Conditions: Fecal Incontinence Bile Acid Malabsorption
Keywords: Incontinence Diarrhea Bile acid Microbiome
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 88
Healthy volunteers: no

Brief summary:
Compare the effects of a combination of colesevelam and clonidine to placebo on bowel symptoms in patients with urge or combined type of FI.

Detailed description:
Fecal incontinence (FI) is a common symptom that can significantly impair quality of life. There is very limited, mostly uncontrolled, evidence to support the approaches currently used to manage FI. The alpha-2 adrenergic agonist clonidine decreased the frequency of loose stools in FI patients with diarrhea. Among patients with diarrhea, clonidine decreased the proportion of days with FI; however results were not statistically significant. Uncontrolled studies suggest that the bile acid binding resin colesevelam also increased stool consistency in patients with functional diarrhea. In this study, the investigators propose to compare the effects of a combination of colesevelam and clonidine to placebo on bowel symptoms in patients with urge or combined type of FI.

Arms:
arm 1: Participants in this arm will receive placebo for 4 weeks. arm 2: Participants in this arm will receive a combination of colesevelam (1.875 gm twice daily) and clonidine (0.1 mg oral twice daily) for 4 weeks.

Interventions: intervention 1: Colesevelam intervention 2: Clonidine intervention 3: Placebo
Intervention descriptions: intervention 1: Participants who satisfy symptom criteria in Phase 2 will be randomized in a 1:1 ratio to receive either a combination of colesevelam (1.875 gm twice daily) and clonidine (0.1 mg oral twice daily) or an identical placebo for 4 weeks. intervention 2: Participants who satisfy symptom criteria in Phase 2 will be randomized in a 1:1 ratio to receive either a combination of colesevelam (1.875 gm twice daily) and clonidine (0.1 mg oral twice daily) or an identical placebo for 4 weeks. intervention 3: Placebo will be identical in appearance to the active drug.

Protocol document:
Prot_SAP_000.pdf: 
 
A Placebo Controlled Study of 
Colesevelam in Fecal 
Incontinence 
 
NCT# 02628626 
 
October 13, 2017 


Colesevelam in FI        1 of 16 
 
Title of the study: 
A Placebo Controlled Study of Colesevelam in Fecal 
Incontinence 
Staff 
Principal 
investigator 
Adil  E. Bharucha 
Co-Investigators 
Subhankar Chakraborty, Gopanandan Parthasarathy, David 
Prichard, Bridget Neja, Margaret Breen Lyles, Pratyusha 
Tirumanisetty 
Coordinator 
Kelly Feuerhak 
Statistician 
Alan Zinsmeister PhD 
 
Abstract 
 
Background: Available therapeutic options for idiopathic fecal incontinence (FI) are limited 
and unsatisfactory.  There are no controlled studies documenting the efficacy of medications 
used to treat FI.  We observed that oral clonidine significantly reduced diarrhea in patients 
with urge FI; while clonidine reduced the frequency of FI, effects were not statistically 
significant.  Small uncontrolled studies suggest that the bile acid binding resin colesevelam 
also improves functional diarrhea.  However, there are no controlled studies that have 
investigated the effect of colesevelam in FI.  In this placebo controlled study, we plan to 
investigate the effects of combining colesevelam with clonidine on symptoms in women with 
FI.  The primary aim of our study is to investigate the hypotheses that compared to placebo, 
colesevelam and clonidine will reduce the frequency of FI and rectal urgency.  Additionally, 
we will also investigate the stool and mucosa associated microbiota in these patients at 
baseline and after treatment.  The role of microbiota in pathogenesis of FI is unknown and 
we hope this study will offer us valuable insight into this question.  Sixty women (18-80 y) 
with symptoms of urge or combined (i.e. urge and passive) FI will be recruited to this study 
and treated with a combination of colesevelam and clonidine or placebo for 4 weeks.  The 
primary outcome will be the proportion of patients who achieve at least 50% reduction in the 
frequency of FI (number of FI episodes per Diem) at the end of 4 weeks of treatment.  


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A. Specific Aims 
Fecal incontinence (FI) is a common symptom that can significantly impair quality of life.  1  
The current management of FI, particularly in patients with moderate to severe symptoms, is 
unsatisfactory.  Our previous study showed that clonidine, a α2- agonist significantly 
decreased the proportion of loose stools in patients with urge or combined FI who also had 
diarrhea.  While clonidine reduced the frequency of FI, effects were not statistically 
significant.  A retrospective analysis of patients with bile acid malabsorption (BAM) observed 
that colesevelam, a bile acid binding resin improved subjective symptoms of FI in more than 
70% patients.2  A prospective placebo controlled study on the impact of colesevelam in 
patients with FI is however lacking.   
While BAM and alterations in fecal and colonic microbiota have been implicated to 
contribute to functional diarrhea and IBS respectively3, 4, the contribution of these 
disturbances to the pathogenesis or management of FI has not been evaluated.  A better 
understanding of the same is necessary to guide the management of FI.  Likewise, while IBS 
has been associated with gene polymorphisms5-7, no studies have evaluated the association 
between gene polymorphisms and FI.  Finally, the colonic mucosal microbiota are currently 
assessed by obtaining mucosal biopsies during a flexible sigmoidoscopy.  It is conceivable 
that the mucosal microbiota can also be sampled with an rectal catheter.  However, the 
characteristics of the colonic mucosal microbiota profile obtained with sigmoidoscopy and a 
catheter have not been compared.  
The central hypothesis of this study is that a combination treatment with clonidine 
and colesevelam is better than placebo in reducing stool frequency and rectal urgency in FI.  
Additional hypotheses are that FI is associated with (i) BAM, (ii) alterations in the fecal and 
colonic mucosal microbiota, which are modified by treatment with colesevelam and clonidine 
and (iii) single nucleotide polymorphisms (SNPs) of selected genes.  
 
In order to investigate these hypotheses we propose five aims as follows:  
Aim 1: Compare the effects of combination treatment with colesevelam and clonidine vs. 
placebo on bowel symptoms in FI  
Aim 2: Investigate the prevalence of BAM in patients with FI 
Aim 3: Evaluate the fecal and colonic mucosal microbiota in patients with FI and the effects 
of treatment with colesevelam and clonidine 
Aim 4: Compare the colonic mucosal microbiota obtained with mucosal biopsies during a 
flexible sigmoidoscopy and an rectal catheter in FI  
Aim 5: Investigate SNPs associated with FI through microarray analysis of genomic DNA 
obtained at baseline.   
 
B. Background and Significance 
 
Fecal incontinence, the involuntary leakage of stool from the anus, is a common 
symptom not only in nursing home residents, but also in the community.  8  Our observations 
(2800 women) from Olmsted County demonstrate that one of 10 adult women and one of five 
women aged ≥ 50 years have had one or more episodes of FI, unrelated to an episode of 
acute diarrhea.  1  Almost one of 15 adult women have moderate to severe FI.   
 
There is very limited, mostly uncontrolled, evidence to support the approaches 
currently used to manage FI.  9-11  Based on observations of responsiveness to placebo in 
controlled clinical trials, conservative measures (e.g., dietary modification, behavioral 
measures to suppress rectal sensation and not including biofeedback therapy) improved 
symptoms in approximately 25% of patients.  12  Amitriptyline and loperamide improved 
diarrhea and rectal urgency in uncontrolled studies.  13-15  However, the use of these agents 


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Principal Investigator: Adil E. Bharucha  
is often limited by constipation.  Biofeedback therapy is better than pelvic floor exercises 
alone in patients who do not respond to conservative measures.16  In the only controlled 
clinical trial of medical therapy for FI, the effects of oral clonidine (0.1 mg twice daily) for 4 
weeks on symptoms were not significantly different versus placebo in all comers with FI.  
However, clonidine significantly decreased the proportion of loose stools in FI patients with 
diarrhea.  Among patients with diarrhea, clonidine decreased the proportion of days with FI; 
however results were not statistically significant.17 Uncontrolled studies suggest that the bile 
acid binding resin colesevelam also increased stool consistency in patients with functional 
diarrhea.18 19 A retrospective chart review and questionnaire suggested that colesevelam 
improved diarrhea and fecal incontinence in patients with cancer and diarrhea due to 
suspected bile acid malabsorption.2 However, among patients with functional diarrhea, the 
effects of bile acid binding agents on stool consistency were relatively modest.  For example, 
in a study from Mayo Clinic, colesevelam reduced the Bristol score from an average of 4.8 
before to 4.4 after therapy.  While colesevelam increases stool consistency by binding bile 
acids, clonidine does so by improving net absorption of fluids and electrolytes.20 Clonidine 
can also increase rectal capacity and reduce rectal sensation.21 Therefore, we propose to 
compare the effects of a combination of colesevelam and clonidine to placebo on bowel 
symptoms in patients with urge or combined type of FI.  
 
At present, FI is attributed to bowel disturbances, particularly diarrhea, and anorectal 
dysfunctions.17 Among older women with FI, our data strongly suggest that diarrhea and 
rectal urgency are more important risk factors than obstetric anal sphincter injury.22, 23 
Because many women with FI also have bowel disturbances (i.e., irritable bowel syndrome 
[IBS]), it is important to understand the contribution of peripheral dysfunctions (e.g., BAM, 
altered colonic microbiota), which are observed in IBS, to FI. For example, while several 
small studies observed differences in the microbiota in feces between health and irritable 
bowel syndrome,24 we recently observed major differences in the mucosal microbiota profile 
between healthy and constipated women.25  However, no studies have evaluated the fecal 
and colonic mucosal  microbiota in FI.  Currently, the mucosal microbiome is evaluated by 
obtaining mucosal biopsies during flexible sigmoidoscopy.  It may be possible to assess the 
mucosal microbiota with an rectal catheter, which is a noninvasive technique.  Hence, we 
propose to compare the fecal and colonic mucosal microbiota obtained with flexible 
sigmoidoscopy and an rectal catheter in patients with FI. 
 
In the community, the prevalence of FI in men is similar to that in women (8.9% and 
7.7% respectively).26  However, in our practice, a majority of patients with FI are women, who 
have had several vaginal deliveries.  By contrast, the 3 main risk factors for FI in men are: 
proctitis secondary to radiotherapy for prostate cancer, a rectal evacuation disorder, and 
iatrogenic anal sphincter injury (e.g., due to surgery).  Hence, the pathophysiology of FI is 
differs between men and women.  Therefore, and to ensure a more homogenous composition 
of the patient group, this study is limited to women.   
 
  
C. Research Design and Methods 


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1. 
Overall experimental design.   
This study will consist of four phases (Figure 1) 
1. Phase 
1 
(Initial 
screening) 
Patients 
who 
consent to participate in 
the study will visit with the 
investigator during which 
a physical examination 
may 
be 
performed.  
During this visit they will 
be asked to complete 
questionnaires designed 
to determine whether or not they have FI, the type of FI, screen for anxiety or depression and 
assess quality of life at baseline.  Those who have diarrhea but not FI as assessed by 
the questionnaires (i.e. Bristol stool type 6 or 7, or >3 bowel movements/day on 
average) will be excluded from the study.  Eligible patients who give consent will undergo 
a urine pregnancy test, a 12 lead EKG, anorectal manometry (if not done at Mayo Clinic in 
the last 6 months), spot stool sample for microbiota analysis, blood draw for genotyping, 
assessment of the a rectal mucosal microbiota with a rectal catheter, and an optional flexible 
sigmoidoscopy.  Patients will be asked to record their dietary intake for 72 hours prior to 
collecting the stool sample.   
Of these tests in phase 1, only the pregnancy test and EKG will determine whether 
patients proceed to phase 2.  The remaining tests will be used to assess specific aims 2-5.  
Anorectal manometry will help clarify whether these patients have FI because of weakness 
of the anal sphincter or rectal hypersensitivity.  The spot stool sample and rectal mucosal 
catheter will be used to assess the microbial diversity in FI. Among patients who have a 
flexible sigmoidoscopy, the mucosal microbiota will be compared on mucosal samples and 
a rectal catheter.  In an attempt at (i) avoiding situations where we recognize subjects are 
not eligible to participate when they present at the screening visit and (ii) reduce the time 
taken for screening visits, we propose, when feasible, to ask participants to complete the 
questionnaires and a 2 week bowel diary before they come for their screening visit.  Subjects 
will return these questionnaires to us by regular or electronic mail.  If subjects are not 
subsequently consented/enrolled into the study, these items will be destroyed. [This bowel 
diary is identical to that approved for use later in the study.]  
 
 
2. Phase 2 (Completion of bowel diaries) 
Patients, who have FI, as documented by the questionnaire during phase 1, are not 
pregnant, and do not have EKG exclusion criteria documented below will enter phase 2.  
During this phase, patients will fill out daily bowel diaries to ascertain if they have FI 
symptoms of sufficient severity.  The duration of this phase is variable and ranges from 2-4 
weeks as clarified below.  Only those patients who meet all the following criteria at the earliest 
weekly review of the bowel diaries starting at week 2 i.e., (i) completion of at least 5 out of 7 
days of the diary in the preceding week and 10 out of 14 in the preceding 2 weeks, (ii) 
experience at least 1 episode of FI per week averaged over 2 weeks, (iii) have a Bristol stool 
form score ≥ 3, and (iii) an average stool frequency of ≥1/day will enter the treatment phase.  
Patients who have not filled out the minimum number of days of bowel symptoms will be 
asked to fill out bowel diaries for an additional 2 weeks.  Those who satisfy criteria at the end 
Figure 1.  Study design 
 


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Principal Investigator: Adil E. Bharucha  
of the two additional weeks will proceed to Phase 3.  Otherwise their participation in the trial 
will be terminated and they will exit the study.  
 
3. Phase 3 (Treatment Phase) 
Patients who satisfy symptom criteria in Phase 2 will be randomized in a 1:1 ratio to 
receive either a combination of colesevelam (1.875 gm twice daily) and clonidine (0.1 mg 
oral twice daily) or an identical placebo for 4 weeks.  The randomization, to be prepared by 
Dr. Zinsmeister, will be balanced on age (<50 vs. ≥ 50 y) and BMI (<30 vs. ≥ 30 kg/m2).  
Patients will fill daily bowel diaries for the entire duration of the treatment phase and mail 
them in every week.  Patients will be contacted periodically (by phone or email) to assess 
compliance with medications, with filling bowel diaries and for any adverse events.  At the 
end of the treatment phase, patients will be asked to fill questionnaires pertaining to their 
quality of life and satisfaction with treatment.  During the treatment phase, patients will be 
allowed to use rescue medication in the form of loperamide to circumvent the potential for 
withdrawal from the study due to overwhelming symptoms.  Patients are allowed to take 
loperamide (2mg/dose) up to 3 unit doses in a 24 hour period, 5 unit doses in a 48 hour 
period or 10 unit doses in any 7-day period.  Patients will need to record their loperamide 
usage in the bowel diary.   
 
4. Phase 4 (Post treatment phase) 
This phase, which lasts 4 weeks, will begin after patients take their last dose of 
medication.  All patients who complete at least 1 week of treatment will enter this phase.  
During this phase, study staff will contact patients periodically by phone or email to enquire 
how they are doing.  Patients will fill out daily bowel diaries for 4 weeks and return them 
weekly for review.  At the end of 4 weeks, they will be asked to complete post-treatment 
questionnaires to assess quality of life, anxiety and depression.  They will also be asked to 
collect a single spot stool sample and mail them in.  The questionnaires will be used to 
investigate if patients had worsening of symptoms after stopping therapy.  The spot stool 
sample will be used to analyze the effect of treatment on the fecal microbiota.  Participants 
will be asked record their dietary intake for 72 hours prior to collecting the stool sample and 
mail in the food record.  
 
 
2. Human subjects.  
Eligibility criteria for each phase of the study are tabulated below. 
 
A. Phase 1 
Inclusion Criteria: 
i) Females aged 18-80 years with urge predominant or combined (i.e. urge plus passive) 
FI, as defined by a validated questionnaire, for ≥ 1 year duration will be eligible to 
participate.27  
 
Exclusion Criteria:   
(i) History of clinically serious cardiovascular or pulmonary disease or EKG showing 2nd 
degree atrioventricular block or higher. 
(ii) Current or past history of colon or rectal cancer, scleroderma, inflammatory bowel 
disease, ischemic colitis, small bowel obstruction, congenital anorectal abnormalities, 
≥ Grade 2 rectal prolapse, history of rectal resection or pelvic irradiation 


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Principal Investigator: Adil E. Bharucha  
(iii) Neurological disorders – Spinal cord injuries, dementia (Mini-Mental status score 
<21), multiple sclerosis, Parkinson’s disease, peripheral neuropathy  
(iv) Conditions precluding safe use of clonidine, i.e., symptomatic hypotension, or systolic 
blood pressure of <100 mm Hg on initial visit in Phase 1 of study  
(v) Colon resection greater than limited resection (e.g. sigmoid colectomy for diverticulitis 
is eligible but right hemicolectomy is not)  
(vi) Patients who are currently on or anytime in the past have been on colesevelam for FI  
(vii) 
Malabsorptive bariatric procedures (i.e., Biliopancreatic diversion with or 
without duodenal switch and Jejunoileal bypass), gastric bypass surgery (i.e., Roux 
en Y gastric bypass) will be excluded. Those patients who have undergone 
restrictive procedures (e.g.  vertical banded gastroplasty, laparoscopic adjustable 
gastric banding) however will be eligible to participate 
(viii) 
 Currently pregnant or nursing women Prior history of intolerance to clonidine 
or colesevelam 
(ix)  History of sphincteroplasty, sacral nerve stimulator, previous injection of Solesta for 
treatment of FI 
(x)  Medications  
• .   
• Relative – opioid analgesics , other antihypertensive agents (i.e. if there is 
concern about synergistic effects and hypotension).  Patients using drugs with 
anticholinergic effects at high doses (e.g. nortriptyline greater than 50 mg/day 
or amitriptyline greater than 25 mg/day) will be excluded at the discretion of the 
physician.  Patients who use lower doses will be eligible to participate provided 
the dose will be stable during the study  
 
B. Phase 2 
Inclusion Criteria: 
i) 
Females aged 18-80 years with urge predominant or combined (i.e. urge plus 
passive) FI for ≥ 1 year, as defined by questionnaire 
 
     Exclusion criteria: 
(i) Positive urine pregnancy screen 
 
     C. Phase 3 
     Inclusion criteria: 
(i) Completion of at least 5 out of 7 days of the diary in the preceding week and 10 out of 
14 in the preceding 2 weeks 
(ii) At least 1 episode of FI per week averaged over 2 weeks 
(iii) Average Bristol stool score of 3 or higher 
(iv) Average stool frequency of ≥1/day 
 
     Exclusion criteria (if at least one is satisfied): 
(i) Missing data in bowel diaries, i.e. if patient did not record bowel symptoms data for 
more than 2 days in 1 week or 4 days over 2 weeks  
(ii) Greater than 6 liquid [Bristol 6 or 7]) stools daily 
(iii) Average of less than 1 bowel movement daily 
(iv) Average Bristol stool score <3 as assessed from analysis of bowel diaries 
 
D. Phase 4 


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Principal Investigator: Adil E. Bharucha  
Inclusion criteria: 
(i) All patients who complete at least 1 week of treatment with study drugs or placebo 
     Exclusion criteria: 
(i) Patients who completed less than 1 week of treatment with study drugs or placebo 
 
 
3. Study procedures 
a) History and physical examination  a physical examination will not be performed, at the 
discretion of the investigator, when this has been performed in the past 3 months at Mayo 
Clinic.  The MMSE test will be administered as described previously.  28 The maximum score 
is 30 and a score of less than 21 is suggestive of dementia. 
b) Anorectal manometry will only be performed when this has not been performed in the 
past 6 months at Mayo Clinic.  After rectal cleansing with 1-2 magnesium citrate 
(Fleets,C.B. Fleet, Lynchburg, 
VA) enemas, anorectal functions 
will be assessed using standard 
and established techniques in our 
laboratory 29.  Anal pressures will 
be measured at rest, during 
squeeze, simulated evacuation, 
and a Valsalva maneuver by 
high-resolution 
manometry.  
Since 
clinical 
observations 
suggest that FI is also associated 
with a rectal evacuation disorder, 
rectal balloon expulsion will also 
be assessed.  
c) Flexible 
sigmoidoscopy 
will be performed in the Clinical 
Research Unit after 1-2 Fleet’s 
enemas.  This is an optional 
procedure and will be performed 
in Phase 1 of the study after the 
rectal catheter and if an anorectal 
manometry is performed, after 
that procedure also.  Using 
standard 
biopsy 
forceps, 
8 
mucosal biopsies will be obtained 
each from the sigmoid colon and 
the rectum.  Eight biopsies will 
provide 
adequate 
tissue 
for 
microbial DNA extraction.  If any 
abnormality 
is 
seen 
on 
sigmoidoscopy, 
additional 
biopsies will be obtained for 
histology.  The colonic mucosal 
Figure 2. Mega-Brush. A. Undeployed brush with blue 
sheath and the foam compressed inside a gelatin sheath. B. 
The foam is pushed out but not yet deployed C. Foam 
deployed. The foam shown in the figure has 20 pores per 
inch. We will be using one which is softer with 80 pores 
per inch.  


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microbiota will be compared to that evaluated with a rectal catheter.  
d) Rectal catheter.  This test will be done in all patients either just prior to anorectal 
manometry or during the initial visit in patients who do not have a manometry.  The Mega 
Brush ® (provided by Capnostics LLC., Figure 2) is a mass cytology cell retriever system 
comprised of a foam sphere made of biocompatible material that is used to obtain specimens 
from the anorectum.  It is FDA cleared (FDA K944614/A) for collection of mucosal specimen 
with an endoscope. .  The foam is enclosed in a flexible outer tube and the tip covered by a 
gelatin cap.  The diameter of the foam is 30 mm when fully deployed and the foam has 80 
pores per sq. inch.  We propose to introduce the catheter under direct vision during the rectal 
examination. Once the catheter is in the rectum, the capsule will be pushed out into the 
rectum and the cap allowed to dissolve over about 5 minutes. This will deploy the foam 
sphere. The foam sphere will be rotated 1-2 times in the rectum very gently with the help of 
the sheath. The catheter will then be slowly withdrawn.  . 
e) Calculating relative abundance of bacterial 16s rRNA to human DNA: Quantitative PCR 
will be used to assay the quantity of bacterial 16s rRNA in the stool sample.  This will be 
normalized to a single copy human gene (e.g. TNF-alpha) as described in literature 30.  The 
relative proportion of bacterial to human DNA will be calculated by first generating a standard 
curve by dilution series using purified plasmids containing a representative target (e.g. E.coli 
16s rRNA or human TNF-alpha).  Purified plasmids will be amplified in parallel with patient 
samples and a standard curve generated to assess number of copies of the target in each 
sample. 
f) Fecal and colonic mucosal microbiome analysis: Microbial DNA will be extracted from 
stool and tissue samples using standardized methods [18].  Phylotype profiles of the 
microbiome from colesevelam and placebo treated FI populations will be generated using deep 
rDNA hypervariable tag sequencing of the hypervariable V3-V5 region of the SSU rRNA gene, 
which has been validated for use with human microbiomes and is one of the methods of choice 
for the HMP.  With the longer reads from the MiSeq (250x250 paired end reads), our 
sequencing will include both the V3-V5 RGTs making for a more optimal phylogenetic analysis 
[19].  Phylotype profiles of the mucosa-associated microbiome will be generated using rDNA 
sequencing of the hypervariable V3-V5 region, which has been validated for standard use by 
the human microbiome project.  The 500 base pair reads will ensure a better optimal 
phylogenetic identification.  Barcoding samples prior to sequencing will yield approximately 
20,000 reads/sample, ensuring detection of both dominant (core microbiome) and poorly 
represented taxa (variable microbiome).  Identifying the existent taxa, diversity, and ecological 
relationships within each sample requires that we process large volumes of 16S DNA 
sequence data.  In order to accomplish this task we will follow protocols that we have 
quantitatively shown to be optimal.  Reads will be aligned using our own custom multiple 
alignment tool (TORNADO v2.0) that has been optimized the production of taxonomic calls 
and alignments for downstream analysis such as OTU clustering and phylogeny from paired 
end reads [20].  Sequences will be culled according to initial quality files using QIIME, pre 
clustered in order to reduce the number of unique reads due to sequencing errors using 
mother, and aligned using the new version of the TORNADO pipeline for paired end reads.  
TORNADO v2.0 will then be used to obtain the taxa calls that will be used in later correlation 
analysis.  Second, quantitative PCR assays targeting functional genes mcrA and mrtA 
(methanogens) dsrA (sulfate-reducing bacteria) and hydA (hydrogen-producing bacteria) will 
be performed on both stool and mucosal samples following published protocols [21].  Similarly, 
different sulfate-reducing genera will be quantified with validated 16S primers.  The abundance 


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Principal Investigator: Adil E. Bharucha  
of functional genes will be correlated to physiological parameters.  Subsequent QPCR assays 
on microbial groups that are significantly distinct in sequencing analyses may also be 
performed. 
 (i) Nutritional Assessment: Habitual dietary intake will be assessed at baseline using an 
electronic graphical food frequency questionnaire (FFQ) (Viocare Technologies, Inc. 145 
Witherspoon Street, Princeton, NJ 08542).  This questionnaire is based on the Women's 
Health Initiative FFQ and has been adapted by VioCare in an electronic computer-
administered format to evaluate dietary intake over a three month period.  It employs the 
Minnesota NDS nutritional analysis database for nutrient analyses.  The FFQ has been 
validated and compares well with nutrient intake validated against food records and four 
24-hr dietary recalls (29, 30).  (ii) Participants will keep food records of their actual dietary 
intake for 72 hours prior to collecting the stool specimens for microbiome analysis.  The 
food records will be analyzed for energy, protein, fat, carbohydrate and fiber content by the 
CRTU nutrition staff using the ESHA Food Processor software (Version 10.15) developed 
by ESHA Research, Salem, Oregon 97309-1028.   
g)   Drug therapy Patients will be treated with colesevelam (1.875 gm) and clonidine (0.1 
mg) twice daily or matching placebo for 4 weeks.  Patients will need to discontinue any 
antidiarrheal medications from the start of Phase 1 through end of Phase 3.  Loperamide 
tablets (2 mg) will be provided for rescue when patients experience intolerable diarrhea.  
Patients can take up to 3 unit doses in a 24 hour period, 5 unit doses in a 48 hour period or 10 
unit doses in any 7-day period.  They will be required to record their loperamide usage during 
the treatment phase.  
h) Genome wide DNA analysis.  We are currently exploring for SNPs associated with FI 
by genotyping 175 women with FI and 175 age- and gender-matched controls from Olmsted 
County (IRB 22-01), using a chip with > 500,000 SNPs spaced on average 1 every 5 kb in 
the genome.  Towards our long-term objective of uncovering associations between SNPs 
and FI, we propose to extract DNA from 20 ml of blood, to be drawn from study participants.  
The tubes will be spun down in the Clinical Research and Trial Unit immediately and the 
plasma stored at minus 80 degrees celsius.  The buffy coat will be stored at minus 80 degrees 
Celsius until DNA is extracted.  DNA extraction will be conducted at the BAP Lab.  Genome-
wide analysis will be conducted using Illumina 610 QUAD microarray or comparable 
approaches.  All patients who qualify for the study based on physician assessment in Phase 
1 will undergo genotyping.  Genotype-phenotype correlations will be examined using these 
patients and other patients in ongoing studies. 
i) Analysis of Differential Gene Expression by RNA Seq analysis.  In order to investigate 
if there is any relationship between the global gene expression in a patient and their response 
to treatment for FI, we will perform RNA Seq analysis with help of the Gene Expression Core 
Facility at Mayo Clinic Rochester.  We will collect 7.5 ml of blood in PAX gene tubes at the 
time of the initial visit.  RNA will be extracted from whole blood immediately and stored at 
minus 80 degrees celsius.  Further processing of RNA will be done by the Gene Expression 
core facility at Mayo Clinic, Rochester.  
Summary of Blood Collections.  The total blood collected for this study is 27.5 ml.   
Item 
Test ID 
Lab 
Tube (amount required)  
DNA for SNPs 
31669 
CRU Lab 
Use buffy coat from EDTA tubes (one 
tube of 20 ml) 


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RNA 
extraction 
and 
processing 
31669 
BAP Lab 
PAX gene tubes (7.5 ml) 
 
j)  Symptom assessments.   
The following instruments will be used to evaluate symptoms, co-morbid conditions, and 
impact on QOL. 
(i) Fecal Incontinence Severity Index (FISI) – which is a validated 4-item scale used to 
assess the frequency of 3 different types of FI.  31 
(ii) Fecal Incontinence Quality of Life (FI-QOL) – is a 30-item validated questionnaire that 
assesses the effects of FI on QOL.  32 
(iii) Hospital Anxiety and Depression (HAD) Scale 33 
(iv) Health-status questionnaire (Radosevich DM, Wetzler H, Wilson SM. 1994.  Health 
Status Questionnaire (HSQ) 2.0: Scoring Comparisons and Reference Data.  Health 
Outcomes Institute, Bloomington, MN.)  is a self-report inventory designed to measure 
a patient’s overall health and functioning.  
(v)  Fecal incontinence and Constipation Assessment (FICA) Scale – This validated 
instrument, developed in our program, has 4-items (frequency, type, amount of 
leakage, and presence of urgency) used to rate the severity of FI.  34  It is the only 
instrument that incorporates the amount of leakage, which is an important yardstick of 
the severity of FI.  
(vi) Satisfaction with treatment: In order to determine if patients were satisfied with the 
treatment received, we will use a previously validated visual analog score.  
FICA score will be calculated from the bowel questionnaire.  FISI, HAD, HSQ and Satisfaction 
with treatment score will be calculated from the General Questionnaire that will be 
administered pre and post-treatment (Table 1). 
 
Table 1.  Symptom assessment tools and their administration during the study 
 
 
Phase 1 
Phase 2 
Phase 3 
Phase 4 
Duration 
Up to 1 week 
2-4 weeks 
4 weeks 
4 weeks 
Bowel questionnaire 
X 
 
X 
X 
Quality 
of 
life 
questionnaire 
X 
 
X 
X 
Hospital 
anxiety 
and 
depression 
questionnaire 
X 
 
 
X 
Daily bowel diaries 
 
X 
X 
X 
Satisfaction 
with 
treatment 
 
 
X 
 
 
4. 
Data and statistical analysis  
i. 
Power Statement:   
Based on the observed difference in the frequency of FI (i.e., mean number of FI episodes 
per subject per diem) between treatment and placebo groups in the clonidine study, we 
estimate that approximately 33 subjects in each treatment group (i.e. colesevelam and 


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clonidine vs placebo) will provide 80% power, using a two sided alpha level of 0.05, to detect 
at least a 50% difference in the frequency of FI between treatment groups.17 
ii. 
Data Analysis Plan:  
a) Statistical analysis 
An Excel (Microsoft, Redmond, WA) spreadsheet of treatment assignments (balanced on 
age and BMI) will be generated (by Dr. Zinsmeister) and sent to the research pharmacy.  
Study personnel will be blinded until the study is completed.  
Bowel diaries will be analyzed as follows: Stool frequency, form, urgency and use of rescue 
medications will be averaged first for each day and then over 1-2 weeks (two weeks in Phase 
1 and one week in Phase 3) in each subject.   
We will compare response to treatment between colesevelam/clonidine and placebo 
between for the treatment period using the values during Phase 1 as covariates and 
separately, post-treatment.  The analyses will use an α value of 0.05 for each comparison.  
Treatment groups will compared using analysis of covariance (ANCOVA) with the 
corresponding baseline (Phase 1) value as the covariate.  Data will be analyzed per intent-
to treat analysis using all subjects randomized.  Missing values will be imputed as described 
previously and an adjustment in the ANCOVA error degrees of freedom will be made.  
Separate ANCOVA models will be used to evaluate whether treatment effects were 
influenced by the presence or absence of diarrhea at baseline.  All tests will be two tailed.  
Endpoints    
The primary and secondary endpoints have been well described in our study of  
clonidine in FI and will be employed in this study as well34 
 
a) Primary endpoint: 
Previously, we have shown that in patients whom the frequency of FI (measured as 
mean number of episodes of FI per person per Diem) decreased by at least 50%, the 
corresponding FICA severity score exceeded the minimum clinically-important 
difference (i.e., the smallest change detected by an instrument that is considered 
clinically significant) in at least 75% of the cases.  This means that the majority of 
people who report at least 50% improvement in FI frequency have a clinically 
significant improvement.  Hence in this study, we will use a 50% reduction in the 
frequency of FI episodes as our primary end point. 
  
Secondary endpoints: 
The following secondary end points will be compared between placebo and colesevelam 
groups 
a) Change in the number of days with FI  
b) Volume of FI- this will be graded as small (staining only), moderate (requiring change 
of underwear) or large (requiring change of all clothes).  We will calculate the 
proportion of FI episodes of each type and compare the proportion before and during 
treatment. 
c) Composition of leaked stools (measured as percentage of all incontinent bowel 
movements that were type 5-7 on the Bristol stool scale)- average Bristol stool form 
will be calculated from bowel diaries and will be compared before and during 
treatment. 
d) Number of episodes of passive and urge incontinence per week 
e) Rectal urgency (proportion of total and incontinent bowel movements preceded by 
urgency) 
f) Proportion of complete bowel movements 


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g) Time for which a bowel movement can be deferred after occurrence of urgency 
h) Change in Bristol stool form 
i) Change in proportion of FI episodes that were diarrhea (Bristol stool score 5-7) 
j) Patient reported severity of bowel symptoms as assessed by VAS 
k) Severity of FI (FISI score) 
l) Impact of FI on quality of life (FI-QOL score) 
m) Impact on symptoms of anxiety and depression (HAD score) 
n) Satisfaction with treatment measured by the visual analog scale  
o) Proportion of days where patient used loperamide 
 
For the above end points, we will obtain information either from weekly bowel diaries or 
questionnaires administered during each of the phases of this study as outlined in Section 
2.  
 
Using standardized approaches established in our laboratory, mucosal microbiota profiles 
will be compared between rectal catheters and  colonic mucosal biopsies obtained at 
flexible sigmoidoscopy.  
 
iii. 
Subject Safety.  Only subjects who are eligible based on pre-defined criteria, an 
interview and a physical examination, and screening laboratory tests will be enrolled.  
The consent form details issues related to privacy, withdrawal from studies, and use 
of blood for genetic analyses among other items.  The eligibility criteria are structured 
to exclude patients who have a greater risk of side effects with clonidine.  Interventions 
(clonidine, colesevelam and anorectal tests) pose a low risk to participants.  In our 
previous study, clonidine was well tolerated by women with FI; dry mouth was the only 
side effect that occurred more frequently with clonidine than placebo.  17  There is no 
known drug interaction between clonidine and colesevelam.  Patients who are positive 
for depression by the HAD score will be offered referral to Psychiatry.  If the patient 
endorses suicidal thoughts then we will first and foremost offer support.  If the patient 
is on Mayo campus and in department, we will then have a co-worker page 911 and 
ask for Security.  The study coordinator will contact the principal investigator 
immediately to alert them that of the situation.  The study staff will stay with the patient 
until Security arrives at the location.  If the patient is not on Mayo campus, then the 
study staff will keep the patient on the line and have a co-worker page 911.  Study 
procedures are conducted by trained and experienced personnel.  Monitoring for side 
effects will be performed by study personnel.  The PI will also review side effects.  
Each study is reviewed and analyzed; study discussions and communications are 
generally by e-mail and stored.   
iv. 
Data Integrity.  Only subjects who are eligible based on pre-defined criteria, an 
interview and a physical examination will be enrolled.  Transcription of data is accurate 
and complete.  Calculations will be standardized and accurate.  The study technician 
will monitor data after every study.  The PI will also review data after every 10 studies.  
v. 
Subject Privacy.  Subjects will be consented in a private room.  Adequate time for 
questions will be provided.   
vi. 
Data Confidentiality.  All records are maintained in password protected electronic files.  
Hard copies are filed in private offices.  
vii. 
Product Accountability.  Research Pharmacy will obtain and dispense medication.  
Unused medication will be collected from subjects at the end of the study and 
destroyed.  


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viii. 
Study Documentation.  Documentation will be per established guidelines during the 
study (hard copy and electronic data capture).  These files will be sampled annually. 
ix. 
Study Coordination.  Quarterly debriefing will be conducted to ensure expectations 
are clear and if educational needs exist. 
 
D.  Human Subjects   
Only subjects who satisfy stringent inclusion and exclusion criteria and provide written 
informed consent will be included in the protocol.  The consent form details issues related to 
privacy, withdrawal from studies, and use of blood for genetic analyses among other items.  
Additional details as follows: 
1. Anorectal assessments are extremely safe; discomfort, if any during rectal distention is 
mild and tolerable and the risk of rectal perforation in an uninflamed rectum is negligible.   
2. Clonidine is generally well tolerated at this dose.  In a recent study, we observed that the 
most common side effect with clonidine was dry mouth (about 70% vs. 4.5% in placebo).  
Other adverse effects included drowsiness (23% vs 14% in placebo), light headedness 
(27% vs.9% in placebo) and fatigue (36% vs. 23% in placebo) although they did not reach 
statistical significance.  17 
3. Colesevelam is generally well tolerated at this dose.  The most commonly reported side 
effects from Colesevelam are constipation, diarrhea, dyspepsia, hypoglycemia (mild and 
well tolerated), headache, abdominal cramps, nausea, and flatulence and back pain.  
Their frequency ranges from 8%-40% in different studies.  35-37 
 
Patients will be appropriately screened and subjects with an increased risk of 
complications will be excluded from the study.  All side effects will be recorded at the follow-
up visit.  Since the effects of clonidine and colesevelam in pregnant and nursing women are 
unknown, women who are pregnant or nursing will be excluded from the study.  Women of 
child-bearing potential will be advised to employ contraceptive measures during the study 
and a pregnancy test will be checked before beginning clonidine or colesevelam.   
 
Patients who cannot tolerate clonidine 0.1 mg bid will be switched to 0.1 mg once daily 
(qhs)  Clonidine will be discontinued if:- 
(i) 
Subjects have syncope, or lethargy that interferes with safe performance of the 
volunteer’s activities of daily living; or 
(ii) 
if the systolic blood pressure drops to < 90 mmHg for patients with a systolic BP 
between 90 and 130 mmHg, or if systolic BP falls to < 100 mmHg for subjects with a 
pre-treatment systolic BP > 130 mmHg; or 
(iii) 
if a subject becomes pregnant during the study.   
Patients who cannot tolerate three tablets of 625 mg colesevelam twice daily will have 
dose reduction by 1 tablet daily until they are able to tolerate the medication.  Colesevelam will 
be stopped if a subject becomes pregnant during the study. 
Data Safety and Monitoring Plan.  The DSMP utilized will adhere to the protocol 
approved by the Mayo Clinic IRB.  We propose the following plan: - 
Data quality and management: The principal investigator will review all data collection 
forms on a three-monthly basis for completeness and accuracy of the data as well as 
protocol compliance.  
Adverse events grading: The common grading scale listed below will be used to grade AEs: 
0 No adverse event or within normal limits or not clinical significant 
1 Mild AE, did not require treatment 
2 Moderate AE, resolved with treatment 
3 Severe AE, resulted in inability to carry on normal activities and required 
professional medical attention 


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Principal Investigator: Adil E. Bharucha  
4 Life threatening or disabling AE 
5 Fatal AE 
Attribution scale: An adverse event includes both, an expected side effect that is of a 
serious nature, or an unexpected side effect/ event regardless of severity.  All events will be 
graded as to their attribution (unrelated to protocol, or possibly, probably, or definitely 
related to protocol).  Any event that is reported to either the principal investigator or his 
designated research associates by the subject or medical staff caring for the subject and 
which meets the criteria will be documented as such. 
Data Monitoring.  The majority of data generated from these protocols will be from 
analyses performed in our laboratory or the immunochemical core laboratory.  Standard 
quality control procedures are in place for each assay.  The frequency of data review for 
this study differs according to the type of data and can be summarized in the following 
table: 
 
Data type 
Frequency of 
review 
Subject accrual (adherence to protocol regarding 
demographics, inclusion/exclusion) 
Weekly 
Adverse event/safety rates (injuries) 
Weekly 
Annual report 
Yearly for IRB  
 
4.  DNA and RNA analyses.  DNA, RNA and plasma samples will be anonymized before 
being submitted to the genotyping or RNA Seq assays.  Specific information is provided in the 
consent form regarding storage and future use of the biologic sample.  These results will not 
be added to the written record.     
 
 
 
 
 
 
E. 
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Wedlake L, Thomas K, Lalji A, Anagnostopoulos C, Andreyev HJ. Effectiveness and 
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