Conditions: Constipation
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 74
Healthy volunteers: yes

Brief summary:
This study is designed to better understand the effects of effects of stress, relaxation, and a medication alfuzosin on bowel control and emptying in healthy people and patients with bowel problems.

Detailed description:
Normally, bowel emptying requires relaxation of the anal sphincter (i.e., lowermost end of intestinal tract) and pelvic muscles. Some people cannot relax these muscles normally and experience constipation. Alfuzosin is a medication which is approved to treat bladder but not bowel problems.

Arms:
arm 1: In Part A, subjects randomized to this arm will receive a single dose of oral alfuzosin immediate release (IR) 2.5 mg.

In Part B, only constipated patients will receive oral alfuzosin (10 mg extended release (ER)) capsules. arm 2: Subjects randomized to this arm will receive a single placebo capsule identical to the study drug.

Interventions: intervention 1: Alfuzosin intervention 2: Placebo
Intervention descriptions: intervention 1: oral alfuzosin immediate release (IR) 2.5 mg (Part A) or oral alfuzosin extended release (ER) 10 mg (Part B) intervention 2: placebo capsule identical to the study drug

Protocol document:
Prot_SAP_000.pdf: 
 
 
 
 
 
 
 
Effects of Alpha-1 Antagonists, 
Stress and Relaxation on Anal 
Pressures 
NCT 01834729  
June 20, 2016 


 
 
1
Title of the study 
Effects of Alpha-1 Antagonists, Stress and Relaxation on Anal 
Pressures 
Principal investigator 
Adil  E. Bharucha – Gastroenterology  
Co-Investigators 
Kim J. Bailey - Psychology 
Amir Lerman and Rebecca (Becky) Nelson - Cardiology 
Jessica Noelting, MD, Internal Medicine 
Christine Kirt - Gastroenterology 
Michael J. Zaccarielo - Psychology 
Alan Zinsmeister – Biostatistics 
Michael Camilleri, MD – Gastroenterology 
David. O. Prichard – Gastroenterology 
Gopanandan Parthasarathy – Gastroenterology 
Subhankar Chakraborty – Gastroenterology 
Pratyusha Tirumanisetty - Gastroenterology 
 
Coordinator 
Kelly Feuerhak 
 
Abstract 
Modification on 6/20/16 shown in blue  
The sympathetic nervous system provides tonic excitatory input mediated by 
adrenergic α1 receptors to the internal anal sphincter. Defecatory disorders (DD) are 
attributed to inadequate rectal propulsive forces and/or impaired anal/pelvic floor relaxation 
during defecation. Anecdotally, many patients with DD have anxiety, but the effects of 
anxiety on anal sphincter pressures are unknown. DD are ideally managed by pelvic floor 
retraining (biofeedback therapy). However, a majority of patients with DD do not have 
access to biofeedback therapy and at best, 70% of patients respond to biofeedback 
therapy.  There are no pharmacological options for DD. 
This proposal will assess the contribution of adrenergic α1 receptors and separately 
anxiety/relaxation to anal pressures in healthy and DD.  The major objectives are to (1) 
study the effects of adrenergic α1A
 receptor antagonists on anal resting pressure, with the 
intent of developing pharmacological approaches to manage DD in future, (2) evaluate the 
effects of anxiety and mental relaxation on anal pressures, and (3) compare rectal 
compliance in DD and healthy subjects.  Our overall hypothesis is that anxiety increases 
while mental relaxation reduces anal resting pressure via α1 adrenoreceptor-mediated 
increased and reduced sympathetic tone respectively.  The specific aims of this study are 
to evaluate the following hypotheses in healthy subjects and patients with DD: (i) α1
 
receptors contribute to anal resting pressure but not the squeeze pressure increment; (ii) 
Mental stress will increase anal resting but not squeeze pressure via α1 adrenoreceptors; 
(iii) Mental relaxation will reduce anal resting pressure via α1 adrenoreceptors; hence the 
α1 adrenoreceptor antagonist will attenuate the reduction in anal resting pressure resulting 
from mental relaxation; (iv) Rectal compliance is higher and the contractile response to 
sinusoidal oscillation is lower in DD than in healthy people; and (v) Alfuzosin will improve 
bowel symptoms in chronic constipation.  
Anal resting and squeeze pressures will be evaluated at baseline, after 
randomization to the highly selective α1 adrenoreceptor antagonist alfuzosin (2.5 mg) or 


 
 
2
placebo and finally after randomization to active stress or mental relaxation in 36 healthy 
women and 36 female patients with defecatory disorders.  Thereafter, bowel habits will be 
recorded at baseline (2 weeks) then during treatment with placebo or alfuzosin ER 
(Uroxatral™ 10 mg daily) for 2 weeks.  The most significant anticipated findings are that 
alfuzosin and mental relaxation will reduce anal resting pressures, thereby providing 
additional options for patients with DD.  


 
 
3
A. Specific Aims 
The anorectum maintains fecal continence and is responsible for defecation 1.  
Defecatory disorders (DD) result from inadequate rectal propulsive forces and/or impaired 
anal/pelvic floor relaxation during defecation 2.  Pelvic floor retraining by biofeedback 
therapy is the cornerstone for managing defecatory disorders.  However, in the United 
States, pelvic floor retraining is only available at a handful of centers and not covered by 
many insurance programs.  A substantial proportion of patients with defecatory disorders 
do not respond to pelvic floor retraining.  Hence, additional options for managing DD are 
necessary.  High anal pressures at rest and during defecation may impair rectal evacuation 
in DD.  Conceptually, increased anal resting pressure may be attributed to the internal or 
external anal sphincters, which are responsible for approximately 70% and 30% of anal 
resting tone respectively.  In humans, monkeys and dogs, the internal sphincter is 
functionally innervated by sympathetic motor nerves.  Ongoing sympathetic activity may 
contribute to as much as 50% basal anal pressure 3,4 predominantly via α1 
adrenoreceptors.  Moreover, in vitro studies suggest that stimulation of sympathetic nerves 
to the monkey IAS can double spontaneous contraction 5.   
Similar to other functional GI disorders, many patients with defecatory disorders have 
anxiety.  However, the effects of anxiety on anal pressures have not been evaluated. We 
postulate that anxiety may increase anal resting pressure via sympathetic stimulation of the 
internal sphincter and/or predispose to conscious or subconscious voluntary contraction of 
the external anal sphincter, which may also increase resting tone.  Hence, our overall 
hypothesis is that anxiety increases anal resting pressure via α1 adrenoreceptors while 
mental relaxation reduces anal resting pressure.  α1- adrenergic stimulation also affects 
hypothalamic stress-mediated release of CRF 6 and the startle response, possibly via CRF 
release from brainstem nuclei mediating startle 7.  Indeed, prazosin reverses the excessive 
startle response induced by alpha-1 agonists  8.  In this study, we propose to use the α1- 
adrenergic antagonist alfuzosin which is highly selective for urological tissues relative to 
the prostate, has a low risk of orthostatic hypotension and penetrates the brain poorly.     
Finally, rectal contraction contributes to the desire to defecate. There is limited 
evidence, more in adolescents than in adults, that chronic constipation is associated with 
increased rectal compliance. Increased rectal compliance is associated with rectal 
hyposensitivity 9.  In children and adolescents, increased rectal compliance is associated 
with severe symptoms but does not predict treatment failure 10, 11.  However, it is unclear if 
increased rectal compliance causes or is consequent to chronic constipation or if increased 
rectal compliance reflects reduced rectal contractile response to distention.  We now 
propose to explore this question by using approaches that we have extensively utilized to 
evaluate rectal pressure-volume relationships in fecal incontinence i.e., distention with a 
barostat and separately with sinusoidal oscillation 12-14.  
Psychological factors such as stress or depression are known to influence the natural 
history of chronic gastrointestinal illnesses such as inflammatory bowel disease (IBD) [15] 
and irritable bowel syndrome (IBS) [16] via the gut–brain axis. These conditions, and also 
constipation are also associated with dysbiosis. [17] Furthermore, the gut microbiota-brain 
axis appears to be bidirectional.[18] It is thus important to also evaluate the gut microbial 
profile in patients and controls to identify potential interactions between gut microbiota and 


 
 
4
the response to stress, in health and in constipation. We propose to assess this using 16s 
rRNA analysis of stool samples.     
This study will evaluate the following specific aims in healthy subjects and in patients 
with defecatory disorders: 
 
1) To assess the effects of a highly-selective α1A
 receptor antagonist alfuzosin on anal 
resting pressure, squeeze increment, and rectoanal gradient during simulated evacuation.  
Our hypothesis is that α1
 receptors contribute to anal resting pressure but not the squeeze 
pressure increment in healthy people and defecatory disorders.   
 
2) To study the effects of mental stress on anal resting and squeeze pressures via α1 
adrenoreceptors.  Our hypothesis is that mental stress will increase anal resting but not 
squeeze pressure via α1 adrenoreceptors in healthy people and defecatory disorders.  
 
3) To evaluate the effects of active mental relaxation on anal resting and squeeze 
pressures.  Our hypotheses is that mental relaxation will reduce anal resting pressure via 
α1 adrenoreceptors; hence the α1 adrenoreceptor antagonist will attenuate the reduction in 
anal resting pressure resulting from mental relaxation in healthy people and defecatory 
disorders.  
 
4) To compare rectal compliance and the response to sinusoidal oscillation.  Our 
hypotheses are that rectal compliance is higher and the contractile response to sinusoidal 
oscillation is lower in defecatory disorders than in healthy people.  
 
5) To explore the effects alfuzosin on bowel symptoms in patients with chronic 
constipation.  Our hypotheses are that alfuzosin will improve bowel symptoms in chronic 
constipation.  
 
6) To evaluate the role of microbiota in the relationship between stress/relaxation 
and defecatory mechanisms. Our hypothesis is that the susceptibility to stress is 
associated with differences in fecal microbiota. B. Research Design and Methods 
This study comprises 2 parts.  Part A will compare the effects of oral alfuzosin (IR 2.5 
mg) and placebo on anorectal functions on 1 day in all subjects (controls and patients).  
Part B will compare the effects of oral alfuzosin ( 10 mg ER) and placebo on bowel habits 
in constipated patients (but not in healthy subjects).  36 healthy women and 36 women with 
defecatory disorders will be enrolled in this study.  This protocol is limited to women only 
because the vast majority of patients with constipation and defecatory disorders in our 
practice are women.  
 
1. Human subjects.  
a)  Inclusion Criteria for Controls (Part A only) 
i. Healthy women volunteers aged 18-80 years  
ii. Able to provide written informed consent before participating in the study 


 
 
5  
iii. A bl e t o c o m m u ni c at e a d e q u at el y wit h t h e i n v e sti g at or a n d t o c o m pl y wit h t h e 
r e q uir e m e nt s f or t h e e ntir e st u d y. 
 
b)  I n cl u si o n Crit eri a f or P ati e nt s  ( w o m e n a g e d 1 8-8 0 y e ar s)  ( P art s A a n d B) 
i. C o n sti p ati o n.  W o m e n wit h c hr o ni c c o n sti p ati o n f or 1 y e ar wit h a n y 2 or m or e of 
t h e f oll o wi n g s y m pt o m s f or 3 m o nt h s or l o n g er, i. e. < 3 b o w el m oti o n s/ w e e k, 
str ai ni n g ≥ 2 5 % of ti m e, h ar d or l u m p y st o ol s ≥ 2 5 % of ti m e, i n c o m pl et e 
e v a c u ati o n ≥ 2 5 % of ti m e, f e eli n g of a n or e ct al bl o c k a g e  ≥ 2 5 % of ti m e . 
ii. A bl e t o pr o vi d e writt e n i nf or m e d c o n s e nt b ef or e p arti ci p ati n g i n t h e st u d y  
iii. A bl e t o c o m m u ni c at e a d e q u at el y wit h t h e i n v e sti g at or a n d t o c o m pl y wit h t h e 
r e q uir e m e nt s f or t h e e ntir e st u d y. 
 
c )  E x cl u si o n Crit eri a f or C o ntr ol s ; It e m s i n di c at e d wit h a n a st eri s k (*) ar e al s o  e x cl u si o n 
crit eri a f or p ati e nt s  
i. Cli ni c al e vi d e n c e of si g nifi c a nt c ar di o v a s c ul ar, r e s pir at or y, r e n al, h e p ati c, 
g a str oi nt e sti n al, h e m at ol o gi c al, n e ur ol o gi c al, p s y c hi atri c or ot h er di s e a s e t h at 
m a y i nt erf er e wit h t h e o bj e cti v e s of t h e st u d y a n d/ or p o s e s af et y c o n c er n s. *   
ii. C urr e nt s y m pt o m ati c ort h o st ati c h y p ot e n si o n or hi st or y of h y p ot e n si v e r e s p o n s e 
a s d efi n e d b y a r e d u cti o n of ≥ 3 0 m m H g i n s y st oli c or ≥ 2 0 m m H g i n di a st oli c 
B P. * 
iii. C urr e nt s y m pt o m s of a f u n cti o n al g a str oi nt e sti n al di s or d er a s s e s s e d b y 
q u e sti o n n air e 1 5 , 1 6   
i v. P ut ati v e ri s k f a ct or s f or p el vi c fl o or tr a u m a, i. e. si x or m or e v a gi n al d eli v eri e s, 
birt h w ei g ht > 4 5 0 0 g m s ( m a cr o s o mi a), or k n o w n 4t h d e gr e e p eri n e al t e ar  
v.  I n ab ilit y t o wit h dr a w m e di c ati o n s pri or t o t h e b a s eli n e p eri o d a n d t hr o u g h o ut th e 
st u d y ( e x c e pt a s pr ot o c ol d efi n e d r e s c u e m e di c ati o n s):  
•  M e di c ati o n s t h at s u b st a nti all y alt er GI tr a n sit  * i n cl u di n g l a x ati v e s, m a g n e si u m 
a n d al u mi n u m c o nt ai ni n g a nt a ci d s, pr o ki n eti c s, er yt hr o m y ci n, n ar c oti c s, a nti -
c h oli n er gi c s, tri c y cli c a nti d e pr e s s a nt s, S N RI a n d n e w er a nti d e pr e s s a nt s  
•  S el e cti v e s er ot o ni n r e u pt a k e i n hi bit or ( S S RI) a nti d e pr e s s a nt s ar e p er mi s si b l e 
at l o w, st a bl e d o s e s. All m e di c ati o n s s h all b e r e vi e w e d a n d di s/ a p pr o v e d b y 
t h e pri n ci p al i n v e sti g at or o n a c a s e b y c a s e b a si s. * 
•  P ot e nt C y p 3 A 4 i n hi bit or s s u c h a s k et o c o n a z ol e, itr a c o n a z ol e a n d rit o n a vir, 
nitr at e s a n d p h o s p h o di e st er a s e i n hi bit or s  *  
N O T E:  st a bl e d o s e s of t h yr oi d r e pl a c e m e nt, e str o g e n r e pl a c e m e nt, l o w d o s e 
a s piri n f or c ar di o pr ot e cti o n, a n d birt h c o ntr ol ( b ut wit h a d e q u at e b a c k u p 
c o ntr a c e pti o n a s dr u g -i nt er a cti o n s wit h birt h c o ntr ol h a v e n ot b ee n 
c o n d u ct e d) ar e p er mi s si bl e. * 
vi.  St a bl e d o s e  of t h yr o xi n e will b e p er mitt e d  * 
vii.  Pr ol o n g e d Q - T c i nt er v al > 5 0 0 m s e c o n E C G wit hi n t h e l a st t hr e e m o nt h s * 
viii.  E sti m at e d gl o m er ul ar filtr ati o n r at e ( e G F R) < 6 0 m L/ mi n ut e . *  B a s e d o n 
g ui d eli n e s a n d r e c o m m e n d ati o n s fr o m t h e N ati o n al Ki d n e y Di s e a s e E d u c ati o n 
Pr o gr a m ( N K D E P) of t h e N ati o n al I n stit ut e s of H e alt h ( NI H) a n d t h e Ki d n e y 
Di s e a s e O ut c o m e s Q u alit y I niti ati v e ( K D O QI) of t h e N ati o n al Ki d n e y F o u n d ati o n , 
t h e a n e G F R u si n g t h e M o difi c ati o n of Di et i n R e n al Di s e a s e ( M D R D) St u d y 


 
 
6
equation is more accurate than a creatinine clearance calculated from serum 
and urine measurements.  The formula is eGFR (mL/min/1.73 m2) = 175 x (Scr)-
1.154 x (Age)-0.203 x (0.742 if female) x (1.210 if African American).  Based on 
our extensive experience in clinical practice and research studies, it is 
anticipated that all potentially eligible participants will have normal serum 
creatinine.  
ix. History of allergies to α1 adrenoreceptor antagonist * 
x. Active rectal inflammation, cancer; perianal sepsis; history of pelvic radiation, 
rectosigmoid surgery or inflammatory bowel disease*  
xi. Pregnant women, prisoners and institutionalized individuals* 
xii. Persons with a latex allergy.  
 
2. Overall experimental design.  Study Assessments are shown in Table 1.  During the 
screening visit, a clinical assessment , a medical history  and physical exam, including 
height, weight and “vital signs” (blood pressure, temperature, heart and breathing 
rates), Draw a blood sample, and test urine for pregnancy if you are a female able to 
become pregnant, along with, questionnaires (bowel questionnaire, Hospital Anxiety 
and Depression Scale 17, a health-status questionnaire 18), screening laboratory tests to 
exclude renal insufficiency, an ECG and a urine pregnancy test if necessary, will be 
completed.    
 
 
THE REST OF THIS PAGE IS INTENTIONALLY BLANK 


 
 
 
7
Table 1. 
Schedule for Study Assessments and Monitoring 
 
 
Screening 
Acute Study 
Bowel Symptoms (Alfuzosin – ER 10 mg) 
 
Days –30 to 1 
Day 1 
Baseline Period (Days 3-16) Treatment Period (Days 17-30) 
Procedure 
 
 
Daily  
Other 
Daily  
Other 
Informed consent 
X 
 
 
 
 
 
Review eligibility 
X 
 
 
 
 
 
Physical examination 
X 
 
 
 
 
 
Concomitant medication review 
X 
X 
 
 
 
 
Vital signs, weight  
X 
X1 
 
 
 
 
Serum creatinine, urine pregnancy test, 
ECG (12-lead) 
X 2 
 
 
 
 
 
Stool sample 
X 3 
 
 
X 3 
 
 
Anorectal study 
 
X 
 
 
 
 
Randomization to treatment  
 
X 
 
 
 
 
Study treatment administration 
 
X 
 
 
 
X3 
Adverse event assessment 
 
X 
 
X4  
 
X4 
Stool diary (Appendix 1) 
 
 
X 
 
X 
 
Abdominal pain (Appendix 2) 
 
 
X 
 
X 
 
Global assessments (relief, severity) 
(Appendix 3) 
 
 
 
Weekly 
 
Weekly 
Global assessment – satisfaction with 
treatment, (Appendix 4) 
X 
 
 
X (day 17) 
 
X (day 31) 
Other abdominal symptoms (Appendix 5) 
 
 
 
Weekly 
 
Weekly 
Telephone contact 
 
 
 
X 4  (q3-5 days) 
 
X 4 (q3-5 days) 
 
1. 
During the acute study, heart rate and blood pressure will be recorded at hourly intervals. Height (cm) and weight (kg) will be recorded at the 
screening visit but not on Day 1.   
2 
Urine pregnancy test to be performed for women of childbearing potential within 2 weeks of Day 1. Serum creatinine and ECG (12-lead) to 
have been done within three months of Day 1.   
3.  A single stool sample can be collected either before the acute study visit, or 3 days after the acute study visit, but before the treatment period. 


 
 
 
8
4. 
Patients will be treated with placebo or alfuzosin ER (10 mg) once daily during this phase. While the protocol is planned for the treatment 
period to follow directly the baseline period, if needed for scheduling, there can be up to a 5-day interval between the baseline and treatment 
period.   
5. 
Patients will be contacted by telephone every ~3-5 days between clinic visits to address questions, ascertain side effects and remind subjects 
to complete diaries.  The final phone call will be within 5 days after completing the treatment period. Subjects will be asked to return unused 
medication by mail. 


 
 
 
9
Figure 1. Experimental Design for Anorectal Study.  During the stress/relaxation epoch, 
subjects will receive 2 stress and 1 relaxation intervention in randomized order. The duration of 
this epoch includes two 10 minute rest periods between the 3 interventions. The manometry 
catheter will be removed and limited ambulation will be permitted while we are waiting for drug 
concentrations to increase.  Combined, this wait period and the second equilibration period are 70 
minutes long, which is within the range of tmax for alfuzosin (1.1 – 1.5 hours).  PAT = peripheral 
arterial tonometry   
 
Wait for drug effect
Drug or placebo
0           50        100      150        200       250      300
350    
Minutes
Equilibration
Pre Stress
Stress & relaxation
Rectal compliance
PAT
Wait for drug effect
Drug or placebo
0           50        100      150        200       250      300
350    
Minutes
Equilibration
Pre Stress
Stress & relaxation
Rectal compliance
PAT
3. Experimental procedures.  Items a-d below exclusively pertain to Part A, 
a) Anorectal sensorimotor functions.  After rectal cleansing with 1-2 sodium phosphate 
(Fleets,C.B. Fleet, Lynchburg, VA) enemas, anorectal resting and squeeze pressures 
and  rectal compliance and sensation will be evaluated before and after randomization 
to alfuzosin (2.5 mg oral) or placebo (Figure 1) in the Charlton 7 CRU. During each 
epoch (i.e., baseline or drug), pressures will be evaluated before and after patients are 
randomized to one of 6 sequences (orders) of 3 interventions (i.e., 2 stressors and 
active relaxation).  The same stressor/relaxation sequence will be applied during pre-
drug and post drug epochs. Anorectal assessments, which will be performed with 
standard techniques, include:  
i. Anal resting and squeeze pressures using high resolution manometry, similar to 
the procedure employed in the clinical lab and in recent studies 19;  
ii. Rectal compliance and sensation using a barostat/ polyethylene balloon.  A 
conditioning distention from 0-40 mmHg in 4 mm steps at 15 second intervals will 
be performed.  Ten minutes thereafter, the balloon will be distended from 0-44 
mmHg or maximum tolerated pressure in 4 mmHg steps; sensory thresholds for 
first sensation, desire to defecate, and urgency will be recorded 13, 20 
iii. Rectal ramp distention. Because the contractile response to distention is 
influenced by the rate of distention 20, a ramp distention to 200 mL over 1 minute 
will be performed; this will be sustained for 2 minutes.  While (ii) is useful for 
characterizing quasi-static pressure-volume relationships representing colonic 
tone, ramp distentions can uncover the active contractile response to distention. 
iv. Rectal sinusoidal oscillation will be performed at 5 cpm for 20 minutes after 
distention to 125 mL.  This paradigm is more sensitive than barostat-driven 
distentions for uncovering increased rectal stiffness in fecal incontinence 21 but 
has not been evaluated in constipation  


 
 
 
10
v. Rectal balloon expulsion will also be assessed in the seated position to 
confirm presence (or absence) of an evacuation disorder.     
b) Mental stressors and relaxation method. The mental stress testing protocol will be 
administered under the supervision of a licensed clinical psychologist who is familiar 
with these tests and routinely administers the same. Two stress interventions and a 
relaxation protocol will be provided in randomized order with 10-minute equilibration 
periods. Two different mental stress tasks of 6-minute duration will be performed. Each 
test is designed to address a different mental skill set. This was designed specifically to 
prevent mental fatigue and keep subjects motivated. All tasks will have intra-test levels 
of varying difficulty and will be externally paced, with the goal of stressing subjects as 
much as tolerated as judged by the psychologist administering the tests. It is known that 
external task pacing accentuates the induced mental stress.22 The aim is to keep 
subjects engaged and focused. The tasks, which will be performed in randomized order 
are as follows: 
i. A computerized version of the Stroop word-color conflict.23,24  Stroop word-color 
conflict test consists of three colored words (red, blue, and green) displayed in 
random order on a computer screen. Each word could appear in either its own 
color (visually concordant), or in one of the other 2 colors (visually discordant). 
The subject’s task will be to enunciate the color of the word, not the actual word 
itself. A rectangular box will surround the stimulus color-word, and this task will 
also be externally paced, with the subject having to keep pace with the 
advancing box.    
ii. Number subtraction of increasing difficulty (1 digit from 2 digits up to 3 digits from 
3 digits) testing math skills.  
iii. Relaxation will be induced by asking patients to view a CD obtained from Mayo 
Patient education. In essence, this CD encourages patients to close their eyes, 
be comfortable, monitor their breathing, and relax the entire body. 
Before and at the end of each task, subjects will be asked to rate their stress and 
arousal on four 10 mm long visual analog scales labeled tired-energetic, peaceful-
tense, worried-relaxed, and active-drowsy 25.   
 
c) Blood pressure and heart rate measurements will be taken 30 seconds before, 2 
minutes into, and at the end of each 6-minute mental stress task.  Subjects will have 5-
minute baseline rest periods between each mental stress task, during which they will be 
instructed to relax. At the end of the mental stress tasks, the reactive hyperemia 
protocol will be repeated. Double product will be calculated as systolic blood pressure 
multiplied by heart rate.  
 
d) Peripheral arterial tonometry (PAT) will provide an independent biological marker of 
mental stress, which causes vasoconstriction, and the α1 antagonist, which causes 
vasodilatation. There is a characteristic PAT signal response to mental stress, with 
diminution of the signal amplitude during stress.  A ratio of the stress PAT amplitude to 
baseline PAT amplitude (PAT score) of 0.8 or less is considered a positive response to 
mental stress and considered a marker for future stress-related cardiovascular events.26  
 


 
 
 
11
e) Drugs.  After carefully reviewing the pharmacokinetic properties and side effects 
of all α1 adrenoreceptor antagonists, we have opted to use alfuzosin, because it (i) is 
highly selective for urological tissues relative to the vascular system, (ii) has a low risk 
of orthostatic hypotension and (iii) penetrates the brain poorly 27, 28.  For example, in 
isolated human tissues, alfuzosin displays the highest selectivity ratio for the prostate 
over the vascular tissue (ratio, 144) compared with tamsulosin (90), doxazosin (51), and 
terazosin (19) .  In experimental animal models, the primary effect of alfuzosin is a 
urethral relaxant activity and not an antihypertensive effect.  Administered intravenously 
to conscious normotensive rats at doses 3 to 10 times higher than those necessary to 
induce significant urethral relaxation, alfuzosin displays the lowest hypotensive activity 
compared with doxazosin, terazosin, and tamsulosin. 
Alfuzosin (2.5 mg) has a mean bioavailability of 64%, tmax of 1.0 – 1.5 hours and 
a terminal thalf of 3-5 hours 27, 28. The drug is extensively metabolized by the liver with 
only 11% of the administered dose excreted unchanged in the urine. This immediate 
release formulation, which is also manufactured by Sanofi-Aventis, not approved in the 
US but widely available in other countries, will be imported from UK (Xatral™, 2.5 mg) 
under an investigator-initiated IND (117098) from the FDA because the long tmax for 
alfuzosin ER limits its use in this acute study.   
f) Stool collection.  Using stool kits and standardized instructions, patients will collect 
stool samples according to the procedure in the Appendix.  Stool samples will be frozen 
and stored in a -20°C freezer.  The first stool specimen will be collected (a) without a 
laxative in controls and (b) if necessary, after their usual laxative regimen in patients.  
In Part B, the effects of alfuzosin extended-release (10 mg, Uroxatral) on bowel 
symptoms will be evaluated.  Since such use satisfies the requirements of 21 CFR 312.2 
(b) (1), an IND is not required.  We confirm that all the following apply to Part B of this 
study:  
a) 
The investigation is not intended to be reported to FDA as a well-controlled study in 
support of a new indication for use nor intended to be used to support any other significant 
change in the labeling for the drug; and  
b) 
The investigation is not intended to support a significant change in the advertising 
for the product; and  
c) 
The investigation does not involve a route of administration or dosage level or use in 
a patient population or other factor that significantly increases the risks (or decreases the 
acceptability of the risks) associated with the use of the drug product; and  
d) 
The investigation will be conducted in compliance with the requirements for 
institutional review set forth in 21 CFR 56 Institutional Review Boards and with the 
requirements for informed consent set forth in part 21 CFR 50 Protection of Human 
Subjects; and  
e) 
The investigation will be conducted in compliance with the requirements of 21 CFR 
312.7 - Promotion of Investigational Drugs. 
 
f) Genome wide DNA analysis.  Towards our long-term objective of uncovering 
associations between SNPs and DD, we propose to extract DNA from 10 ml of blood, to 
be drawn from study participants. DNA extraction will be conducted at the BAP Lab.  
Genome-wide analysis will be conducted using Illumina 610 QUAD microarray or 


 
 
 
12
comparable approaches.  Genotype-phenotype correlations will be examined using 
these patients and other patients in ongoing studies. 
 
g) Sequencing and analytical methods.  Using resources available in the UIUC-Mayo 
consortium, two complementary approaches will be utilized to analyze stool specimens from 
each subject . First, microbial DNA will be extracted from stool and tissue samples using 
standardized methods [33].  Phylotype profiles of the microbiome from control and 
constipated 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 [34].  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 300 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. Paired end reads will be stitched, aligned, and classified 
using a custom pipeline (TORNADO v2.0).[35] Briefly, low base quality reads will be either 
trimmed or discarded,[36] and these reads will not be classified as a bacteria kingdom[37] or 
matched to the bacteria 16S rRNA secondary structure.[38] To evaluate the microbial 
diversity and abundance, UPARSE will be used for Operational Taxonomical Units (OTU) 
clustering,[39] and FastTree for phylogeny.[40] The 16S data will be clustered into OTUs at 
97% sequence similarity, and the taxonomy will be assigned using the Ribosomal Database 
Project classifier. h) Effects of alfuzosin in bowel habits (Part B).  During the first 2 weeks, 
bowel habits will be recorded at baseline.  During the next 2 weeks, patients will be treated 
(double-blind) with placebo or alfuzosin (10 mg) ER once daily, which is FDA-approved dose 
for urinary symptoms.  Symptom assessments are shown in Table 1 and in Appendices 1-5.  
Per eligibility criteria, subjects will discontinue medications which can affect GI transit during 
the baseline and treatment periods.  Patients who need rescue medication, after three full 
days without a bowel movement, can receive 1-2 bisacodyl 5 mg tabs or a bisacodyl 
suppository (10 mg) or up to Fleet’s enemas.  In addition, patients who need relief of mild 
pain can use analgesic drugs such as NSAIDs and COX-2 inhibitors for up to two doses per 
week.  During the procedure, intake of acetaminophen up to 1.5g per day in divided doses 
will be permissible. 
 
4. Data and statistical analysis  
a) Part A.  Anal manometry will be analyzed by established techniques and 
summarized as anal resting and squeeze pressures, rectal and anal pressure and 
rectoanal gradient during evacuation 19, 29.  Rectal pressure-volume relationships will be 
summarized by the Prhalf during rectal compliance, rectal capacity, pressure and volume 
sensory thresholds for first sensation, desire to defecate, and urgency 14.  During 


 
 
 
13
sinusoidal oscillation, average rectal pressures and the variability in pressures over 
20 min will be summarized 14.  
The analysis will be based on analysis of covariance (ANCOVA) models to assess 
the pre-dug period for intervention effects with subject status and intervention type as 
the primary factors (a check for order effects of the intervention sequences will also be 
made, and the corresponding pre-stress response will be included as a covariate). 
Models with a subject status by intervention type interaction will also be examined.  
During period 2, subjects will have been randomized to drug or placebo (balanced on 
subject status) and use the same intervention sequence as the pre-drug period). The 
analysis of the post drug period will also be based on ANCOVA models incorporating 
subject status, intervention type, treatment group, and corresponding interactions (a 
check for order effects will again be made). The corresponding responses from the pre-
stress interval from period 1 and VAS scores for stress and arousal will be used as 
covariates, 
b) 
Part B.  
i. Endpoints.  Symptoms and Signs of Chronic Constipation. The effects of 
alfuzosin compared to placebo will be assessed by evaluating  
• Bowel habits using  a daily diary  (Appendix 1) to record stool consistency 
(Bristol Stool Form scale), frequency, ease-of-passage and sensation of 
complete bowel emptying (yes/no)  
• Daily Abdominal Pain Scores (Appendix 2) collected as (a) most severe daily 
score and (b) overall average daily score).  
• Global Patient Treatment Assessments reporting outcomes on a weekly basis 
(Appendix 3) including overall relief and severity of constipation 
• Global Patient reported outcomes on a 2-week basis (Appendix 5) 
• Patient reported outcomes of other abdominal symptoms (Appendix 6)  
Two outcomes will be derived from primary data.  
- 
Weekly rate of SBMs:  a bowel movement (BM) is considered an SBM if no 
laxative, enema or suppository was taken in the preceding 24 hours. 
- 
Weekly rate of CSBMs:  if the patient indicated that the SBM was associated with 
a sensation of complete bowel emptying, the SBM will be counted as a CSBM.   
ii. Analysis.  Daily bowel diary responses will first be averaged over study periods 
(baseline and treatment) and in the case of stool frequency, per week.  These 
responses will also be assessed using ANCOVA models, with the corresponding 
baseline value as the primary covariate.  For stool frequency, the two weekly 
values will be assessed using a repeated measures ANCOVA model with the 
overall average baseline stool frequency value as a covariate.  The global 
assessment scores (adequate relief and severity) will first be summed over the 
two treatment weeks, and then assessed using the Wilcoxon rank sum test.  
Individual adverse events (AEs) will be summarized by treatment group and the 
overall total frequency (per subject) of AEs assessed using the Wilcoxon rank 
sum test.  The analyses will follow the intent to treat (ITT) paradigm with all 


 
 
 
14
patients randomized included in the analyses.  Patients with missing values for 
a particular endpoint will have their missing values imputed using the 
corresponding overall mean value from all patients with non-missing data for that 
endpoint.  An adjustment in the ANCOVA error degrees of freedom (subtracting 
one for each imputed value) will be made to account for the imputation.  A similar 
adjustment for the Wilcoxon rank sum test (adjusting the degrees of freedom for 
the t-approximation) will also be examined. 
 
5. Sample size assessment.  The sample size assessment is based on the variation in 
anal resting pressures in healthy subjects and patients with defecatory disorders and 
the effect of the α1 receptor antagonist (indoramin) on anal resting pressure in a 
previous study 30.  The CV (%) in anal resting pressure evaluated by the same 
technique in healthy women (<50 years old) and patients with defecatory disorders (<50 
years old and resting pressures >90) was 20% (mean=93.7{SD=18.5}) and 11% 
(mean=102.9{SD=11.7}), respectively. In period 2 the sample size of 36 controls and 36 
patients will yield 18 subjects per treatment arm (drug vs. placebo) in each subject 
group (control vs. patients). This should provide 80% power to detect a treatment effect 
size of roughly 15-20%, based on a two sample t-test with a two-sided alpha level of 
0.05 separately in each subject group.  The effect size is defined as the difference in 
group means as a percentage of the overall (both groups) mean value. The previous 
study suggested an effect size of 47% for drug effects 30.  For part B, data pooled from 
constipation subjects imply differences between treatment groups of ~4 stools per 
week, differences in stool consistency scores of ~1.1 and differences in ease of 
passage scores of 0.8 could be detected with 80% power (2-sided alpha level of 0.05) 
31.  It is anticipated that the ANCOVA models will provide similar power for somewhat 
smaller differences by incorporating the baseline values as covariates.  
  
6. Anticipated results and interpretation.  We anticipate that: (i) alfuzosin will reduce anal 
resting pressure and increase the rectoanal gradient during simulated evacuation but 
not affect the squeeze increment; (ii) alfuzosin will attenuate the augmentation of anal 
pressure by mental stress; (iii) mental relaxation will reduce anal resting pressure to a 
greater extent after placebo than alfuzosin; and (iv) rectal compliance and the 
contractile response to sinusoidal oscillation will be lower in chronic constipation than in 
healthy subjects. 
 
7. Potential pitfalls and precautions taken.  The anorectal procedures to be used in this 
study are established in our laboratory. While the effects of alfuzosin on anal pressures 
are unknown, the dose used in this study is safe and relaxes the urethral sphincter to 
increase urinary flow. Peripheral arterial tonometry will provide an independent 
biological marker for effects of stress and alfuzosin.  We recognize the given the 
phenotypic variability in defecatory disorders 32, it is conceivable that only a subset of 
patients will respond to an α1 antagonist.  To maximize the likelihood of a response, 
patients with high anal resting pressure by digital rectal examination or manometry will 
be recruited for initial studies.  Established perturbations administered in a standardized 
format will be used to modulate arousal and anxiety and the effects of these 


 
 
 
15
perturbations will be evaluated by standardized and validated VAS instruments.  
While the sample size estimate is primarily designed to detect acute effects on 
anorectal functions, it is also sufficient to identify clinically-significant effects on bowel 
symptoms (eg, bowel symptoms) 31.   
 
8. Innovative aspects, significance, and future directions.  The concepts that mental stress 
can increase anal resting pressure via α-1 adrenergic receptors and the application of 
α-1 antagonists to manage these disorders are novel.  Alternative approaches to pelvic 
floor retraining, which is the cornerstone for managing defecatory disorders, are 
necessary because (i) many insurance programs do not cover pelvic floor retraining; 
and (ii) our experience suggests that a considerable proportion of patients do not 
respond to pelvic floor retraining in clinical practice.  If alfuzosin reduces anal resting 
pressures and increases the rectoanal gradient during defecation, further studies with 
the long-acting formulation, either alone or as an adjunct to biofeedback therapy, will be 
considered.  Likewise, further studies to evaluate the effects of mental relaxation on 
symptoms and to understand the extent to which the effects of biofeedback therapy are 
mediated by mental relaxation in patients with DD may be considered.  Conceivably, 
the response to these approaches may vary among patients and the acute response to 
these perturbations may predict response to chronic therapy.  
 
C.  Human Subjects   
1.  
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 potential risks of studies among 
other items.  The anorectal assessments are extremely safe.  Subjects may experience 
the urge to defecate during rectal distention.  Consistent with all our studies, distention 
will be terminated if patients experience severe discomfort.  The listed side effects of 
alfuzosin include faintness/dizziness, vertigo, malaise, headache, hypotension 
(postural), nausea, abdominal pain, diarrhea, dry mouth, and asthenia.  Hypotension is 
a listed side effect.  However, even with chronic administration, the risk of hypotension 
is low. For example, in a study comparing alfuzosin 2.5 mg immediate release tid with 5 
mg sustained-release twice daily in 2442 patients, the incidence of hypotension at 1 
year was 1.6%; there were no clinically significant changes in systolic and diastolic 
blood pressure or heart rate between baseline and 24 months, i.e., -3.1mmHg, -
1.4mmHg, and - 0.05 bpm, respectively 33. In the largest experience of 13,389 patients 
(average age 67 years old) treated with alfuzosin 2.5 mg tid for benign prostatic 
hypertrophy for 3 months, two-thirds of the adverse events leading to discontinuation 
were vasodilatory and occurred in 2.7% of the patients: vertigo/dizziness (1.4%); 
malaise (0.6%); hypotension (0.4%), and headache (0.4%) 34.  While alfuzosin ER is 
approved by the FDA, the 2.5 mg immediate release formulation is not.  Hence, this 
formulation will be imported from Europe after obtaining an IND from the FDA. 
 
2. Data and Safety Monitoring Plan (DSMP)  


 
 
 
16
a) 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.  
Study procedures are conducted by trained and experienced personnel. Interventions 
(alfuzosin and anorectal tests) pose a low risk to participants. Monitoring will be 
performed during and after the study by CRU RNs and the Study Coordinator.  The PI 
will also review side effects. UPIRTSOs and non-UPIRTSOs will be reported per 
protocol to IRB and FDA (IND required for alfuzosin).  Each study is reviewed and 
analyzed; study discussions and communications are generally by e-mail and stored.  
Study will be stopped if any rectal perforation occurs.  
b) 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 6 studies.  
c) Subject Privacy. Subjects will be consented in a private room. Adequate time for 
questions will be provided.   
d) Data Confidentiality. All records are maintained in password protected electronic 
files. Hard copies are filed in private offices.  
e) Product Accountability. Research Pharmacy will obtain and dispense medication. 
Unused medication will be collected from subjects at the end of the study and 
destroyed.  
f) Study Documentation. Documentation will be per established guidelines during the 
study (hard copy and electronic data capture).  These files will be sampled annually. 
g) Study Coordination. Annual debriefing will be conducted to ensure expectations are 
clear and if educational needs exist. 
 
 
 
 


 
 
 
17
 
 
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Collins, S.M., M. Surette, and P. Bercik, The interplay between the intestinal microbiota and 
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1480-91. 
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Bharucha, A.E., et al., Functional Anorectal Disorders. Gastroenterology, 2006. 130: p. 
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Scoring Comparisons and Reference Data. . Health Outcomes Institute, Bloomington, MN., 
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Women: Effects Of Age And Significance Of Rectoanal Gradient. American Journal of 
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Andrews, C., et al., Rectal sensorimotor dysfunction in women with fecal incontinence. 
American Journal of Physiology - Gastrointestinal & Liver Physiology, 2007. 292(1): p. 
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Bharucha, A.E., J. Edge, and A.R. Zinsmeister, Do myogenic mechanisms explain increased 
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1): p. A 112. 
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35. 
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sequence data. Bioinformatics, 2014. 30(15): p. 2114-20. 
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Bioinformatics, 2009. 25(10): p. 1335-7. 
39. 
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Disorders Using High Resolution Anorectal Manometry Gastroenterology, 2013. In press. 
42. 
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43. 
Zinsmeister, A., D. Burton, and M. Camilleri, Pharmacodynamic and clinical endpoints for 
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Lukacs, B., J.C. Grange, and D. Comet, One-year follow-up of 2829 patients with moderate 
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20
Appendix 1.    Sample Stool Diary Card 
 


 
 
 
21
Appendix 2.    Daily Diary:  Abdominal Pain Questionnaire 
 
 
None 
 
0 
Very Mild 
 
1 
Mild 
 
2 
Moderate 
 
3 
Moderatel
y Severe 
4 
Severe 
 
5 
Very 
Severe 
6 
1. Describe your most severe abdominal pain in 
the last 24 hours? 
□ 
□ 
□ 
□ 
□ 
□ 
□ 
2. Describe your overall average abdominal 
pain in the last 24 hours? 
□ 
□ 
□ 
□ 
□ 
□ 
□ 
 


MAYO CONFIDENTIAL 
Appendix 3.    Weekly Global Constipation Questionnaire 
 
- 
Global Patient reported outcomes on a weekly basis: 
- 
Overall relief of constipation  
- 
Severity of constipation  
 
 
Completely 
Relieved 
0 
Considerably 
Relieved 
1 
Somewhat 
Relieved 
2 
Unchanged 
3 
Somewhat 
Worse 
4 
Conside
Worse 
5 
1. 
Over the last week, 
describe the overall relief 
of your constipation 
symptoms? 
□ 
□ 
□ 
□ 
□ 
□ 
 
 
 
 
No 
Symptoms 
0 
Very Mild 
 
1 
Mild 
 
2 
Moderate 
 
3 
Moderately 
Severe 
4 
Sev
 
5 
2. 
Over the last week, describe 
the overall severity of your 
constipation symptoms? 
□ 
□ 
□ 
□ 
□ 
□ 
 


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Appendix 4.    Bi-Weekly Global Satisfaction with Constipation Treatment 
Questionnaire (adapted from PAC-QoL Question 28) 
 
- 
The following question is designed to measure the impact constipation has had 
on your daily life during the past two weeks.  For this question, please tick one 
box. 
 
 
This question asks about how 
satisfied you are. 
Not at all 
 
0 
A little bit 
 
1 
Moderately 
 
2 
Quite a 
bit 
3 
Extremely 
 
4 
1. To what extent, during the past 2 
weeks, have you been satisfied 
with your treatment? 
□ 
□ 
□ 
□ 
□ 
 


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Appendix 5.    Patient Reported Outcomes for Other Abdominal 
Symptoms 
 
INSTRUCTIONS: The questions in this diary are designed to measure the severity of 
various abdominal symptoms. Please complete the diary each week as directed, 
thinking about the 24-hours prior to completion of the diary on a particular day   
For each question, please choose the one response that best describes your 
experiences during the past 24 hours. 
1. 
How would you rate your bloating at its worst in the past 24 hours? 
0 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
No 
bloating 
 
 
 
 
 
 
 
 
 
 
Worst 
possible 
bloating 
2. 
How would you rate your abdominal pain at its worst in the past 24 hours? 
0 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
No 
abdominal 
pain 
 
 
 
 
 
 
 
 
 
Worst 
possible 
abdominal 
pain 
3. 
How would you rate your nausea at its worst in the past 24 hours? 
0 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
No nausea 
 
 
 
 
 
 
 
 
 
 
Worst 
possible 
nausea 
 
4a. 
How would you rate your vomiting at its worst in the past 24 hours? 
0 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
No 
vomiting 
 
 
 
 
 
 
 
 
 
 
Worst 
possible 
vomiting 
 
4b. 
How many times have you vomited in the past 24 hours? 
_____________  times (If no vomiting, enter 0) 
 
 
 
 
 
 
 
 


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APPENDIX 6 
 
Stool Specimen Collection Instructions 
 
Attaching the collection container 
1. Open the kit 
 
2. Lay the collection container on a flat surface with the labeled side facing 
up.  Fold up the two cardboard sides (Figure 1). 
 
 
 
3. Remove the backing from the tape on each of the cardboard sides. 
 
4. Insert the collection container into the toilet bowl and attach the tape to 
the top of the toilet seat toward the back half of the bowl (Figure 2).  The 
cardboard sides should be up against the bottom of the toilet seat. 
 
  
5. Shape the paper dish (middle part of the collection container) into a bowl 
by gently pushing down the center. 
 
Collection a sample 
1. Do not urinate into the collection container.  (You may wish to urinate 
before attaching the collection container to the toilet seat.) 
 
2. Have a bowel movement into the paper dish 
 
3. Take out the collection tube and unscrew the cap.  Use the spoon 
attached to the cap to scoop a marble-sized sample.  Insert filled spoon 


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back into the tube and tightly screw the camp onto the collection tube 
(Figure 3). 
 
 
 
Disposal 
1. Remove the paper dish holding the stool by gently lifting up the four 
attachment sites.  Flush the paper dish and stool. 
 
2. Remove the cardboard frame from the toilet seat and discard it in the 
wastebasket. 
 
3. Wash your hands thoroughly with soap and water. 
 
 
Returning the sample to Mayo Clinic 
1. Be sure the collection tube cap is tightly fastened. 
 
2. Place the collection tube containing your sample into the small white bag. 
 
 
 
 
If you are at Mayo Clinic: 
At your earliest convenience, return the white bag containing your specimen 
to: Station S/Specimen Collection Cart 
Monday through Friday, 7 a.m. to 5 p.m. 
 
If you are mailing your stool specimen to Mayo Clinic: 
At your earliest convenience, mail your specimen to Mayo Clinic using the 
prepaid mailer provided.