Multistrain Probiotic for Functional Constipation

Primary Endpoint: Whole gut transit time

The primary endpoint of this clinical trial is whole gut transit time, which will be
assessed using abdominal x-rays (supine anteroposterior kidney-ureter-bladder, KUB) on days
0 and 42. Each subject will ingest 24 radiopaque Sitz markers each day for 6 consecutive
days at the same time of the day prior to abdominal x-rays on days 0 and 42, also taken at
the same time of the day that the markers were taken. The number of markers present in the
right, left, and rectosigmoid colon will be summed to yield a total marker count. Whole gut
transit time will be calculated using the classic film estimate as described in the
following formula:

WGTT = ni x (t/N) 19, 20

where ni is the number of markers observed on x-ray, t is the time between marker ingestions
in hours, and N is the total number of markers ingested each day. Thus, in this study, t/N
equals 1 (24 markers per capsule/24 hours between marker ingestions), and WGTT is,
therefore, equal to the total marker count 19, 20.

Marker counts will be identified by a single radiologist at each site who will remain
blinded to subject treatment assignment.

The methodology above is safe, noninvasive, and the two abdominal x-rays required for this
study collectively expose each subject to radiation doses of approximately 140 millirem
(equivalent to 1.4 millisievert). The overall lifetime risk that this radiation dosage may
cause cancer is approximately 1 in 4,000 for women aged 18 years, 1 in 6,000 for men aged 18
years, 1 in 20,000 for women aged 70 years, and 1 in 25,000 for men aged 70 years. Similar
abdominal x-ray methodology has been utilized in similar studies in adults aged 18 years and
older 21, 22.

Secondary Endpoint: Patient Assessment of Constipation Quality of Life (PAC-QoL) The PAC-QoL
(Appendix A) is a 28-question survey that measures the impact that constipation has on daily
life over the past 2 weeks. The questions are comprised of four subscales (worries and
concerns, physical discomfort, psychosocial discomfort, and satisfaction) and an overall
scale. Multinational studies have demonstrated that the PAC-QoL is internally consistent,
reproducible, valid, and responsive to improvements over time 23. Subjects will complete the
PAC-QoL at days 0 and 42.

Secondary Endpoint: Wexner Constipation Score (WCS) The Wexner Constipation Score (WCS)
(Appendix B) is an 8-question survey that assesses frequency of bowel movements, straining,
incomplete evacuation, abdominal pain, time needed for defecation, assistance for
defecation, unsuccessful attempts, and duration of constipation. The WCS is a validated
questionnaire since WCS scores correlate well with objective physiologic findings in
constipated patients 24. Subjects will complete the WCS at days 0 and 42.

Secondary Endpoint: Gastrointestinal Symptom Rating Scale (GSRS) The GSRS (Appendix C) is a
15-item instrument designed to assess common GI symptoms 25. It has five subscales (reflux,
diarrhea, constipation, indigestion, and abdominal pain) with subscale scores ranging from 1
(no discomfort) to 7 (severe discomfort). Higher scores represent higher symptom burden.
Subjects will complete the GSRS at days 0 and 42.

Secondary Endpoint: Stool frequency Throughout the study, subjects will record the number of
defecations per day in a diary throughout the study. The stool frequency endpoint will be
the absolute change in weekly stools in each group from the second week of the run-in period
to the sixth study week.

Secondary Endpoint: Stool Consistency Stool consistency will be rated with the Bristol Stool
Scale Form 26 (Appendix D). Subjects will grade each stool using a daily diary throughout
the study. The stool consistency endpoint will be the mean change in each group from the
second week of the run-in period to the sixth study week.

Secondary Endpoint: Fecal Probiotic Count Stool samples will be collected at baseline and
end of study to assess fecal probiotic count via live culture, which will include total
lactobacillis count and total bifidobacterium count. At the Day 0 visit following
randomization, subjects will be provided two separate stool collection kits—one to be used
at baseline and one to be used at the end of the supplementation period.

After receipt of the kits, subjects must provide the first stool sample before taking the
first dose of study product, regardless of the length of time needed to have a bowel
movement. The stool sample can be provided at the study site on Day 0 or the subject may
collect their first stool thereafter and ship the sample in a provided refrigerated
container to the laboratory for analysis. Once the first stool sample is provided, the
subject may then begin consuming study product.

During the final 3 days of the supplementation period (i.e. Day 40, 41, or 42), subjects
must provide another stool sample and ship the sample in a provided refrigerated container
to the laboratory for analysis. This must be completed before returning to the site for the
Day 42 assessments.

Secondary Endpoint: Adverse Events Adverse events (AEs) will be assessed throughout this
clinical study. The main safety endpoint will be the proportion of subjects in each group
that report one of more AEs at any time during the study. Please see Section 6 for
additional details on the AE assessment.

Ancillary Outcome: International Physical Activity Questionnaire (IPAQ)-short version The
International Physical Activity Questionnaire (IPAQ)-short version is a validated
questionnaire suitable for quantifying physical activity levels in adults (Appendix E) 27.

Ancillary Outcome: 24-hour Food Recall A 24-hour food recall will be completed in order to
quantify total calories, carbohydrate (g), fat (g), protein (g), fiber (g), and liquid
intake (ml). The food recall must take place on a weekday (Monday through Thursday).
Subjects must record all foods and beverages consumed as well as the quantity of each
(recorded as weight or volume).

Ancillary Outcome: Concomitant medication use Throughout the study, subjects will record use
of any concomitant medication and, if required, the need for rescue medication each day in a
diary.

Inclusion Criteria:

1. Age 18 to 65 years

2. Body mass index 18.5 to 39.9 kg/m2

3. Meets the criteria below over the last 3 months with symptom onset at least 6 months
prior (Rome III definition of functional constipation 1):

- Meets 2 or more of the following criteria:

- Straining during at least 25% of defecations

- Lumpy or hard stools in at least 25% of defecations

- Sensation of incomplete evacuation for at least 25% of defecations

- Sensation of anorectal obstruction/blockage for at least 25% of defecations

- Manual maneuvers to facilitate at least 25% of defecations (e.g. digital
evacuation, support of the pelvic floor)

- Fewer than three defecations per week

- Loose stools are rarely present without the use of laxatives

- Insufficient criteria for irritable bowel syndrome

4. Agree to use contraception throughout study period, unless postmenopausal or
surgically sterile (females only)

5. Able to understand the nature and purpose of the study including potential risks and
side effects

6. Willing to consent to study participation and to comply with study requirements

7. Successful completion of 2-week run-in period, defined as:

- Must meet the Rome III definition of functional constipation during this 2-week
period

- Completion of all study-related questionnaires

Exclusion Criteria:

1. Major gastrointestinal complication (e.g. Crohn's disease, ulcer, cancer)

2. Prior abdominal surgery that, in the investigator's opinion, may confound study
outcomes

3. Clinically significant systemic disease (e.g. cancer, diabetes, CAD)

4. Consumption of probiotics or prebiotics within 2 weeks of randomization

5. Antibiotic use within 4 weeks of randomization

6. Laxative or other constipation medication use within 2 weeks of randomization

7. Eating disorder

8. Known allergies to any substance in the study product, including lactose intolerance

9. Pregnant or breastfeeding women

10. History of alcohol, drug, or medication abuse

11. Participation in another study with any investigational product within 3 months of
randomization

12. Any condition that could, in the opinion of the investigator, preclude the subject's
ability to successfully and safely complete the study or that may confound study
outcomes