 
 
Expected missing data and drop-outs  
We anticipate droput/missingness due to  
1) side effects to colesevelam. We presume this to be ‘missing at random’. 
Drop-out from this cause could dilute the observed effect of colesevelam. This reflects real-world 
effect and is accounted for by analysing both an effectiveness (ITT) and an efficacy (PP) estimand. 
2) lack of effect from placebo. Presumably somewhat dependent on baseline diarrhoea severity,  
which is controlled for in the modelling. 
Drop-out from this cause probably has no effect on the primary endpoint. 
3) due to the (large) pill size. We presume this will be ‘missing at random’. 
Drop-out from this risks diluting the observed effect of colesevelam. This is only specific to the trial 
context and not future clinical practise. We take this into account in a sensitivity analysis excluding 
these cases. 
 
Statistical analysis plan for ancillary endpoints 
Diagnostic validation endpoints 
 Placebo-controlled diarrhoea ITT remission rate for colesevelam defined by the Hjortswang 
criteria in patients with BAD defined by fasting FGF19 ≤ 60 pg/mL 
 ROC analyses to assess the negative predictive values of C4 < 15 ng/mL, FGF19 > 204 
pg/mL, SeHCAT >10%, respectively with diarrhoea remission defined by Hjortswang criteria 
as gold standard test. 
 Repeatability of baseline fasting C4 and FGF19 assessed with marginal limits of agreements 
(Bland-Altman plot) comparing visit 2 samples with ‘optimal’ visit 1 samples (ie. fulfilling 

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visit 2 sampling criteria: fasting, not using statins within a week, no alcohol consumption 
within 24h) 
 Exploratory assessment of the effect of sampling time, statin use, and alcohol intake on C4. 
This is a regression model of C4 at visit 2 explained by: C4 at visit 1, sampling time, alcohol 
consumption, statin use, respectively. 
 ROC-AUC for all visit 1 C4 samples versus SeHCAT ≤ 10% as gold standard 
 Placebo-controlled subjectively assessed "good response” ITT rate in the primary endpoint 
population 
 Diagnostic values for subjectively assessed "good response” in the colesevelam-arm  
o C4 > 46 ng/mL 
o SeHCAT ≤10% 
 
Descriptive statistical analyses 
 correlations between each of FGF19, C4, and SeHCAT and baseline mean stools and 
number of watery stools 
 correlations between each of  FGF19, C4, and SeHCAT and change in mean stool number 
and number of watery stools in the colesevelam population 
 
Exploratory analyses of bile acid species in plasma and feces 
We will explore the distribution of bile acid species in feces and plasma regarding bile acid 
diarrhoea both defined with C4 and SeHCAT. Explorative statistical modeling will be done to 
analyse the diagnostic and prognostic performance of bile acid parameters. Bile acid species will 
primarily be assessed as sums of primary and secondary species given as a percentage of the total 
amount of bile acids. Percentages of the total amount of bile acids have less variability than 
absolute amounts (43). Secondary analyses of single bile acid species and conjugation forms either 
on absolute values or percentages may be performed given a relevant signal in the primary 
exploratory analysis. 
Elucidation of further aspects of treatment effect 
 Placebo-controlled ITT diarrhoea remission rate defined by the Hjortswang criteria for 
colesevelam in patients with BAD by C4 ≥ 46 ng/mL AND  
o fecal primary bile acids >10%, and >15% 
 Logistic regression model predicting remission in the colesevelam group for 
o C4 controlled for baseline mean stools/day 
 Sensitivity, specificity, PPV and NPV for C4 > 46 ng/mL 
 Model characteristics reported with mean stools/day 
o SeHCAT controlled for baseline mean stools/day 
 Sensitivity, specificity, PPV and NPV for SeHCAT ≤10%  

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 Six-month follow up on the aetiology of diarrhoea  
 Six -month follow up on medication 
 Exploratory regression analyses to determine if fecal or plasma bile acid profile may be a 
predictor for diagnosis and for treatment response. 
 Placebo-controlled change in fecal bile acids during treatment with colesevelam. 
 
Endpoints for Patient Reported Outcomes 
These will be reported in a separate paper. Health-related quality of life (HRQoL) (Questionnaires) 
correlated to diarrhoea defined by the Hjortswang criteria 
 The change in Health-related quality of life (HRQoL) in the primary endpoint population by  
o the Short Form 36 version 2 (SF36v2) items (colesevelam vs. placebo) 
 Physical component score and Mental component score  
o Each of the 4 items in the SHS 
 Correlation between HRQoL and Bristol stool scale diarrhoea. 
 Correlation between HRQoL and stool number per week. 
 Correlation between changes in HRQoL and primary endpoint 
 Description of sexual dysfunction with HRQoL in the population  
 Six-month follow up on HRQoL 
 
Power calculation  
Based on the primary endpoint we assume  
- a remission rate of 67% for colesevelam and 27% for placebo (36, 38).  
- two-sided α = 0.05, and β = 0.20 (ie. 80% power); and 1:1 allocation. 
Thus, we need 23 subjects with BAD in each arm (G.Power 3.1: z-test of two independent 
proportions).   
As we include all patients with diarrhoea regardless of aetiology, the fraction or prevalence of BAD 
amongst these eligible patients is crucial.  Of 71 subjects in our VABAD study, 59 patients had 
diarrhoea by Hjortswang criteria and deemed by C4 ≥ 46 ng/mL, 16 of the 59 had BAD (27%). In 
comparison with SeHCAT, Borghede et al. found that 41% of patients referred for SeHCAT had 
primary BAD defined with SeHCAT (44).  
 
Thus to include 2 x 23 patients with BAD defined with C4 > 46 ng/mL, we need 
 
2 x 23 BAD subjects
27% BAD subjects among patients with diarrhoea  = 170 𝑝𝑎𝑡𝑖𝑒𝑛𝑡𝑠 with diarrhoea. 
 
 

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Methods 
Bristol stool form scale 
Bristol stool scale classifies stool from hard lumps (1 on the scale) to watery (7 on the scale). A 
one-week stool diary is usually collected in clinical trials (45, 46). Time and form of stools are 
noted and further information on pain, urge and incontinence can be obtained. In patients 
undergoing diagnostic workup for diarrhoea, those with an organic cause of diarrhoea more often 
have ≥3 stools per day and watery stool consistency (47)  compared with functional diarrhoea. 
 
Response criteria in chronic watery diarrhoea – Hjortswangs criteria 
Hjortswang et al. have validated that disease activity defined by stool consistency and frequency 
correlates with reduced quality of life determined by health-related quality of life (HRQoL) 
questionnaires in patients with collagenous colitis  (45, 48) and lymphocytic colitis (49).  
 Activity is defined as ≥ 3 stools per day or ≥ 1 watery stool (BSF 6-7) per day as the mean of 
seven (six) days.  
 Remission is defined as <3 stools per day and < 1 watery stool per day as the mean of 
seven (six) days.  
In conjunction with these definitions, it was recently documented that both stool frequency and 
consistency correlated with an organic cause of chronic diarrhoea, including BAD, with consistency 
being the most powerful predictor (47). As there are no validated activity criteria for BAD and 
chronic diarrhoea is the major symptom, we will apply Hjortswang’s criteria for activity and 
remission. 
Diarrhoea remission rates are calculated from comparison of the baseline Study Diary 1 (i.e. Study 
Day 1 – 7) with the endpoint part of the Study Diary 2 (i.e. Study Day 14 – 20). 
 
Patient-reported outcomes  
Short Health Scale and Short Form 36 version 2 
The Short Health Scale (SHS) is a simple index using four 0 – 100 VAS scales to estimate symptom 
burden, social function, disease anxiety, and well-being (50). The SHS correlates to the more 
elaborate HRQoL questionnaires both with the severity of symptoms and global assessment and 
has been validated in English and in Swedish in patients with both inflammatory and functional 
bowel disorders (45, 48, 50-53). 
The SHS has not been validated in BAD nor in Danish. Therefore, we will compare the result of an 
SHS questionnaire with the more elaborate SF36v2 and disease activity defined by the Hjortswang 
criteria. 

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Gastrointestinal Symptom Rating Scale 
The Gastrointestinal Symptom Rating Scale (GSRS) quantifies gastrointestinal symptoms on 15 
Likert scales grading symptoms from 1 (mild) to 7 (severe). Added questions specify symptom from 
the upper and the lower gastrointestinal tract. The GSRS questionnaire is validated in Danish and 
widely recognised (54). 
 
Health-related quality of life and sexual dysfunction 
Sexual dysfunction from chronic diarrhoea due to inflammatory bowel syndrome is common but 
often the problem goes unnoticed. The questionnaires Female Sexual Distress Scale (FSDS), 
Female Sexual Function Index (FSFI), and International Index of Erectile Function (IIEF) address the 
issue in general and the novel IBD-SEX questionnaire address sexual dysfunction from 
inflammatory bowel disease (IBD) and chronic diarrhoea (55, 56). The IBS-SEX questionnaire is 
validated in Danish language and validation in Danish patients with IBD is ongoing. 
Patient-reported “good treatment response” 
A diagnostic therapeutic trial is a common practice instead of testing for BAD. Patients with 
suspected BAD are treated with a sequestrant and assess the subjective effect. We ask the 
subjects in the intervention group if they had good response. In order to compare this with the 
dichotomous Hjortswang response criteria, answers are: 
Question: “Do you think that the treatment relieved your diarrhoea?” 
a) “No, it was not sufficiently effective”; b) “Yes, it was sufficiently effective”. 
 
In Danish 
“Synes du behandlingen fjernede din diarré?” 
a) ”Nej, den var ikke tilstrækkelig effektiv”; b) ”Ja, min diarré forsvandt under behandlingen”. 
 
Blinding  
Initial blinding: Over-encapsulation makes colesevelam and placebo tablets identical. These are 
produced, packed, and uniquely numbered at the Hospital Pharmacy of the Capital Region, from 
where packages are distributed to each study centre. 
 
Maintaining blinding: Due to the expected treatment effect and possible side effects, maintaining 
subject blinding could be difficult. This potential bias cannot be avoided.  
To avoid investigators to be unblinded, dose titration and AE registration during treatment are 
performed by an independent study nurse. 
 
We assess subject blinding early in the treatment period and again at treatment end. We assess 
investigator blinding at treatment end. Each assessment the question is: “Do you think you/the 

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subject is given a) Colesevelam, b) Don’t know, c) Placebo. With this information, we quantify both 
initial and maintained blinding to estimate the size of potential bias (57-60). 
 
The blinding continues until the last subject has completed the clinical study phase and all data 
entries have been checked and locked. At this time point, the blinding is unveiled, all subject are 
informed, and the treatment is documented in the medical chart.  
Emergency unblinding  
Sealed envelopes for emergency unblinding are enclosed with each delivery from the Pharmacy to 
a study centre for the local primary investigator with a copy to the Sponsor. The local investigator 
or Sponsor may use such an envelope to unblind the treatment at any time and without 
restrictions if it is deemed necessary. The study subjects are also instructed to keep the contact 
information for the primary investigator who is contacted in case of any emergency that could 
warrant unblinding of the study intervention. 
In case of unblinding, two dated signatures are needed on the envelope; in an emergency, these 
need not be from investigator or subinvestigator. Signing the envelope states that it will be 
opened, and beforehand is intact and untampered. 
 
Randomisation 
A randomisation list is made by the Hospital Pharmacy of the Capital Region by computer 
randomisation distributing consecutive unique numbers in variable block sizes of two, four, or six 
to one of the two treatment arms (www.randomization.com). The different block sizes hinder 
deduction of the allocation of the ultimate slots in a block and secure a 1:1 allocation. 
The Hospital Pharmacy of the Capital Region keeps the randomization lists separately and 
exclusively until study end. 
 
Biochemistry  
All samples are prepared and immediately frozen for later bulk analysis once inclusion is finished. 
FGF19 is analysed by commercially available ELISA assay (R&D Systems, MN, USA). C4 is analysed 
with liquid chromatography-tandem mass spectrometry (LC-MS/MS) in the Department de Chimie, 
Sorbonne Universités, Paris (Dominique Rainteau) (61). C4 will also be analysed in the department 
of Clinical Biochemistry, Rigshospitalet, Copenhagen (Svend H. Hansen). This founds future clinical 
availability of the C4 analysis in Denmark. Therefore, we will use the Copenhagen C4 
measurements for the endpoint analyses. Ancillary analyses comparing C4 from the two 
laboratories will be done. 
Routine blood samples are analysed in the hospital laboratories of participating centres. These 
include ALT, ALP, amylase, HDL, LDL and total cholesterol, triglycerides, bilirubin, glucose with 
sampling time specified elsewhere.  In total, these samples comprise 24 mL blood from each 

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subject. We accept differences in laboratory reference values and procedures in between study 
centres as these analyses do not comprise endpoints. 
 
Biobank  
Study Biobank: Frozen plasma samples for FGF19 and C4 are stored in a biobank for analysis once 
the last participant has finished the study. After analysis, surplus plasma is re-frozen and stored for 
a period of maximum 15 years, but only if the subject has given consent to the biobank for future 
scientific work described below. Analysis of bile acid species including C4 is done in Paris (see 
cooperating departments, page 3). Biobank for future scientific work: This is created for future, 
yet unspecified scientific work. 22mL blood is required to be stored as 4mL plasma and 10mL 
EDTA-blood. Furthermore, a stool sample is collected for this biobank. Separate informed consent 
is obtained for the biobank. Consent may be withdrawn and the samples are then destroyed. This 
biobank is stored for a period of 15 years and then destroyed. The biobank will not be given to the 
third party. Data security is monitored by the Danish Data Security Agency. 
Study medication 
Colesevelam  
Colesevelam is registered for treating hypercholesterolemia; however, it is extensively used off-
label as a sequestrant treating bile acid diarrhoea (13, 14, 37, 38). The treatment effect is 
comparable to that of colestyramine and it is considerably better tolerated (13, 38). Colesevelam 
tablets are bought from Sanofi-Aventis by the Hospital Pharmacy of the Capital Region of 
Denmark. The Pharmacy encapsulates colesevelam and placebo tablets (Capsugel® DBcaps®, size 
AAA) in accordance with Good Distribution Practise and Good Manufacturing Practise and 
documents this. 
Purpose: the active arm of the double-blinded randomised intervention.  
Dose: We strive for a standard dose of three capsules each of 625mg twice daily taken before 
breakfast, lunch, or dinner. To avoid drop-out due to dose-related adverse reactions such as 
nausea, constipation, and vomiting we use dose escalation and titration. 
Start dosage: 2 tablets twice daily for two days. If the subject has no or limited side effects, the 
dose is increased to 3 tablets twice daily. In case of severe constipation, nausea, or vomiting the 
dose may be decreased (to one capsule twice daily). Minimum dose is one capsule twice daily. 
Dosing once daily is not allowed. All subjects are contacted by telephone on day 2–3 to ensure 
dose escalation if possible. If the dose is changed the subjects is contacted again 2–3 days 
afterwards to follow up. 
Communication regarding dose and registration of AEs is kept between the subject and a study 
nurse does not partake in endpoint registration nor in reporting of the study. If the nurse needs 
advice from a doctor, this is first sought from a doctor who is not affiliated with the study; ie. not 
from an investigator. In matters regarding possible serious adverse events, the decision of 
emergency unblinding, study participation etc. the investigator must be involved. 
 

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Side effects to colesevelam: See the exert below from Summary of Product Characteristics 
(updated 28.11.2018). 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Placebo 
Matrix placebo tablets (17mm size) with same capsule (Capsugel® DBcaps®, size AAA) making 
these identical to the colesevelam capsules. 
 
The tablet matrix does not affect bowel function. Constituents are as follows per tablet: 
 Lactose monohydrate 330mg 
 Potato starch 335mg 
 Gelatine 12mg 
*Description of selected adverse events (continued from exert) 
The background incidence of flatulence and diarrhoea were higher in patients receiving placebo in the 
same controlled clinical studies. Only constipation and dyspepsia were reported by a higher percentage 
among those receiving Cholestagel (colesevelam), compared with placebo. 
 
The incidence of intestinal obstruction is likely to be increased among patients with a history of bowel 
obstruction or removal. 
Cholestagel in combination with statins and in combination with ezetimibe was well tolerated and the 
adverse reactions observed were consistent with the known safety profile of statins or ezetimibe alone. 

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 Magnesium stearate 3.5mg 
 Talc 31.5 mg 
 
The placebo capsules (Capsugel® DBcaps®, size AAA) dissolve in the stomach giving a negligible 
delay in availability of colesevelam of 2–3 minutes.  
  
No side effects are expected from the placebo tablets. 
Study plan 
Pre-screening – Written invitation 
This has two steps: 
1. Eligible participants as deemed by inclusion and exclusion criteria are either given or send 
the written invitation by the Gastroenterological department when referred for SeHCAT or 
send from the Clinical Physiological Nuclear Medicine department to referred patients. All 
pre-screened participants are anonymously registered on a pre-screening list.  
 
2. At the first SeHCAT visit a sub-investigator asks the potential subject, if the written 
invitation is received, read, and understood. Further, we ask if they consider participation 
and/or would like further information from the investigator (doctor). 
The investigator assess eligibility, informs and may include the patient as specified under “Ethics”. 
If an eligible subject opts to participate, this visit (first SeHCAT visit) is called Study visit 1.  
 
Study visit 1 – Inclusion and start of baseline registration 
Study Day 1.  
After inclusion, a blood sample for the study biobank is drawn. This sample is used to assess the 
concordance between repeated tests, and we register the given conditions for this sample to 
explore how sampling time, fasting, medication etc. affect the C4 sample. 
Case Record Form 
At inclusion the (sub)investigator creates an electronic Case Record Form for the study subject to 
document: 
 Medical history 
o Physical status 
o Medication of interest: antidiabetic, analgetic, anti-hypercholesteremia, anti-
diarrhoea, laxatives, systemic glucocorticoids, recent (3 mo.) systemic antibiotics.    
o Prior medical and surgical history 

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o Results within 12 months of any diagnostic workup regarding diarrhoea 
 Microbiological stool samples 
 Fecal-calprotectin 
 Fecal-elastase 
 Biochemical analyses for lactose intolerance, coeliac disease, thyrotoxicosis 
 Endoscopies, regarding coeliac disease and inflammatory bowel disease 
including histology 
 Breath tests for malabsorption or bacterial overgrowth 
o Use of tobacco and alcohol 
 Baseline blood analyses (ALT, ALP, bilirubin, amylase) 
 Weight (measured) and height 
The (sub)investigator needs access to the subject’s medical file and chart. This is specified in 
participant information and is given in the written informed consent. 
  
All subjects complete the baseline questionnaires (Q-base) consisting of  
1) Short Form 36 version 2 (SF36v2 two week recall) on HRQoL  
2) Gastrointestinal Symptom Rating Scale (GSRS)  
3) Short Health Scale focused on the gastrointestinal effect on HRQoL. 
 
We ask all subjects if they as a voluntary supplement would opt to answer the questionnaires 
addressing sexual dysfunction due to chronic diarrhoea. If yes; we send the questionnaire to the 
subject to answer at home before Visit 2 and again at the six-month follow-up. 
All subjects are given: 
1. the baseline diary  
2. a kit for stool sampling at home 
Optional stool sample for biobank for future scientific work 
The subject may opt to take a collection kit for stool sample at home on study day 6 or 7 to make 
75-Se radiation negligible. The sample is immediately placed in the subjects’ -18 – -23°C freezer. 
The subject brings the sample on study visit 2 for placement in a - 80°C freezer. If the subject 
continues to treatment, he/she may opt for a second sample taken on treatment day 18–20 and 
deliver it on Visit 3. 
Stool sampling is not mandatory for study participation and is only planned if it is feasible 
according to the individual circumstances. 
 
 

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Notification to the institution that referred for SeHCAT 
The (sub)investigator e-mails the institution that referred for SeHCAT with instructions to contact 
the investigator if they find participation inappropriate. This notification is only optional on the 
condition that the subject consents to the notification. 
Study visit 2 – Assessment of eligibility by stool diary results 
Study Day 8. Concurrent with the second SeHCAT visit, all subjects meet in the fasting state, the 
only exception is drinking still water. Subjects must not ingest alcohol on the day before visit 2. 
Fasting blood samples are drawn from all subjects, no later than 10:00 AM.  
The blood samples include: 4mL plasma for C4 and for FGF19 analysis (total 8mL). Routine samples 
for: p-triglycerides, p-cholesterols (total, High and Low-Density Lipoproteins), fasting p-glucose. 
Any violation of alcohol abstinence the day before study visit 2 and of pausing statin/fibrate use 
leads to exclusion as a protocol violation. Minor violations of fasting (drinking thin fluids like coffee 
or tea, also with milk added) are noted in the CRF and the patient can participate in the study. All 
other violations lead to exclusion or rescheduling for fasting blood sampling another day. 
 
The (sub)investigator collects the baseline study diary and tallies this.  
Study Diary 1 – baseline 
Subjects are screened for baseline diarrhoea as defined by Hjortswang’s criteria:  
 ≥3 stools per day or ≥ 1 watery stool per day (Bristol Stool form 6 or 7) as a mean of seven 
days (minimum six days).  
The baseline week is the week between the first and second SeHCAT retention test. 
 
The result of the baseline stool diary determines whether the subject objectively has diarrhoea as 
specified above. 
Subjects without diarrhoea:  
These subjects do not proceed to the intervention, thus, we do not register adverse events for 
these subjects. 
 
SeHCAT results are registered once available and the subjects are reminded of the six-month 
follow-up on the subject’s medical chart and the aetiology of the subject’s health complaints. 
The follow-up is done by telephone and the questionnaires are distributed electronically, or in 
special circumstances by mail. 
After the six-month follow-up, study participation ends for the subjects without diarrhoea. 
 

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Subjects with diarrhoea:  
This is the ITT population. 
Adverse Event registration starts now (study day 8) and includes all subjects with diarrhoea.  
The (sub)investigator dispatches the relevant double blinded treatment packet according to the 
randomisation sequence. 
Start of intervention and possibility for delay of intervention 
If the time/hour of the day for visit 2 enables the subject to take two doses of the medication this 
day, the intervention may start on visit 2, which subtracts one day from the time schedule below. 
For logistic reasons in the collaboration between the Department of Nuclear Medicine and the 
Department of Gastroenterology at the local study centre, the treatment start may be delayed for 
a maximum of seven days. In case the start is postponed, the postponed days are not counted as 
study days. 
Study Diary 2 – treatment period 
This diary registers the number of daily stools and consistency according to the Bristol stool form. 
The participants also note any AEs. The Study Diary 2 is commenced at Study Visit 2 and entails 
three periods 
1. Study Visit 2 
 
 
(Study day 8)  
2. Run in period of five days 
 
(Study day 9  –  13) 
3. Registration of Primary endpoint   
(Study day 14 – 20) 
These periods are not separated in the diary. The diary also forms the subject’s study participation 
card, with contact information to the relevant investigator or study nurse. 
On study day 9, subjects set an “X” in the diary according to what treatment they think they are 
receiving: a) Colesevelam, b) Don’t know, c) Placebo. This is used to assess initial blinding. 
 
Telephone consultation: Start of intervention 
Study day 10 
The intervention has started on study day 9. The study nurse calls the subject to note possible 
early AEs, aid compliance, and to titre the dose. If the nurse suspects an SAE, she contacts the 
investigator. 
Titration of intervention dose 
If the subject experiences lack of effect or the adverse symptoms constipation, vomiting, or 
nausea that could be caused by too high a dose of colesevelam the study nurse may taper or 
increase the daily dosage to three or one capsule(s) twice daily. 

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Whenever the dose is changed, a follow-up telephone consult is planned within 2–3 days.  
Telephone consultation: Start of endpoint period – compliance and AEs 
Study day 14 (± 1 day) 
The study nurse calls the subject to note possible early AEs, aid compliance, and to titrate the dose 
if indicated. This consult is mandatory regardless of follow-up on any prior dose titration. 
Study visit 3 – Treatment end and repeated questionnaires (Q-2) 
Study day 21 (+1–3 days). The treatment ends at the end of day 20.  
Before (same day or 1-2 days ahead of) the clinical contact on visit 3, the investigator answers if 
he/she has had any contact with the subject, perhaps through the study nurse: yes/no. Further if 
yes, what he/she thinks the allocated treatment was: “a) Colesevelam, b) Don’t know, c) Placebo. 
The (sub)investigator then in the order below: 
 distributes repeated questionnaires (Q-int) 
o SF36v2, SHS, GSRS 
 If the subject participates in the voluntary questionnaire on HRQoL and 
sexual dysfunction, this questionnaire is distributed again by e-mail 
o In the questionnaire, the subject is asked to mark which treatment he/she thinks to 
have received. This will assess the maintenance of blinding. 
 collects the Study Diary 2 and checks this for deficiencies 
 obtains a history of AEs and deem if any AE the nurse has noted are related to the 
intervention, ie. AR. This can only be done by a doctor 
 collects surplus medication, counts the number of return capsules and assess compliance  
 orders follow up blood analysis to assess biochemical side effects (sampled in 3 – 4 days) 
o ALT, ALP, bilirubin, amylase, 
 schedules study end telephone consultation 
 confirms agreement for the six-month follow-up 
The six-month follow-up is included in the study consent. This follow-up is identical to the one 
described above for subjects without diarrhoea. 
End of the clinical study phase 
Study day 26 (± 3 days). AE registration continues for minimum 72 hours after the end of 
treatment. Colesevelam is not absorbed from the gastrointestinal tract. Normal gastrointestinal 
transit is 36 – 48 hours leaving minimal if any traces of colesevelam at this time. If visit 3 (above) is 
scheduled after these 72 hours, the AE registration at visit 3 is final. The investigator may opt to 
only telephone the patient is an end-of-trial blood samples is abnormal. Otherwise the 
investigator telephones the subject (this also applies to subjects in open-label treatment): 
 Assess biochemical AEs 
o Informs the subject of these results 

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 Takes a history of AEs 
 Takes action if needed (blood sampling, extended AE registration etc.) 
The blinding is not unveiled at this time. 
The central study nurse notes what he/she thinks the treatment was (questions as above). 
Six-month follow-up 
The local (sub)investigator notes the result of the diagnostic work-up of diarrhoea. 
The questionnaires (SF36v2, SHS, GSRS) are distributed electronically. This questionnaire also asks 
if, what, and how any medicine against diarrhoea is taken. 
If the subject has consented to answer the questionnaires regarding sexual dysfunction these are 
also distributed via e-mail. 
Analysis of primary and secondary endpoints does not wait until the follow-up is finished. 
Adverse events 
All adverse events (AEs) and adverse reactions (ARs) are registered by an AE/AR table in the study 
Case Record Form and documented at study visits as specified above.  
Serious AEs (SAE) and serious ARs (SAR) are defined as an AE or AR that is life-threatening or 
leading to death, that leads to hospitalization or prolonging of hospitalization, that causes 
seriously or sustained disability or incapacity to work, or causes a congenital anomaly.  
All SAEs and SARs will be reported to sponsor as soon as possible and within 24 hours. A SAR that 
is not expected is deemed Suspected Unexpected SAR (SUSAR). If the event is life-threatening or 
deadly sponsor reports to the DMA and to the Ethical Committee immediately and within 7 days 
with follow up within further 8 days. 
All other SUSARs are reported to DMA within 15 days of sponsors knowing. 
A SAR is deemed SUSAR if not listed in the AE table for colesevelam.  
All SAR’s are reported to the DMA and the Ethical Committee in yearly reports and after study 
completion (sooner than 3 months from the last visit, last patient). 
Schedule  
Inclusion starts in the second half of 2018 and is estimated to complete within 18 months. Primary 
data collection ends two weeks after the last participant is included. The questionnaire follow-up 
ends six months after this time point.  
Rights  
Sponsor is senior author on the planned publications, and the coordinating investigator is the first 
author. All protocol authors and the site investigators as listed on page 1 and page 2 have had the 

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opportunity to comment and influence the protocol. These persons are authors on the planned main 
publication. All rights are specified in agreements between each study centre and Sponsor. 
Planned publications  
The main publication reports the primary and secondary endpoints described above, and 
authorship is described in detail clinical trial agreements. 
Secondary publications based on tertiary or ancillary endpoints and from the biobank for future 
scientific work will have at least one author from each study centre. Authorship is based on the 
Vancouver criteria. 
Ethics 
The study is conducted in accordance with the Helsinki declaration with guidelines from the 
International Conference on Harmonization of Good Clinical Practice (ICH-GCP). The Danish 
Medicines Agency, DMA (Lægemiddelstyrelsen) is applied for permission to using colesevelam and 
placebo. The GCP units as specified on page 3 monitor the study. 
Registers with personal data are subject to the Danish Data Protection Agency, and will be handled 
in accordance with ”Lov om behandling af personoplysninger”. Data registers are subject to the 
“Region Sjællands paraplygodkendelse” and are applied as such.  
The study is registered in the ClinicalTrials.gov register.  
Pregnant women are excluded. This study entices no significant risks or strain for the study subjects. 
The study gives crucial information on the correlation between both the SeHCAT scintigraphy and 
the biochemical diagnostics to the effect of colesevelam treatment. If our thesis is confirmed, bile 
acid diarrhoea may be diagnosed exclusively on basis of a blood test thus eliminating the need for 
radiation exposure and providing the possibility for easy and early diagnosis at local facilities thereby 
making it possible for many more patients to be identified and treated. Academically, it will be 
possible to re-examine individuals with BAD to describe changes over time. This improves the 
diagnostic algorithm and provides great benefit to both future patients and potentially also to study 
subjects. 
We offer subjects reimbursement for documented extra expenses for transportation.  
Recruiting 
Patients referred for SeHCAT at study centres are eligible. Referring doctors/investigators may hand 
out the written information directly or (sub)investigators at SeHCAT centres may send the written 
invitation alongside the SeHCAT appointment. The potential participants are given the folder: 
”Forsøgspersoners rettigheder i et sundhedsvidenskabeligt forskningsprojekt”. (Your rights as a 
participant in a research project). They are informed that this enquiry is regarding a medical 
scientific study, that participation is voluntary, and that it will have no influence on current and 
future examinations and treatment, whether the patient decides to participate in the study or not. 

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Information and consent 
The potential participant is invited to bring a bystander to the first SeHCAT day. A sub-investigator 
asks for interest in participation or in an information meeting, which then is arranged on the same 
day. The meeting is held in an office without time constraints. The written material is explained and 
questions are answered. It is the responsibility of the investigator (doctor) that the potential 
participant understands the information and is qualified for giving informed consent. If the potential 
participant after the information meeting need further time for reflection a maximum of 24 hours 
may be given. Informed consent is given on the criteria above. The doctor signs to guarantee this. 
 
Participation is voluntary and consent may be withdrawn at any time with no reason given. 
Consent for biological material for the biobank is given separately on the same occasion. 
Likewise regarding consent for the investigator to contact the referring institution. 
If the study reveals important information on the health of the subjects, he or she is informed unless 
the subject in the informed consent specifically wished not to be informed. 
 
Results 
Upon conclusion of the study participants are offered a short synopsis of its results in plain Danish. 
Participants may beforehand decline to this. All data and information are kept in accordance 
with:”Lov om behandling af personoplysninger” and ”Lov om patienters retsstilling”. 
The results - positive, negative or inconclusive - will be published in international peer-reviewed 
journals. 
 
Risks and nuisances 
In total 50 mL full-blood is drawn with routine sterile technique and this is not considered a risk.  
Colesevelam is well known as an off-label treatment for BAD. Common side effects are headache 
and constipation (10%) but it is generally well tolerated. See full description of side effects above. 
Longtime treatment with colesevelam may give malabsorption of fatty nutrients and vitamins, but 
this is irrelevant in the short context of this study. 
 
Data security and access 
Anonymized data are kept in Microsoft Access with password protection and audit trail and in IBM 
SPSS. Register with social security number (CPR) and all other personal data is kept in a file on 
SharePoint Teamsite under Region Zealand with logged access only for (sub)investigators with 
responsibility for data collection or data analysis. Backup discs are kept in a secure and locked facility 
within the department. Study data and metadata is anonymized after study end and saved in a 
repository for public access. The biological material is saved anonymized and registered in the 

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above-mentioned files and is destroyed 10 years after study end. Registers are under the supervision 
of the Danish Data Protection Agency. 
Data from the participants’ medical chart 
The participants’ medical chart data are reviewed for information on diarrhoea work-out and to 
screen for inclusion and exclusion criteria. This includes biochemical tests and the shared 
medicines chart (FMK). 
For monitoring of study safety and conduct the (sub)investigators, Danish Medicinal Agency, and 
the GCP units need access to the subjects’ medical chart. 
 
Primary data responsibility 
Lars Kristian Munck and Christian Borup 
Investigators with data access for analysis 
Signe Wildt, Jüri Johannes Rumessen, Morten Dahl, Jesper Graff 
Investigators at participating centres, as fully listed on page 2 and 3: Bente Sonne, Camilla 
Nøjgaard, Hans B. Timm, Søren Peter German Jørgensen, Tine Nygaard Gregersen, Trine Borup 
Andersen, Anna Zaremba, Lars Vinter-Jensen, Dominique Rainteau. 
 
Economy 
The study is investigator-initiated. The “Overlæge Johan Boserup and Lise Boserup Foundation” 
has donated DKK 120.000 for biochemical analysis and assistance. The Civilengineer H.C. 
Bechgaard & wife Ella Mary Bechgaard’s Fund has donated DKK 50.000.  
Production of colesevelam and placebo is funded by the Axel Muusfeldt Foundation (DKK 198.505) 
and the Aase and Ejnar Danielsen Foundation (DKK 100.000). 
Approximately DKK 900.000 is budgeted for PhD salary, of which the Region Zealand Scientific 
Fund has granted DKK 352.600.  
The ”Fabrikant Vilhelm Pedersen og hustrus mindelegat” donated DKK 2.000.000 after 
recommendation by The Novo Nordisk Foundation. 
Funds are administrated by the Zealand University Hospital.  
Both central and local investigators have no personal economic gain by the study. 
Compensation and insurance in case of injury caused by the study 
Sponsor and local sites are public institutions and are as such covered by the general patient 
compensation patienterstatningen. 
 
 
 

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Appendix 1: Study overview chart 
 
Study overview chart: SeHCAT: 75Selenium tauro-homocholic acid retention test, AE: adverse event