Safety, Tolerability, and Biosignature of Humanized Prebiotics in Healthy Adults
Phase: NA
Conditions: Intestinal Health
Sponsor: University of North Carolina, Chapel Hill

 
Safety, Tolerability, and Biosignature of Humanized Prebiotics in 
Healthy Adults 
 
 
NCT number NCT06068894 
Document Date 03/26/2024 
 

Interventional Study 
 
Study Title: Safety, Tolerability, and Biosignature of Humanized Prebiotics in Healthy Adults 
 
 
UNC Principal Investigator (PI): Sylvia Becker-Dreps 
UNC PI Department/Division: Family Medicine 
 
Number of Sites: 1 
Lead Site: N/A  
 
Lead Site Principal Investigator:  
Co-Investigator(s) and Department/Division:  
 
M. Andrea Azcarate-Peril, Associate Professor of Medicine, Division of Gastroenterology and 
Hepatology at the University of North Carolina in Chapel Hill  
 
Dr. Jose Bruno-Barcena, Professor of Plant and Microbial Biology at North Carolina State 
University 
Dr. Sarah McGill, Associate Professor of Medicine at the University of North Carolina in Chapel 
Hill  
Dr. Jeff Roach, Senior Scientific Research Associate at the University of North Carolina in Chapel 
Hill  
Dr. Kai Xai, Assistant Professor at the University of North Carolina in Chapel Hill  
 
 
 
Study Statistician: Kai Xia 
 
Sponsor(s): 
 
Name/Address: NC TraCS Institute 
Source(s) of Funding 
 
Name/Address: NC TraCS Institute 
 
I HAVE READ AND APPROVE THIS VERSION OF THE PROTOCOL.   
[electronic signature accepted]  
 
Principal Investigator:      
 
 
 
 
 
 
Date:  
 
 
 
Statistical Co-investigator:      
 
 
 
 
 
Date:  
 
 
 
 

Safety, Tolerability, and Biosignature of Humanized Prebiotics in Healthy Adults 
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Summary of Changes from Previous Version [If applicable]: N/A 
 
Previous  
Version No. 
Affected  
Section(s) 
Summary 
 of Revision(s) 
Reason  
for Change(s) 
6 
 
REDUCE VOLUME OF BLOOD COLLECTION TO 10ML; 
CHANGE TO VIRTUAL CONSENT PROCESS; UPDATED DIET 
HISTORY QUESTIONNAIRE PLANS AND MEAL 
JOURNALING. 
NO NEED FOR ADDITIONAL 
BLOOD; VIRTUAL CONSENT IS 
NEEDED DUE TO IDS 
REQUIREMENT TO PROVIDE 
SIGNED CONSENT IN 
ADVANCE OF DISPENSING 
NUTRITIONAL SUPPLEMENT 
7 
 
1. CHANGE GASTROINTESTINAL SYMPTOM SCALE FROM 
GSSC TO PROMIS 
2. INCLUSION AND EXCLUSION CRITERIA WERE MADE 
CONSISTENT THROUGHOUT DOCUMENT AND ADDED 
SEVERE/PERSISTENT SYMPTOMS FROM THE PROMIS 
ASSESSMENT. 
PROMIS IS A BETTER 
EVIDENCE-BASED TOOL FOR 
ASSESSMENT OF 
GASTROINTESTINAL 
SYMPTOMS (SPIEGEL, ET AL. 
AM J GASTROENTEROL. 
2014) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Table of Contents 
 
Statement of Compliance .................................................................................................................................... 6 
Abbreviations and Definitions of Terms .............................................................................................................. 7 
1. 
Protocol Synopsis ..................................................................................................................................... 8 
2. 
Introduction: Background and Scientific Rationale ................................................................................ 9 
2.1. 
Background Information ...................................................................................................................... 9 
2.2. 
Supporting Pilot / Unpublished Data ................................................................................................. 10 
2.3. 
Scientific Rationale ............................................................................................................................. 11 
3. 
Objectives .............................................................................................................................................. 12 
3.1. 
Specific Aim 1 ..................................................................................................................................... 12 
3.2. 
Specific Aim 2 ..................................................................................................................................... 12 
3.3. 
Specific Aim 3 ........................................................................................ Error! Bookmark not defined. 
3.4. 
Specific Aim 4 ........................................................................................ Error! Bookmark not defined. 
4. 
Study Design .......................................................................................................................................... 12 
4.1. 
Treatment Design ............................................................................................................................... 14 
4.2. 
Experimental / Observational Design ................................................................................................ 14 
4.3. 
Measurement Design ......................................................................................................................... 14 
4.4. 
Outcome Measures ............................................................................................................................ 14 
5. 
Study Participants .................................................................................................................................. 15 
5.1. 
Number of Participants ...................................................................................................................... 15 
5.2. 
Eligibility ............................................................................................................................................. 15 
5.3. 
Strategies for Recruitment and Retention ......................................................................................... 16 
5.4. 
Consent Process ................................................................................................................................. 16 
6. 
Study Intervention ................................................................................................................................. 17 
6.1. 
Intervention – Test Article (if applicable) ........................................................................................... 17 
6.2. 
Assignment Procedures...................................................................................................................... 20 
6.3. 
Intervention – Procedural (if applicable) ........................................................................................... 21 
6.4. 
Concomitant Therapy (if applicable) .................................................................................................. 21 
6.5. 
Rescue Medications and Procedures (if applicable) .......................................................................... 21 
6.6. 
Compliance Checks ............................................................................................................................ 21 
6.7. 
Withdrawal / Discontinuation of Enrolled Participants ..................................................................... 21 
6.8. 
Voluntary Withdrawal (Drop-Out) of Enrolled Participants ............................................................... 22 
7. 
Study Procedures and Schedule ............................................................................................................ 22 
7.1. 
Table of Events ................................................................................................................................... 22 
7.2. 
Enrollment/Pre-Screening (Day -14 to 0) ........................................................................................... 23 

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7.3. 
Screening (Day -14 to 0) ..................................................................................................................... 23 
7.4. 
Study Visits ............................................................................................ Error! Bookmark not defined. 
7.5. 
Phone Contact (Day 0 +7±2, Day 0 + 14±2, Day 0 + 21±2, and Day 0 + 35±2 ) .................................. 23 
7.6. 
Final Visit (Day 0 +28±2) ..................................................................................................................... 23 
7.7. 
Follow-Up Contact (Day 0 + 35±2) ..................................................................................................... 24 
7.8. 
Premature Discontinuation ................................................................................................................ 24 
7.9. 
Collection and Management of Tissue Specimens............................................................................. 24 
8. 
Study Measurements and Evaluations ................................................................................................. 25 
8.1. 
Outcome Measures for Evaluation of Feasibility / Tolerability .......................................................... 25 
8.2. 
Outcome Measures for Evaluation of Efficacy ................................................................................... 25 
8.3. 
Outcome Measures for Evaluation of Safety ..................................................................................... 25 
8.4. 
Other Outcomes in the Causal Pathway ............................................................................................ 25 
8.5. 
Baseline Characteristics of the Participants ....................................................................................... 25 
8.6. 
Variables Representing Treatment .................................................................................................... 26 
9. 
Statistical Analysis Plans........................................................................................................................ 26 
a. 
Strategies that Apply to all the Specific Aims ......................................................................................... 26 
9.1. 
Description of the Study Cohort......................................................................................................... 27 
9.2. 
Aim-Specific Plans .............................................................................................................................. 27 
9.3. 
Planned Interim Analyses (If Applicable) ........................................................................................... 28 
9.4. 
Plans for Coping with Withdrawals and Loss-To-Follow-Up .............................................................. 28 
9.5. 
Sample Size Rationale ........................................................................................................................ 28 
10. 
Safety Monitoring and Management .................................................................................................... 29 
10.1. 
Risk / Benefit Assessment .............................................................................................................. 29 
10.2. 
Assessment of Safety ..................................................................................................................... 30 
10.3. 
Unanticipated Problems, Adverse Events, Serious Adverse Events .............................................. 30 
10.4. 
Safety Monitoring .......................................................................................................................... 32 
10.5. 
Study Suspension / Early Termination of the Study....................................................................... 33 
11. 
Supporting Documentation and Operational Considerations ............................................................. 33 
11.1. 
Regulatory, Ethical, and Study Oversight Considerations .............................................................. 33 
11.1.1. 
Informed Consent Process ............................................................................................................. 33 
11.1.1.1. 
Consent/Assent and Other Informational Documents Provided to Participants ...................... 33 
11.1.1.2. 
Consent Procedures and Documentation .................................................................................. 33 
11.2. 
Study Discontinuation and Closure ................................................................................................ 34 
11.3. 
Confidentiality and Privacy ............................................................................................................ 34 

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To further protect the privacy of study participants, a Certificate of Confidentiality will be issued by the 
National Institutes of Health (NIH).  This certificate protects identifiable research information from forced 
disclosure. It allows the investigator and others who have access to research records to refuse to disclose 
identifying information on research participation in any civil, criminal, administrative, legislative, or other 
proceeding, whether at the federal, state, or local level. By protecting researchers and institutions from 
being compelled to disclose information that would identify research participants, Certificates of 
Confidentiality help achieve the research objectives and promote participation in studies by helping assure 
confidentiality and privacy to participants. .................................................................................................... 35 
11.4. 
Future Use of Stored Specimens and Data .................................................................................... 35 
11.5. 
Key Roles and Study Governance................................................................................................... 35 
11.6. 
Safety Oversight ............................................................................................................................. 36 
11.7. 
Clinical Monitoring ......................................................................................................................... 36 
11.8. 
Quality Assurance and Quality Control .......................................................................................... 37 
11.9. 
Data Handling and Record Keeping ............................................................................................... 37 
11.9.1. 
Data Collection and Management Responsibilities ....................................................................... 37 
11.9.2. 
Study Records Retention ................................................................................................................ 38 
11.10. 
Protocol Deviations ........................................................................................................................ 38 
11.11. 
Publication and Data Sharing Policy............................................................................................... 38 
11.12. 
Conflict of Interest Policy ............................................................................................................... 39 
12. 
Additional Considerations ..................................................................................................................... 39 
13. 
References ............................................................................................................................................. 39 
14. 
List of Appendices .................................................................................................................................. 42 
 
 
 
 

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Statement of Compliance 
This study will be conducted in accordance with the International Conference on Harmonization 
guidelines for Good Clinical Practice (ICH E6) and the Code of Federal Regulations on the Protection of 
Human Subjects (45 CFR Part 46). The statistical analysis plans will be consistent with guidance such as 
the CONSORT Statement [1] or STROBE Statement [2], ICMJE recommendations [3], the 2016 and 2019 
statements of the American Statistical Association [4,5], and recommendations in Nature [6,7].1 All 
personnel involved in the conduct of this study have completed human subjects’ protection training. 
 
 
 
 
1 [1] www.consort-statement.org [2] www.strobe-statement.org [3] www.icmje.org [4]  Wasserstein RL, et al. 
(2016), The ASA's Statement on p-Values, The American Statistician, 70:2, 129-133 [5]  Wasserstein RL, et al. 
(2019), Moving to a World Beyond p < 0.05, The American Statistician, 73:sup1, 1-19 [6]  Amrhein, et al. (2019) 
Scientists rise up against statistical significance, Nature 567, 305-307 [7] Editorial (2019) It’s time to talk about 
ditching statistical significance: Looking beyond a much used and abused measure would make science harder, but 
better. Nature 567, 283-283. 

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Abbreviations and Definitions of Terms 
 
Abbreviation/Acronym 
Definition 
AE 
Adverse Event/Adverse Experience 
CI 
Confidence Interval 
CRC 
Colorectal Cancer  
CRF 
Case Report Form 
CRP 
C-Reactive Protein 
eCRF 
Electronic Case Report Form 
FDA 
Food and Drug Administration 
GCP 
Good Clinical Practice 
GI 
Gastrointestinal 
GOS 
Galacto-oligosaccharides 
Gal 
Galactose 
Glc 
Glucose 
GSSC 
Gastrointestinal Symptom and Severity Checklist 
hGOS 
GOS enriched with LacNAc; humanized GOS 
HIPAA 
Health Insurance Portability and Accountability Act 
IB 
Investigator's Brochure 
IBD 
Inflammatory Bowel Disease 
IBS 
Irritable Bowel Syndrome  
ICF 
Informed Consent Form 
IL 
Interleukin 
INF 
Interferon  
IND 
Investigational New Drug Application 
IRB 
Institutional Review Board 
N 
Number (typically refers to subjects) 
NDA 
New Drug Application 
PI 
Principal Investigator 
QA 
Quality Assurance 
QC 
Quality Control 
SAE 
Serious Adverse Event/Serious Adverse Experience 
SD 
Standard Deviation 
SE 
Standard Error 
SOP 
Standard Operating Procedures 
TNF 
Tumor Necrosis Factor 
UC 
Ulcerative Colitis  
UP 
Unanticipated Problem 
WGS 
Whole Genome Shotgun (Sequencing) 
 
 

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1. Protocol Synopsis 
 
Study Title 
Safety, Tolerability, and Biosignature of Humanized Prebiotics in Healthy Adults  
Objectives 
Objective 1. Validate 15 g/day dose safety and tolerability (including absence of 
GI adverse effects) of GOS and hGOS in healthy adults. 
Objective 2. Establish a biological signature of GOS and hGOS through 
assessment of prebiotics effects vs placebo (powdered corn syrup comprised of 
fructose, glucose, and an inert cellulose material that matched the consistency, 
color, sweetness, and taste of the prebiotics) on (i) abundance of beneficial gut 
bacteria and restoration of the gut microbiome saccharolytic potential, (ii) 
modulation of biomarkers of inflammation and (iii) evaluation of intestinal 
barrier function. 
Target 
Population 
Healthy adult individuals 
Key Inclusion Criteria: Individuals ages 18-55 with BMI of 18.5 to 32.  
Key Exclusion Criteria:  
• 
Formal diagnosis of  IBD i(ncluding ulcerative colitis, Crohn’s Disease) 
colorectal cancer, diabetes 
• 
Celiac disease or IBS diagnosed by a physician 
• 
Lactose intolerance as diagnosed by a hydrogen breath test 
• 
 C. difficile infection within 2 months 
• 
Antibiotic consumption in the previous 2 months 
• 
History of gastric bypass surgery 
• 
Current tobacco smoker 
• 
Pregnant or breastfeeding 
• 
A response of 4 or 5 to questions 2 (abdominal pain), 6 (abdominal 
discomfort), 8 (constipation), 16 (diarrhea), 22 (abdominal distention), 
26 (abdominal bloating), 31 (nausea), or 33 (vomiting).  
Numbers of 
Participants 
                                                                                                                              
Number to be recruited for screening: 55-60 participants 
 
Number of eligible participants enrolled: 48 adult individuals 
Clinical Phase 
Pilot 
 
Intervention 
Daily oral administration of 10 - 15 g of Prebiotic galacto-oligosaccharides (GOS) 
or GOS enriched with lactosamine (LacNAc) for four weeks 
Study 
Description 
We will enroll 48 adult individuals (18-55 y.o.). Subjects will be assigned 
randomly to one of 3 groups (n = 16): GOS, hGOS or placebo (powdered corn 
syrup comprised of fructose, glucose, and an inert cellulose material that 
matched the consistency, color, sweetness, and taste of the prebiotics) to test 
safety and dose tolerability. The prebiotic treatment will last for 4 wk. Before the 
treatment (baseline) and during week 4 of the trial (after the prebiotic 
treatments), we will assess changes in quality and quantity in bacterial 
components of the gut microbiome pre- and post-treatment. Likewise, we will 

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determine serum markers of inflammation, integrity of the intestinal barrier at 
baseline and at week 4. Participants will complete the Diet History Questionnaire 
(DHQ), a freely available food frequency questionnaire (FFQ) at baseline and will 
be asked to maintain a record of their daily diet by using a printed meal tracking 
diary. In addition, the PROMIS Gastrointestinal Symptom Scale will be used for 
potential side effect evaluation at baseline and each week for the duration of the 
study. 
Outcome 
Measures 
Dose safety and tolerability in healthy adults. 
Gut microbiome 
Inflammation 
Integrity of intestinal barrier 
Study Duration 
4 weeks 
 
Subject 
Participation 
Duration 
4-5 weeks 
 
Estimated Time 
to Complete 
Enrollment 
40 weeks 
 
Statistical 
Analysis Plans 
Data will be presented as mean ± SEM for variables that are normally 
distributed, or median (IQR) for variables not normally distributed. Group means 
will be compared by ANOVA and post hoc tests except when data is normally 
distributed (a nonparametric analysis of medians will be performed using the 
Kruskal-Wallis test for non-normally distributed data). Chi-square tests or 
Fisher’s exact tests will be used for incidence data.  
 
For analysis of microbiome taxonomic and functional data we will carry out 
Mann-Whitney-Wilcoxon matched pair tests for pairwise comparisons between 
time points. Unpaired Mann-Whitney-Wilcoxon tests will be used for pairwise 
comparisons between treatment groups. We will apply the Kruskal-Wallis test 
when more than two groups are compared. P values will be corrected for the 
total number of comparisons with false-discovery rate (FDR). 
 
2. Introduction: Background and Scientific Rationale 
2.1. Background Information 
An undefined three-point association exists between altered gut microbiota (dysbiosis), compromised 
gut barrier, and disease phenotype. This relationship is well documented in animal studies but not well 
examined in human studies; hence a ‘triangulation’ is inferred based on the association between two of 
these three factors. A recent review of the mechanisms, measurement, and clinical implications of a 
“leaky gut” in humans1 stated that studies suggest that there are non-pathological situations that may 
be associated with increased intestinal permeability, and these relatively minor perturbations “can be 
reversed with dietary, non-pharmacological approaches”. Approaches to alleviate these conditions, 
including prebiotics and probiotics, in human interventions are lacking. 

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Galacto-oligosaccharides (GOS) are prebiotics that have an FDA Generally Regarded As Safe (GRAS) 
status2. GOS modulate the intestinal microbiota3, 4 and strengthen intestinal barrier function via 
modulation of goblet cell function5, 6. LacNAc is the major building block of gut glycoproteins and human 
milk oligosaccharides7, 8, but its beneficial effects have not been studied as it could not be produced in 
enough quantities at reasonable cost until now. We have shown that short-term feeding of pure GOS 
increases expression of protective mucus-producing MUC2, decreases intestinal permeability, and 
reverses proportions of fecal β-galactosidases and β-glucosidases, reflecting increases in saccharolytic 
bacteria, including Bifidobacterium9. Likewise, GOS enriched with LacNAc, a building block of gut 
glycoproteins (humanized GOS, hGOS) decreases intestinal permeability in old mice. Although LacNAc is 
a biological component of human breast milk and gut mucins, its use as a supplement has not been 
assessed and hence its GRAS status has not been granted yet by FDA. An IND exempt status has been 
obtained for this study. GOS is also effective in the treatment of lactose intolerance10, 11. Therefore, GOS 
and hGOS represent an attractive approach to reducing intestinal permeability, improving the gut 
microbiome, and reducing inflammation in older adults. Our long-term goal is to establish the safety of 
pure prebiotic GOS and hGOS and their effectiveness in (i) reducing intestinal permeability and 
inflammation, (ii) restoring the saccharolytic potential of the gut microbiome, and (iii) alleviating mild 
gastrointestinal manifestations and self-reported food intolerances in older adults in long-term care. The 
objectives of this study are (1) to validate 15 g/day dose safety and tolerability of GOS and hGOS in 
healthy adults, and (2) to establish a biological signature of GOS and hGOS through assessment of 
prebiotics effects vs placebo on (i) abundance of beneficial gut bacteria and restoration of the gut 
microbiome saccharolytic potential, (ii) modulation of biomarkers of inflammation and (iii) evaluation of 
intestinal barrier function.  
2.2. Supporting Pilot / Unpublished Data 
Prebiotics significantly affect gut health and microbiota composition. The impact of recognized 
prebiotics, or functional foods that stimulate growth of gut native beneficial bacteria, on microbiome 
composition and functionality has been documented by us and others3, 4, 11. β(1-4) galacto-
oligosaccharides (GOS) are complex carbohydrates that resist digestion in the upper GI tract, and thus 
arrive to the colon intact and consequently increase the abundance of specific primary and secondary 
metabolizers. Studies have reported the beneficial impact of GOS, alone or in combination with 
probiotics, on bowel function12, 13, diarrhea and abdominal pain10, 11, 14, 15, bloating and flatulence16, 17 in 
adult individuals. N-Acetyl-D-lactosamine (LacNAc, Galβ1,4GlcNAc) is a biologically relevant molecule 
that is a building block of gut glycoproteins, and a precursor of several important blood group epitopes, 
including Lewis A, Lewis B, or sialyl Lewis A, which are involved in biological processes, including 
fertilization and pathogen recognition18. Of relevance for our study, repeating and variably branched 
lactose or LacNAc units, frequently with attached sialic acid and fucose monosaccharides, compose 
complex human milk oligosaccharides (HMOs), which are uniquely abundant in human breast milk but 
not in that of other mammals19-21. Most HMOs are not digested by human enzymes, acting as natural 
prebiotics for the development of a healthy gut microbiota in infancy7, 8, 22. While GOS is not a naturally 
occurring non-digestible carbohydrate, it does share structural similarities with LacNAc, with the latter 
containing nitrogen molecules attached to the disaccharide backbone. Despite the biological relevance 
of LacNAc, no studies have addressed its impact on human gut physiology and the microbiota since the 
chemical methods to generate LacNAc demand multi-step syntheses and are expensive and labor 
intensive. Likewise, current enzymatic methods use galactosyl-transferases, which require UDP-
galactose as a glycosyl donor23, and have high acceptor selectivity, yielding low efficiency with unnatural 
acceptors24. 

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GOS and hGOS beneficially impact the gut microbiome, increasing beneficial bacteria (Bifidobacterium 
and Akkermansia) and saccharolytic potential of the microbiota and reduced intestinal permeability, 
modulating markers of inflammation9. Furthermore, GOS, but not hGOS, increased mucus in the 
intestinal lumen, suggesting different mechanisms of action. Our previous studies in mice have shown 
that short-term feeding of pure GOS increases expression of protective mucus-producing MUC2, 
decreases intestinal permeability, and reverses proportions of fecal β-galactosidases and β-glucosidases, 
reflecting increases in saccharolytic bacteria, including Bifidobacterium9, 25. 
 
In this trial, we will determine changes in the abundance of saccharolytic bacteria as described 9. Briefly, 
we will use a reference study that mined 532 publicly available gut reference genomes and assigned 
them to four different groups (proteolytic, saccharolytic, lipolytic, and generalist bacteria) using 
metagenome analytical methods 26, and the metabolic reconstruction network AGORA 27. In the case 
that the genus is not categorized in the mentioned studies, will refer to the Bergey’s Manual of 
Systematic Bacteriology and previously published reports. 
2.3. Scientific Rationale 
The study will test two prebiotics: highly pure GOS and LacNAc-enriched GOS (hGOS). The intervention 
will include a dose of 15 g daily as previously reported10. Dose was selected based on previous literature 
reports16, 17, 28-30 and the interventions conducted by our group to assess efficacy of GOS in lactose-
intolerant individuals10, 11, 31, 32. The hGOS targeted dose will be composed of 15g of GOS enriched with 
150 mg of LacNac (99:1), which is in agreement with the range found in human breast milk33. Based on 
the concentration detected in breast milk, an infant consumes between 54 and 243 mg of LacNAc per 
day, with the variation depending on stage of lactation and infant age and weight.  
 
Participants will be assigned randomly to one of 3 groups (n = 16): GOS, hGOS or placebo (powdered 
corn syrup comprised of fructose, glucose, and an inert cellulose material that matched the consistency, 
color, sweetness, and taste of the prebiotics)10, 32 to test safety and dose tolerability. The prebiotic 
treatment will last for 4 weeks, since we have shown in adult individuals that a 4-week period allows for 
the observation of changes to the gut microbiome10, 11. The doses of GOS, hGOS and placebo will be 
increased over 28 days beginning with 10 g/day and increasing to 15 g/day. Dose regiment will be 5 
grams twice daily on days 1–10 followed by 7.5 grams twice daily on days 11–28. Doses will be in 
powder form and will be mixed with water or food and taken as directed by the dosing scheme.   
 
High-sensitivity C-reactive protein (hs-CRP) is a recognized marker of systemic inflammation34 and 
specific pro-inflammatory cytokines mediate interaction between immune cells and non-immune cells, 
contributing to the inflammatory status of the intestine35, 36. Based on previous studies with probiotics 
and prebiotics that report potential immunomodulatory effects37-39, we decided to monitor hs-CRP, IL-
1α, IL-1β, IL-2, IL-6, Il-8, IL-12, TNFα, and IFNγ for the duration of the clinical trial. 
 
Before the treatment (baseline) and during week 4 of the trial (after the prebiotic treatment), we will 
assess changes in quality and quantity in bacterial components of the gut microbiome pre- and post-
treatment. Likewise, we will determine serum inflammatory markers, and integrity of the intestinal 
barrier at baseline and at week 4. Subjects will also respond to the Diet History Questionnaire (DHQ), a 
freely available food frequency questionnaire (FFQ)  and will be asked to keep daily diet records. 
 

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3. Objectives 
3.1. Specific Aim 1 
To validate 15 g/day dose safety and tolerability of GOS and hGOS in healthy adults. 
3.2. Specific Aim 2 
To establish a biological signature of GOS and hGOS through assessment of prebiotics effects vs placebo  
on (i) abundance of beneficial gut bacteria and restoration of the gut microbiome saccharolytic 
potential, (ii) modulation of biomarkers of inflammation and (iii) evaluation of intestinal barrier function. 
4. Study Design 
 
This will be a controlled (GOS, hGOS versus placebo), interventional, longitudinal (baseline compared to 
end of study), prospective, single-site pilot study. Subjects will be assigned randomly to one of 3 groups 
(n = 16): GOS, hGOS or placebo. The treatment will consist in 10 - 15 g/day of GOS, hGOS or placebo 
(powdered corn syrup comprised of fructose, glucose, and an inert cellulose material that matched the 
consistency, color, sweetness, and taste of the prebiotics), which will be provided to participants as 
powder that can be added to any non- alcoholic beverage or sprinkled on food for intake. The 
intervention will last for 4 weeks, since we have shown in adult individuals that a 4 week period allows 
for the observation of changes to the gut microbiome. The study will end after the second time-point 
sample collection at 4 weeks. 
 
Dose rationale: The intervention will include a dose of 5 grams twice daily on days 1–10  followed by 7.5 
grams twice daily on days 11–2810.Doses were selected based on previous literature reports16, 17, 28-30 and 
the interventions conducted by our group to assess efficacy of GOS in lactose-intolerant individuals10, 11, 
31, 32. The hGOS preparation will be composed of GOS enriched with LacNac at a concentration of 6.7-
31μg/mL (<1% of the formulation), which is in the range found in human breast milk33 and hence 
considered safe. 
 
Flow diagram  
 
Prior to  
Enrollment 
 
 
 
 
 
 
 
 
 
 
Obtain informed consent. Screen potential participants by inclusion and 
exclusion criteria; obtain medical history, document. Total N = 48 
Baseline: collect blood and stool. Administer 38-point Gastrointestinal 
Symptom and Severity Checklist (GSSC) and Diet History Questionnaire 
Arm 3 Placebo 
N=16 
Arm 1 GOS 
N=16 
Randomize 
Arm 2 hGOS 
N=16 

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Visit 1 
Time Point  
 
                                                                                                      
                                                                     Weekly GSSC data collection 
 
 
 
 
Visit 2 
Time Point  
 
 
 
 
 
 
 
Analyses: (a) Evaluation of 
inflammation and intestinal 
barrier function in serum; (b) 
4 weeks: collect blood and stool. Administer 38-point Gastrointestinal 
Symptom and Severity Checklist (GSSC) Questionnaire (FFQ) 

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4.1. Treatment Design 
Dose rationale. The intervention will include a dose ranging between 10 to 15 g daily as previously 
reported10. Doses were selected based on previous literature reports16, 17, 28-30 and the interventions 
conducted by our group to assess efficacy of GOS in lactose-intolerant individuals10, 11, 31, 32. The hGOS 
preparation will be composed of GOS enriched with LacNac at a concentration of 6.7-31μg/mL (<1% of 
the formulation), which is in the range found in human breast milk33 and hence considered safe.  
 
Regimen. The prebiotics dose will be 5 grams twice daily on days 1–10  followed by 7.5 grams twice daily 
on days 11–28. The placebo (powdered corn syrup comprised of fructose, glucose, and an inert cellulose 
material that matched the consistency, color, sweetness, and taste of the prebiotics) will be 
administered in a blinded matching packet at the same doses. If participants miss taking a dose on a 
given day, they will be directed to put the pouch for that day aside, and on the next day take the next 
numbered pouch. 
 
4.2. Experimental / Observational Design 
Subjects will be assigned randomly to one of 3 groups (n = 16): GOS, hGOS or placebo. 
4.3. Measurement Design 
Blood and stool samples will be collected at baseline (Day 0) and end of treatment (Day 28). Stool 
collection kits will be provided to participants in case samples cannot be obtained during study visits. 
Safety and tolerability will be determined based on the severity and/or frequency of side effect using a 
38-point Gastrointestinal Symptom and Severity Checklist (GSSC). In addition, tolerability will be 
evaluated through occurrence of drop-out and adherence. 
Measurements on blood (10 mL per visit for a total of 20 mL): 
(1) Fasting venous blood samples will be drawn and serum will be used to quantify zonulin using the 
Zonulin (IDK® Zonulin) (Serum) ELISA test (Immundiagnostik AG). 
(2) Determination of levels of biomarkers of inflammation (IL-1α, IL-1β, IL-2, IL-6, Il-8, IL-12, TNFα, and 
IFNγ) as well as high-sensitivity C-reactive protein, a marker of low-grade inflammation (hsCRP) in 
serum. We will determine cytokine levels using the MCYTOMAG-70K (Milliplex) in the Advanced 
Analytics Core (AAC) of the CGIBD. (ii) hsCRP concentration will be measured via hsCRP ELISA (Sigma 
Aldrich). 
Measurements in stool: 
Determination of abundance of beneficial bacteria (Bifidobacterium and Akkermansia species) in the gut 
microbiome of treatment and placebo groups and the microbiome saccharolytic potential using WGS 
sequencing, the most accessible, cost-effective measure currently available. 
4.4. Outcome Measures 
Primary Outcome(s):  
The expected outcome is to confirm a 15 g/day as a safe and tolerable dose of GOS and hGOS in healthy 
adult individuals using the 38-point Gastrointestinal Symptom and Severity Checklist (GSSC).   
 
Secondary Outcome(s):  
Definition of prebiotic biosignature characterized by: 

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• 
Increased abundance of saccharolytic bacteria (Bifidobacterium) and other beneficial 
bacteria (Akkermansia) 
• 
Modulation of biomarkers of inflammation 
• 
Improved intestinal barrier function. 
 
Tertiary/Exploratory Outcome(s): N/A 
 
Other Measures: Baseline Characteristics and Assigned Treatments: N/A 
 
5. Study Participants 
5.1. Number of Participants 
Number to be recruited for screening: 55-60 participants will be recruited for screening. 
Number of eligible participants enrolled: The targeted number of participants is 48. We expect that 
20% of screened individuals will not be eligible or will be unwilling/unable to participate in the study. 
Participants will be adults (male and female, 18-55 years old with no formal diagnosis of IBD, IBS, UC, 
CRC, celiac disease, or diabetes mellitus) recruited at UNC Chapel Hill and within the general population 
in the surrounding area via email communications, social media, and flyers. 
Subjects will not be diagnosed with specific GI conditions (IBD, IBS, UC, CRC) and with no history of celiac 
disease, diabetes mellitus or antibiotic consumption in the previous 2 months. Individuals with a non-GI 
related disease diagnosis might be included in the study.  
5.2. Eligibility 
Eligibility criteria are based on self-reported information from study participants. 
Inclusion Criteria:  
• 
Individuals must be between 18 and 55 years old. 
• 
All participants will not be tobacco smokers and will be well-nourished according to 
standard anthropometric criteria with BMI between 18.5 and 32. 
• 
Individuals must be able to give informed consent. 
• 
Subjects willing and able to: 
• 
consume prebiotics or placebo preparations for a period of 4 weeks 
• 
record daily food consumption using hard-copy food diary provide at the 
baseline vist, and complete the Diet History Questionnaire (DHQ). 
• 
provide stool, and blood (via venipuncture) samples. 
• 
Enrollment will not be restricted based on race, ethnicity, or gender. Subject population will 
reflect the population providing a broad selection of individuals to allow to enroll subjects 
from all races, ethnicities and genders as represented in North Carolina state. 
 
Exclusion Criteria:  
• 
Individuals diagnosed with Inflammatory Bowel Diseases (IBD), Ulcerative Colitis (UC), 
Colorectal Cancer (CRC) , or diabetes mellitus.  
• 
Celiac disease or IBS diagnosed by a physician 

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• 
Lactose intolerance as diagnosed by a hydrogen breath test 
• 
C. difficile infection or treatment within 2 months 
• 
Individuals currently or within the past 2 months on prescribed antibiotics. 
• 
Patients with a history of gastric bypass surgery. 
• 
Any physical or psychological condition that, in the opinion of the investigators, would post 
unacceptable risk to the patient or raise concern that the patient would not comply with 
protocol procedures 
• 
Women that are pregnant or breastfeeding will be excluded from the study. 
• 
A response of 4 or 5 to questions 2 (abdominal pain), 6 (abdominal discomfort), 8 
(constipation), 16 (diarrhea), 22 (abdominal distention), 26 (abdominal bloating), 31 
(nausea), or 33 (vomiting) on the PROMIS Gastrointestinal Symptom Scale. 
5.3. Strategies for Recruitment and Retention 
Recruitment Strategy:  
Subjects will be recruited using study flyers and/or asked to participate in the study via email or phone 
call. Only if the subject agrees to participate and meets enrollment criteria will they be enrolled in the 
study. Subjects will be screened by  the Clinical Research Coordinator on the basis of inclusion and 
exclusion criteria laid out in this study.  Only individuals with no formal diagnosis of IBD, IBS, UC, CRC, 
celiac disease, or diabetes mellitus will be selected as study participants based on a self-report. Also, 
individuals with a history of gastric bypass, current use of antibiotics or use of oral antimicrobials in the 
past 2 months will be excluded. The PI will review subject enrollment at weekly project meetings so that 
any deviation from the anticipated enrollment schedule will be detected quickly. All outreach and 
advertising activities will also be reviewed on a weekly basis. 
 
Retention Strategy:  
The PI and research team will hold weekly project meetings to monitor enrollment and ensure enrolled 
subjects complete all study procedures as intended. Likewise, brief weekly contacts (either by phone, 
email, or text message) with enrolled subjects will follow up regarding protocol adherence, 
gastrointestinal symptoms, and study satisfaction, in addition to any protocol-specific procedures. This 
approach provides the opportunity for early identification of possible reasons for study non-adherence 
or early discontinuation by the subject and to address such issues, if possible. If a subject decides to 
withdraw participation in the study, the date and reason will be recorded and stored in the RedCap 
database. On a weekly basis, the study coordinator will run a report on participants accrual and 
completion status for review by team to identify and resolve any problems or potential issues that arise 
during the course of the study. 
 
Screen Failures:  
During the screening period, inclusion/exclusion criteria for the study participation will be checked / 
tested. Subjects who meet all inclusion criteria and do not meet any exclusion criterion will be eligible to 
be randomized. Those who are not eligible for randomization or dosing will be considered as screening 
failures. In the case of screen failures, the reason will be recorded. We expect a 20% screen failure rate.  
5.4. Consent Process 
Potential participants will be approached via email or text message, and a virtual screening appointment 
will be set up over Zoom. The study will be explained to the subjects by Dr. Becker-Dreps or Clinical 

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Research Coordinator, Kelli Hammond. Potential participants who meet screening criteria and are still 
interested in the study will undergo consent procedures. Consent will be performed virtually using the 
Qualtrics platform. Potential participants will be allowed time to read the consent and all questions will 
be answered. Consent discussion will take place until the subjects are fully aware of all study details and 
all questions have been answered. There is no set time for consent discussion. Subjects will be told that 
they can withdraw from the study at any time without penalty. The subject and investigator and/or 
study coordinator will sign the consent form. An electronic version of the consent will be provided to the 
participants and printed copy will be provided to the subject at the time of the first study visit.  
6. Study Intervention 
 
6.1. Intervention – Test Article (if applicable) 
Description: Prebiotic galacto-oligosaccharides (GOS) have an extensive history of safety and have 
been used for decades to improve overall host health via modulation of the gut microbiota40. Since GOS 
are complex carbohydrates resistant to digestion in the upper gastrointestinal tract that arrive to the 
colon intact, they increase the abundance of specific primary and secondary degraders expanding the 
probiome, autochthonous beneficial members of the intestinal microbiota41. Specific increases of 
Bifidobacterium (the “bifidogenic effect”) hence are fundamental to the beneficial effect of the prebiotic 
that result in increased concentration of colonic short chain fatty acids (SCFAs), particularly butyrate42. 
Over 95% of the microbially generated SCFAs are absorbed by the colonic epithelium43-45. The three 
major SCFAs (acetate, propionate and butyrate) stimulate proliferation of normal crypt cells and 
intestinal barrier integrity. Butyrate and, to a lesser degree, propionate, have been reported to inhibit 
growth of colon cancer cell lines46. Furthermore, butyrate has potent immunomodulatory effects 
influencing activity of HDACs responsible for decreasing dendritic cells IL-12 and IL-6 cytokine 
secretions47, and induces TREG cells and IL-10-secreting T cells through Gpr109a on colonic dendritic cells 
and macrophages48.  
 
The prebiotic GOS and hGOS in this formulation are generated by the β-hexosyltransferase from 
Sporobolomyces singularis, optimized and heterologously expressed to generate high-purity GOS49, 50.  
Commercial GOS are a mixture of mono, di, and oligosaccharides in varying ratios. Although major 
advances that have led to standardization, and better characterization and reporting of such mixtures 
have been made, variability in the GOS composition impacts the reproducibility of studies and 
interventions as well as the ability to predict outcome. GOS are currently produced commercially by 
trans-galactosylation of lactose as donor and acceptor substrate by β-galactosidase enzymes (EC 
3.2.1.23)51. Converting lactose into GOS by β-galactosidases is a kinetically controlled reaction, by the 
competition between hydrolysis and trans-galactosylation. During this conversion, the 
thermodynamically favored hydrolysis of lactose, which generates D-galactose and D-glucose, competes 
with the transferase activity that generates a complex mixture of various galactose-based di- and 
oligosaccharides of different structures52. Vivinal GOS is produced by Bacillus circulans β-galactosidase, 
Bimuno is a GOS blend derived from lactose conversion using Bifidobacterium bifidum NCIMB 41171 
enzymes, and Oligomate is produced by the β-hexosyltransferase from the yeast Sporobolomyces 
singularis.  
 
GOS may have a degree of polymerization (DP) up to 9. However, the enzymatic generation of GOS 
tends to produce low molecular weight oligosaccharides. As shown in Table 1, concentration of 

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disaccharides in commercial formulations can be as high as 52%. DP2 elements may differ in their 
structure. One seminal study53 that reported the generation of GOS by B. circulans showed that the ß-(1-
3), ß-(1-2), and ß-(1-6) linked galactosyl disaccharides (D1-D3) were formed by the trans-glycosylation 
reaction with the ß-galactosidase from B. circulans. Since lactose [the ß-(1-4) linked galactosyl-
disaccharide] was already present in the initial reaction as substrate, the authors used a different 
reaction (containing pNP-Gal and glucose) to confirm lactose synthesis by the enzyme.  
 
In a recent study54, the analysis of 6 commercial GOS products using high-performance liquid 
chromatography (HPLC) size exclusion on a Rezex RSO-oligosaccharide Ag+ column showed that all 
tested prebiotics had the monosaccharides galactose (Gal) and glucose (Glc) together with DP2 (mostly 
lactose) up to DP5 oligosaccharides in detectable levels. Vivinal GOS, and two other GOS products had 
also detectable amounts of DP6 oligosaccharides. Interestingly, except for one GOS product, the DP2-
DP4 fractions of the various products comprised >90% of all GOS components, with most products 
containing a majority of DP2, with exception of one GOS product that had the highest DP4, DP5, and 
DP6 content of all GOS products with very low amounts of Gal and Glc, indicating a DP-based 
purification step. This study is one of the most detailed studies on composition of GOS; however, it does 
not provide the names or sources of 6 of the 7 analyzed compounds. Another study that reported on 
impact of B-GOS (Bimuno, Clasado Limited) on the immune system of 40 elderly individuals stated that 
52% of the product was DP229. In contrast, RP-G28, a purified preparation reported <3.2% of DP2 
oligosaccharides11 (Table 1).  
 
Table 1. Composition of prebiotic GOS commercial formulations 
 
 
RP-G2811 
Vivinal54 
Bimuno*29 
BIOLIGO® GL 
700 ** 
Oligomate  
Degree of 
polymerization  
 
 
 
 
 
DP2 
<3.2% 
42.55 ± 0.22% 
52% 
17.5% 
14.8% 
DP3 
56-59% 
23.63 ± 0.20% 
26% 
38-52% 
34.4% 
DP4 
26-30% 
10.21 ± 0.23% 
14% 
 
 
DP5 
 
3.00 ± 0.17% 
8% 
 
 
DP6 
11-16% 
0.39 ± 0.17% 
 
 
 
Glucose 
<0.1% 
18.52 ± 0.12% 
 
22% 
22.4% 
Galactose 
<0.1% 
1.70 ±0.08% 
 
0.8% 
8.6% 
 
*Bimuno = B-GOS (Clasado) = Clasado BioSciences Ltd. (St. Helier, Jersey, UK) =  
HOST-G904 (Therabiomics, Jersey, Channel Islands) 
** BIOLIGO® GL 5700 GOS (Ingredion) = Purimune (GTC Nutrition, Golden, Colorado [2008]) 
 
To ensure the safety of the human volunteers we followed the FDA guidance for deriving the maximum 
recommended starting dose (MRSD) for clinical trials of new molecular entities in adult healthy 
volunteers. See: U.S. Department of Health and Human Services Food and Drug Administration. (2005). 
Guidance for Industry: Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for 
Therapeutics in Adult Healthy Volunteers. Rockville, MD: Center for Drug Evaluation and Research 
(CDER), 7. 
  
The equation used is as follows: 

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HED (mg*kg-1) = Animal Dose (mg*kg-1) * Animal Km/Human Km 
 
Where the Km factor is a number based on body surface area. In our animal study we used a dose of 
1,500 mg kg−1, based on ~30 mg LacNAc fed to a ~20 g mouse per day25. We used a Km factor of 3 for 
mice and 37 to represent a 60 kg human adult.  
 
We can consistently produce hGOS containing concentrations that exceed 25 g/L of LacNAc with no 
significant variation between batches observed. Therefore, our approach is to normalize to reach the 
proposed dose levels using pure GOS. The active component is in power form that will be obtained after 
lyophilization. Concentration will be confirmed using previously developed HPLC protocols33  
 
Acquisition: Dr. Jose Bruno-Barcena, Professor of Plant and Microbial Biology at North Carolina State 
University and Co-investigator in this proposal, will coordinate with the Golden LEAF Biomanufacturing 
Training and Education Center (BTEC) the cGLP production of the prebiotics galacto-oligosaccharides 
(GOS) and GOS enriched in N-acetyl-D-lactosamine (LacNac, humanized GOS, hGOS) for the clinical 
intervention planned for the study. BTEC has the capabilities, equipment, and expertise for the 
bioprocess cGLP production (See Appendix 1).  
 
Formulation/Packaging/Labeling: GOS, hGOS, and placebo will be supplied as a sterile powder by 
BTEC, dried and portioned into daily dose pouches under sterile conditions at the NC Food Innovation 
Lab, Kannapolis, NC, . Pouches will be labeled and will serve to protect the preparations from light, air, 
and moisture. Product will then be transferred to the UNC Investigational Drug Services (IDS). 
  
 
 
Storage and Stability:  
Protocol Number:  
Study Acronym: GOS Pilot 
  
 
Enrollment ID:                                                                        Lot No.:  
Subject Number:                                                                    Subject’s Initials:  
Date Dispensed:  
Contents: 56 pouches, 5 g each for the first 10 days followed by 7.5 grams each for days 11-28 
of GOS, hGOS or matching placebo, plus two additional packets  (one 5mg and one 7.5mg) for 
inadvertent loss or spillage. 
Directions: Use only as directed. To be taken by mouth with food or beverage.  
This product is an investigational product to be dispensed only by a qualified investigator.  
Storage: Room Temperature 15-30 ℃ (59-86℉)  
Caution: New drug limited by law to investigational (clinical trial) use only. Keep out of reach of 
Children. 
Manufactured By: Golden LEAF Biomanufacturing Training and Education Center (BTEC), 850 
Oval Dr, Raleigh, NC 27606 
Distributed by:  

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The powder prebiotics galacto-oligosaccharides (GOS), GOS enriched in N-acetyl-D-lactosamine (LacNac, 
humanized GOS, hGOS), and placebo can be safely stored in a dry area at room temperature and hold an  
expiration date of 2 years after production. 
 
Preparation and Administration: Individuals will be provided with a total of 58 pouches numbered by 
day (#1 to #56) containing the appropriate daily dose of treatment or placebo. Pouches 57 and 58 will be 
extra pouches in case of loss or spillage. Participants will be instructed to consume one pouch twice 
aday. The premeasured powder can be added to hot or cold non-alcoholic beverages or sprinkled over 
food. 
 
Modification:  Individuals experiencing more than three of the following side effects a day: bloating, 
flatulence, belching, abdominal pain, discomfort, watery stool, and vomiting will be instructed on how 
to spread out the daily dose to minimize side effects. Participants experiencing these side effects be 
asked about frequency and severity. Severe side effects interfering significantly with subject’s usual 
function will result in withdrawal from study. Weekly phone contacts will allow for timely report of 
adverse effects. Participants will also be given the choice to contact the study PI directly in case of 
emergencies. 
Accountability: UNC Investigational Drug Services (IDS) will keep source documentation for 
accountability of the study treatments that will detail receipt, dispensing, and return of used and unused 
prebiotics and placebo. Treatment products will be stored in locked facilities until they are either 
returned to the PI at the end of the study. Prior to dispensing, sachets containing the treatment will be 
visually inspected. If the package appears to have tears or sign of tampering, the sachet will be rejected 
for use but retained until the end of the study. At the end of the study, the study team will determine 
dispensation of all remaining unused treatment packs at the study site. An accountability log will be 
kept, indicating the final disposition of study treatment. This log will be provided to the PI at the end of 
the study.  
 
6.2. Assignment Procedures 
Matching and Stratification:  
N/A 
 
Randomization, Concealment, and Blinding:  
The Excel RAND function will be used for simple randomization of participants into the 3 different 
groups. The PI and designated study personnel are blinded to the randomization and are responsible 
for following the randomization and blinding procedures described in each clinical trial protocol. 
 
Masking (Blinding): 
This is a double-blinded study. The master randomization codes will be kept by IDS with appropriate 
security measures and access control. The research team will be unblinded once all of the samples have 
been collected and processed, before data analysis. 
Unblinding. Circumstances for unblinding will be clearly established before the first patient begins 
treatment. Randomization procedures, if any, will be followed to ensure the code is broken only in 
accordance with the protocol. The study codes will be stored at the study site or may only be available 
directly from IDS. The code will only be broken in the case of an adverse event where it is necessary for 
the PI to know which treatment the individual is receiving before the individual can be treated. If it is 

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necessary to break the blind, the researcher will notify the IRB and document the event, together with 
the reasons for breaking the code.  
6.3. Intervention – Procedural (if applicable) 
Description:  
(1) Subjects will receive enough prebiotic or control powder for the complete 28 days in pouches 
containing the exact daily dose. An additional pouch will be provided for both the 5g and 7.5g 
quantities in case of  loss or spillage. The powder can be added to hot or cold non-alcoholic 
beverages or sprinkled over food. 
(2) Subjects in the study will consume 5 grams of either GOS, hGOS or placebo twice daily on days 
1–10 followed by 7.5 grams twice daily on days 11–2810.  
Training on Procedural Intervention:  
Participants will be provided with detailed instructions of how to prepare and consume the prebiotics or 
control powder. Individuals will be informed on the potential side effects and how to avoid them by 
rationing the treatment over the day if needed or chosen.  
6.4. Concomitant Therapy (if applicable) 
New diagnosis and initiation of a new therapeutic including supplements during the course of the study 
should be disclosed. Antibiotic treatment during the course of the study will lead to discontinuation.  
 
 
6.5. Rescue Medications and Procedures (if applicable) 
N/A 
 
6.6. Compliance Checks 
Participant adherence will be monitored weekly via email and/or text messages through self-reporting 
of number of treatment pouches left. 
 
6.7. Withdrawal / Discontinuation of Enrolled Participants 
At baseline and every week after for the duration of the study, participant will answer a 33 question 
gastrointestinal symptom assessment with the PROMIS Gastrointestinal Symptom Scale (Spiegel, et al, 
Am J Gastrol, 2014). If a subject experience and increase in 2 levels over baseline in their response to 
questions 2 (abdominal pain), 6 (abdominal discomfort), 8 (constipation), 16 (diarrhea), 22 (abdominal 
distention), 26 (abdominal bloating), 31 (nausea), or 33 (vomiting) on the PROMIS Gastrointestinal 
Symptom Scale, they will be a) withdrawn from the study if the participant is in the first 10 days of their 
participation, or b) asked to go back to the lower dose of 5g sachets twice daily and reassessed in 3 days, 
if the participant has passed day 10 of participation.  If participants are able to tolerate the lower dose, 
they will continue participating in the trial albeit consuming the lower dose of the prebiotic treatment. 
Likewise, if a participant develops a condition that is an exclusion criterion or participant noncompliance 
increases risk of decreased study integrity, the participant will be withdrawn from the study. 

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If 5 of the first 20 subjects enrolled experience a change of 2 levels on questions 2, 6, 8, 16, 22, 26, 31, 
33 on the PROMIS Gastrointestinal Symptom Scale (bloating, flatulence, watery stools, abdominal pain) 
compared to baseline or higher adverse event leading to subject discontinuation, we will stop the study 
temporarily and request the medical monitor to do an unblinded assessment of the data. The medical 
monitor (unblinded) will assess subjects who discontinued due to the specified adverse effects to 
determine if they are all in one arm of the study or range across arms.  The medical monitor will assess if 
there is a safety signal in a specific arm or treatment dose over the other that warrants the closure of 
the study. Since the medical monitor will be the person determining continuation or discontinuation of 
the study, the study team will remain blinded and unbiased. 
6.8. Voluntary Withdrawal (Drop-Out) of Enrolled Participants 
Participants may voluntarily withdraw from study at any time, for any reason, with no penalty or loss of 
rights.  Participants that choose to withdraw from the study will be followed up (via text or email 
communication) to determine safety based on frequency and severity of side effects. Reasons for 
discontinuation will be documented in the study RedCap database.  
7. Study Procedures and Schedule 
7.1. Table of Events 
Procedures 
Screening 
–Day -14 to 0) 
Baseline (Study 
Visit 1) 
Phone Contact 1 
(Day 0 + 7±2) 
Phone Contact 2 
(Day 0 + 14±2) 
Phone Contact 3 
(Day 0 + 21±2) 
Final Visit 
(Day 0 + 28±2) 
Follow-up Contact 
(Day 0 + 35±2) 
Premature 
Discontinuation 
Eligibility Assessment 
X 
 
 
 
 
 
 
 
Informed Consent 
X 
 
 
 
 
 
 
 
Medical History 
X 
 
 
 
 
X 
 
X 
Concomitant Medications 
X 
 
 
 
 
X 
 
 
Study Intervention 
 
X 
X 
X 
X 
X 
 
 
Adherence Evaluation 
 
 
X 
X 
X 
X 
 
 
Assessment of Adverse Events 
 
 
X 
X 
X 
X 
X 
X 
35-question PROMIS  
Gastrointestinal Symptom Scale 
(Appendix 1) 
 
X 
X 
X 
X 
X 
X 
X 
Clinical 
Laboratory 
Phlebotomy 
 
X 
 
 
 
X 
 
 
Research 
Laborato
ry 
Immunology - serum 
 
X 
 
 
 
X 
 
 
Microbiome - stool 
 
X 
 
 
 
X 
 
 

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Procedures 
Screening 
–Day -14 to 0) 
Baseline (Study 
Visit 1) 
Phone Contact 1 
(Day 0 + 7±2) 
Phone Contact 2 
(Day 0 + 14±2) 
Phone Contact 3 
(Day 0 + 21±2) 
Final Visit 
(Day 0 + 28±2) 
Follow-up Contact 
(Day 0 + 35±2) 
Premature 
Discontinuation 
Intestinal barrier 
function - serum 
 
X 
 
 
 
X 
 
 
 
 
 
7.2. Enrollment/Pre-Screening (Day -14 to 0) 
Screening  to determine eligibility and if applicable, consent processes will be performed virtually on 
zoom. Within 2 weeks, blood sample collection, food questionnaire and stool collection kit will be 
provided on day 0.  
7.3. Screening (Day -14 to 0) 
Activities to be performed at screening: 
(1) obtain consent,  
(2) schedule study visits 
(3) provide participant instructions 
 
7.4. Visit 1 (Day 0, Baseline) 
(1) schedule study visits/communications 
(2) provide participant instructions 
(3) collect blood sample for research only 
(4) provide food questionnaire link and food diary booklet 
(5) Gastrointestinal Symptom and Severity Checklist 
(6) provide stool collection kit and mailing instructions for return 
(7) provide intervention packets for 28 days 
 
7.5. Phone Contact (Day 0 +7±2, Day 0 + 14±2, Day 0 + 21±2, and Day 0 + 35±2 ) 
Information to be collected at days 7±2, 14±2 and 21±2: 
(1) Follow-Up Phone Script  
(2) Participant’s compliance with intervention (adherence) 
(3) Gastrointestinal Symptom and Severity Checklist - symptom rating 
(4) Record adverse events as reported by subject  
7.6. Final Visit (Day 0 +28±2) 
Visit 2 (V2) will be the final visit of the study. The following will be conducted during this visit: 
 

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(1) Collect blood sample 
(2) Provide food frequency questionnaire 
(3) Provide stool collection kit and mailing instructions for return 
(4) Gastrointestinal Symptom and Severity Checklist 
(5) Record adverse events as reported by subject  
(6) Record subject's compliance with intervention (adherence) 
(7) Collect leftover intervention packets 
 
7.7. Follow-Up Contact (Day 0 + 35±2) 
Participants will be contacted at Day 0 +35±2 to monitor overall status and record adverse events.  
(1) Follow-Up Phone Script  
(2) Gastrointestinal Symptom and Severity Checklist - symptom rating 
(3) Record adverse events as reported by subject  
 
 
7.8. Premature Discontinuation 
If a subject withdraws or the investigator discontinues subject participation after Visit 1, data from that 
visit will be included in the analysis of Objective 1, but not Objective 2 
7.9. Collection and Management of Tissue Specimens 
Sample Preparation:  
Stool samples: After collection, samples should be immediately placed in the provided DNA/RNA 
Shield™ Fecal Collection Tube (Zymo Research Catalog No. R1101). The procedures for sample collection 
and stabilization should be carried out as quickly as possible. Samples can be stored at ambient 
temperature (4°C-25°C) for ≤ 2 years and frozen (< -20°C) indefinitely. In the Core facility, samples will be 
stored at -80°C until processed. 
Blood samples: Samples will be collected on site using BD Vacutainer™ Venous Blood Collection Tubes: 
SST™ Serum Separation Tubes (FisherScientific Catalog No. 02-683-148) and stored at -80°C immediately 
after serum separation. 
Record Keeping and Monitoring: The following procedures will be applied for maintaining sample 
accountability and traceability and for ensuring regulatory compliance. 
1. 
Make sure the cryovial cap is secured tightly 
2. 
Review the patient’s Stool Sample Collection Log 
3. 
Complete the cryovial labels 
 
 
 
 
Patient #: ________________________________ 
 
Visit #  ________ and Date __________________ 
 
Collection place:     Home          or Study Site   

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4. 
Affix a label on each cryovial and put in plastic bag. Store the cryovials immediately in the -80°C 
freezer. 
5. 
Complete the stool sample tracking log (only if sample is kept) 
 
Sample Storage and Security:  
Study personnel will receive, assess for integrity and relevant information, and store stool samples 
mailed by participants. Blood samples will be collected on site (V1 and V2) and placed immediately in ice 
and stored at – 80°C. Stool and blood samples will be stored in the UNC Microbiome Core in a 
designated -80°C freezer (serial #: 0160203301161024, located in Taylor Hall, room 312C). Only study 
personnel will have access to the blood and stool samples. All samples will be stored at UNC MCF until 
the study is completed.  
8. Study Measurements and Evaluations 
8.1. Outcome Measures for Evaluation of Feasibility / Tolerability 
Tolerability will be assessed and recorded by the following parameters: 
(1) Occurrence of drop-out 
(2) Adherence/compliance 
(3) Side effects (bloating, flatulence, belching, abdominal pain, discomfort, watery stool, and 
vomiting) will be monitored every week via text, email, or phone call using the PROMIS 
Gastrointestinal Symptom Scale.  
8.2. Outcome Measures for Evaluation of Efficacy 
N/A 
 
8.3. Outcome Measures for Evaluation of Safety 
The incidence and severity of AEs will be assessed during the study at visit 2 and every week via email, 
phone call, and/or text messages. 
 
8.4. Other Outcomes in the Causal Pathway 
N/A 
 
8.5. Baseline Characteristics of the Participants 
Apparently healthy adults 18-55 years old. All participants will be nonsmoking and well-nourished 
according to standard anthropometric criteria.  
 

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Enrollment will not be restricted based on race, ethnicity, or gender. Subject population will reflect the 
population in the long-term care facility providing a broad selection of individuals to allow to enroll 
subjects from all races, ethnicities and genders as represented in North Carolina state. 
 
8.6. Variables Representing Treatment 
A total of 45 individuals will be randomized 1:1:1 (placebo, GOS, hGOS) to treatment. Allowing for a 20% 
drop out rate, approximately 55 - 60 subjects will be enrolled. 
 
9. Statistical Analysis Plans 
 
a. Strategies that Apply to all the Specific Aims 
(1) To help ensure replicability of the research, the analysis plans will be reviewed and finalized 
prior to collection of data (a priori). For the specific aim, the analysis plan specifies detailed 
steps for obtaining estimates of population parameters (e.g intervention safety) and for making 
inferences.   
(2) Sensitivity analyses will be performed to assess the robustness of the major results as indicated 
by their sensitivity to reasonable perturbations of the choices of the methods and assumptions 
used. Any question about the optimal choice of methods and assumptions are best handled by 
relegating competing approaches to roles in the domain of sensitivity analyses. Results of the 
sensitivity analyses will be used to guide our level of trust in the main results. Best practices for 
dealing with incomplete data will depend on the documented causes of missing, censored, and 
coarsened values. The reasons for missing data values, drop-out, and protocol departures will 
have been documented in/with the database.  
(3) The plan also includes outcome-dependent exploratory analyses to generate new hypotheses. 
(4) Descriptive graphical and tabular methods will be used to characterize the participants, visualize 
the data, and examine relationships among variables. 
(5) To indicate precision, all statistical estimates of population parameters will be tabulated along 
with corresponding confidence intervals (CI) or standard errors (SE). The CI will be interpreted as 
the set of potential values of the population parameter that are most compatible with the 
observed data.  
(6) All hypothesis tests yielding p-values that are deemed to be not statistically significant will be 
reported as being inconclusive. The proposed statistical analysis strategy acknowledges that no 
p-value can reveal the plausibility, presence, truth, or importance of an association or effect--
which is consistent with the statements of the American Statistical Association [4,5], the 
recommendations in Nature [6,7], and guidance, such as the CONSORT Statement [1], STROBE 
Statement [2], and ICMJE guidance [3].2]    
 
2 [1] www.consort-statement.org; [2] www.strobe-statement.org; [3] www.icmje.org; [4]  Wasserstein RL, et al. 
(2016), The ASA's Statement on p-Values, The American Statistician, 70:2, 129-133. [5] Wasserstein RL, et al. 
(2019), Moving to a World Beyond p < 0.05, The American Statistician, 73:sup1, 1-19. [6] Amrhein, et al. (2019) 
Scientists rise up against statistical significance, Nature 567, 305-307. [7] Editorial (2019) It’s time to talk about 
 

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9.1. Description of the Study Cohort 
A table will be used to summarize the participant cohort characteristics (age, gender) as well as basic 
dietary information. Figures will be used to feature microbiome analysis data and inflammatory 
biomarkers.  
9.2. Aim-Specific Plans 
Plans for Aim 1 (A1):  Validate 15 g/day dose safety and tolerability (absence of GI adverse effects) of 
GOS and hGOS in healthy adults. 
 
Method. We will report descriptive statistics for demographics, basic medical history, prescription and 
over the counter, and dietary supplements, including means and standard deviations for continuous 
variables, and percentages and ranges for categorical data. We will report the number and percent of all 
individuals (intent to treat) who had any adverse event (prompted or in response to an open-ended 
question). We will also report the number and percent of events over time, as well as the severity of the 
event. 
 
Statistical analysis for A1. Data will be presented as mean ± SD for variables that are normally 
distributed, or median (IQR) for variables not normally distributed. Binary/categorical outcomes will be 
reported as a percentage with confidence interval.  
 
Plans for Aim 2: Establish a biological signature of GOS and hGOS through assessment of prebiotics 
effects vs placebo on: 
 
(A2.1) Abundance of beneficial gut bacteria and restoration of the gut microbiome saccharolytic 
potential 
 
Method. Determination of abundance of beneficial bacteria (Bifidobacterium and Akkermansia species) 
in the gut microbiome of treatment and placebo groups and the microbiome saccharolytic potential by 
Whole Genome Shotgun (WGS) sequencing55. For assignment of saccharolytic (SAC) and non-
saccharolytic (NON_SAC) phenotypes we will use the reference study by Vieira-Silva et al.26 as 
described9.  
 
Statistical analysis (A2.1). we will perform both exploratory analysis and statistical testing for changes in 
plasma cytokines. Data will be presented as mean ± SD. To find the change of microbiome abundance 
under difference circumstance, Mann-Whitney-Wilcoxon matched pair tests between baseline and 4 
weeks follow-up will be performed for abundance of beneficial gut bacteria in each treatment arm. To 
compare the change of microbiome abundance in three arms, we will apply the Kruskal-Wallis test 
whether the change of microbiome abundance is the same among three groups. Due to multiple testing 
was proposed in A2.1, p values will be corrected for the total number of comparisons with a controlled 
false-discovery rate (FDR) 
 
 
ditching statistical significance:  Looking beyond a much used and abused measure would make science harder, but 
better. Nature 567, 283-283. 
 

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(A2.2) Modulation of biomarkers of inflammation 
 
Rationale. High-sensitivity C-reactive protein (hs-CRP) is a recognized marker of systemic inflammation34 
and specific pro-inflammatory cytokines mediate interaction between immune cells and non-immune 
cells, contributing to the inflammatory status of the intestine35, 36. Based on previous studies with 
probiotics and prebiotics that report potential immunomodulatory effects37-39, we decided to monitor 
hs-CRP, IL-1α, IL-1β, IL-2, IL-6, Il-8, IL-12, TNFα, and IFNγ for the duration of the clinical trial. 
 
Method. Determination of levels of biomarkers of inflammation (IL-1α, IL-1β, IL-2, IL-6, Il-8, IL-12, TNFα, and 
IFNγ) as well as high-sensitivity C-reactive protein, a marker of low-grade inflammation (hsCRP) in serum. We 
will determine cytokine levels using the MCYTOMAG-70K (Milliplex) in the Advanced Analytics Core (AAC) of 
the CGIBD. hsCRP concentration will be measured via hsCRP ELISA (Sigma Aldrich). 
 
Statistical analysis (A2.2). Similar to A2.1, we will perform an exploratory analysis of changes in plasma 
cytokines. Data will be presented as mean ± SD. To find the change of biomarker of interest under 
difference circumstance, paired t-test for matched pair between baseline and 4 weeks follow-up will be 
performed for levels of biomarkers in each treatment arm. To compare the change of biomarker level in 
three arms, we will use ANOVA to test whether the change of biomarker level is the same among three 
groups. Due to multiple testing, p values will be corrected for the total number of comparisons with a 
controlled false-discovery rate (FDR) 
 (A2.3) Evaluation of intestinal barrier function. 
 
Method. Blood samples will be drawn and serum will be used to quantify zonulin using the Zonulin (IDK® 
Zonulin) (Serum) ELISA test (Immundiagnostik AG). Zonulin is a biomarker of impaired intestinal 
permeability, which has been associated with various disorders56, 57. 
 
Statistical analysis (A2.3). Since A2.3 has similar data and analytic goal as A2.2, the analytic approach 
will be the same as what we proposed in A2.2. 
 
9.3. Planned Interim Analyses (If Applicable) 
We will review safety data (adverse events) continuously, however, no formal interim analyses are 
planned. 
9.4. Plans for Coping with Withdrawals and Loss-To-Follow-Up 
Enrolled participants who discontinue treatment/or withdrawn from study will not be replaced. Allowing 
for a 20% drop out rate, approximately 55 - 60 subjects will be enrolled. 
9.5. Sample Size Rationale 
Tolerability: The sample size for tolerability was not determined from power analysis but based on 
multiple previous clinical trials conducted with GOS58-61.  
 
Microbiome: Our previous studies10, 11, 31, 32 conducted with a different but equivalent GOS preparation 
(purified prebiotics) have demonstrated a consistent and statistically significant increased abundance of 
the targeted beneficial bacteria: Bifidobacterium and Akkermansia. The proposed pilot study will recruit 

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60 subjects. After estimated dropout rate of 20%, the final subjects that will complete the subject will be 
N=48, which is 16 for each arm. This sample size is chosen given limited funds available with the pilot 
award supporting this project. 
 
Biomarkers of inflammation: Since this is the first study, we do not have the estimate of effect size so 
the power cannot be computed. Additionally, due to the limitation of the funding with n=20, we do not 
expect to have sufficient power to detect differences over time. In that case, we will perform an 
exploratory analysis of changes in plasma cytokines and biomarkers of inflammation and compare the 
summary statistics such as mean and standard error.  
 
9.6. Missing data 
We expect a 20% drop out during the proposed study. We assume the subjects will drop out randomly 
and all the missing data is missing completely at random (MCAR). In this case, we will implement 
complete-case analysis for the primary statistical analysis. However, we will also explore the possibility 
that the missing data is not MCAR. In the sensitivity analysis, we will compare the drop-out and non-
drop group in terms of demographic characteristics and clinical factors. More specifically, we will 
perform two-group testing between the drop-out and non-drop group using either t-test, Wilcoxon test 
or chiq-square test depending on distribution and type of the variables of interest. If the missing data is 
indeed missing at random (MAR), we will perform multiple imputation method to generate the missing 
values with Rubin’s rule to obtain the unbiased inference, which will be compared with results from the 
primary statistical results.  
 
10. Safety Monitoring and Management 
10.1. Risk / Benefit Assessment 
Potential Risks: Subjects in the GOS and hGOS groups will consume 7.5-15g/day of the 
prebiotics  or placebo preparations as a drink. Possible side effects of the GOS and hGOS groups 
may include mild to moderate bloating, gas (flatulence), belching, abdominal pain or discomfort, 
and vomiting. Individuals will be withdrawn from the study if treatment leads to severe side 
effects.   
 
Potential Benefits: Substances defined as “prebiotics” (a substrate that is selectively utilized by 
host microorganisms conferring a health benefit per the latest definition of the International 
Scientific Association for Probiotics and Prebiotics, ISAPP) have a demonstrated beneficial effect 
in humans and animals. Galacto-oligosaccharides (GOS) in particular have been shown to 
alleviate symptoms of lactose intolerance and other minor gastrointestinal manifestations in 
adequate doses.  
 
A compromised gut barrier, dysbiosis and inflammation are hallmarks of the aging gut with 
consequences that can range from mild GI manifestations like constipation, abdominal pain, and 
flatulence, to disorders of the gut-brain axis and other conditions not directly associated with 
the GI tract. This study will add to the knowledge of beneficial modulation of the microbiota to 
mediate restoration of the gut barrier and biomarkers of inflammation. A positive outcome from 

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this study will further advance research to improve nutritional conditions and overall health of 
older individuals in long-term care.  
 
10.2. Assessment of Safety 
Participants will be withdrawn if during the first 10 days of study participation, they experience a 2 level 
change in one or more of the most reported side effects (bloating, flatulence, watery stools, abdominal 
pain as assessed by questions 2 (abdominal pain), 6 (abdominal discomfort), 8 (constipation), 16 
(diarrhea), 22 (abdominal distention), 26 (abdominal bloating), 31 (nausea), or 33 (vomiting) on the 
PROMIS Gastrointestinal Symptom Scale as compared to their baseline responses. Participants who have 
surpassed 10 days of study participation and experience a 2 level change in one or more of the most 
reported side effects (as described above) as compared to baseline, they will be asked to reduce the 
dose of study product to one sachet of 7.5 mg per day. The study product can be taken all at once or 
distributed between two time points. If the PROMIS Gastrointestinal Symptom Scale does not improve 
upon reducing the dose, the participant will be withdrawn from the study. If 5 or more of the first 20 
subjects enrolled experience a 2 level change in the most commonly reported side effects (bloating, 
flatulence, watery stools, abdominal pain) compared to baseline or higher adverse event leading to 
subject discontinuation, we will stop the study temporarily and request the medical monitor to do an 
unblinded assessment of the data. The medical monitor (unblinded) will assess subjects who 
discontinued due to the specified adverse effects to determine if they are all in one arm of the study or 
range across arms.  The medical monitor will assess if there is a safety signal in a specific arm or 
treatment dose over the other that warrants the closure of the study. Since the medical monitor will be 
the person determining continuation or discontinuation of the study, the study team will remain blinded 
and unbiased. 
 
Participants who choose to withdraw from the study will be followed up (via text or email 
communication) to determine safety and efficacy outcomes. Reasons for discontinuation will be 
documented in the study RedCap database. 
10.3. Unanticipated Problems, Adverse Events, Serious Adverse Events 
 
Unanticipated Problems: An unanticipated problem is any incident, experience or outcome that 
meets all three OHRP criteria (1) unexpected (in severity, specificity, frequency, or nature), (2) 
related or possibly related to the research, and (3) suggests the research places subjects or others at 
greater risk than previously known or recognized.  
 
Adverse Event (AE) Definitions: An AE is “any untoward medical occurrence associated with the 
use of a drug in humans, whether or not considered drug related.”  For the purposes of this 
definition, “untoward” means unfavorable, negative, or harmful.  An AE is any event observed or 
reported that is associated with the intervention, without regard to causality. 
 
Serious Adverse Events (SAE) Definition: An AE or suspected adverse reaction will be 
considered “serious” if, in the view of either the Investigator or Sponsor, it results in any of the 
following outcomes: 
• 
Death; 
• 
Is life-threatening (immediate risk of death); 

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• 
Requires inpatient hospitalization or prolongation of existing hospitalization; 
• 
Results in persistent or significant incapacity or substantial disruption of the ability to 
conduct normal life functions;  
• 
Results in congenital anomaly/birth defect; 
• 
Is an important medical event that may require an intervention to prevent one of the 
outcomes listed above. 
 
Grading the Severity of Adverse Events and Events of ‘Special Interest’:  
The severity or intensity of an AE describes the degree of impact upon the subject and/or the 
need for, and the extent of medical care required to treat the AE. The Investigator will use the 
adjectives MILD, MODERATE, or SEVERE to describe the maximum intensity of the AE.  For 
purposes of consistency, these intensity grades are defined as follows: 
MILD 
Does not interfere with subject’s usual function. 
MODERATE 
Interferes to some extent with subject’s usual 
function. 
SEVERE 
Interferes significantly with subject’s usual function. 
 
Relatedness Definition:  
 
The Investigator will assess relatedness for all AEs (serious and non-serious).  Factors considered 
when assessing the relationship of an event to the investigational product: 
(1) Alternative etiology – Is the event due to the underlying disorder being studied or to 
another known disorder?   
(2) Known relationship – Has the event been observed before in patients treated with this 
investigational product or similar products? 
(3) Temporal relationship – Is there a reasonable temporal relationship between the time 
of onset of the event and the administration of the investigational product? 
(4) Concomitant medication – Is the event a known side effect of a concomitant 
medication? 
 
Based on the previous factors we will consider the following descriptions when assessing 
relationship of an event to the investigational product: 
 
Definite:  There is a reasonable causal relationship between the investigational product and the 
AE, when the event responds to withdrawal of the investigational product (de-challenge) and 
recurs with re-challenge by administration of the investigational product.  
Probable:  There is a reasonable causal relationship between the investigational product and the 
AE.  The event responds to de-challenge.  Re-challenge not required. 
Possible:  There is a reasonable causal relationship between the investigational product and the 
AE.  De-challenge is lacking or unclear.   
Not Likely:  There is a temporal relationship between the investigational product administration 
and the AE, but there is not a reasonable causal relationship between investigational product 
and the event.    
Unrelated:  There is not a temporal or causal relationship to the investigational product 
administration.  
 
The Investigator will record causal relationship in the source documents, as appropriate, and 
report such an assessment in accordance with the SAE reporting requirements, if applicable.   

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Expectedness Definition: An assessment of expectedness will be performed for each AE and 
SAE. Previously reported expected effects of prebiotic administration include abdominal pain 
(cramps), bloating, flatulence (gas) and diarrhea (loose stools >3 times/day).  During the study, 
GI side effects will be expected and only Aes that are meet the definition of severe AE or 
considered an SAE will be reported to the IRB or FDA. 
 
AE and SAE Assessment, Follow-up Procedures: The Investigator will record all directly 
observed AEs and all AEs spontaneously reported by the study subject beginning from the time 
the subject begins use of the investigational product through 7 days after the last dose of the 
investigational product.  Changes in health status after the subject has provided informed 
consent but before the subject begins use of the investigational product shall be recorded as 
changes in the medical history.   
The Investigator will assess subjects at each visit for the occurrence of AEs.  In order to avoid 
bias in eliciting AEs, subjects should be asked the following non-leading question: 
“Have there been any changes in your health since your last visit?”   
 
All AEs (serious and non-serious) reported by the subject must be recorded regardless of if a 
causal relationship with the investigational product is suspected.  Each AE will be assessed to 
determine if it meets the criteria for a serious adverse event (SAE).  If an SAE occurs, expedited 
reporting will follow local and international regulations as appropriate.   
 
Reporting and Documentation Procedures: AE and SAE reporting periods will extend from 
the signature of the informed consent through the End of the Study Visit or until 7 days after the 
last dose of the investigational product. If an SAE occurs, Dr. Becker-Dreps will be notified within 
24 hours of awareness of the event by the Investigator.   This timeframe also applies to adding 
new information (follow-up) on previously forwarded SAE reports. In the rare event that the 
Investigator does not become aware of the occurrence of an SAE immediately, the Investigator 
will report the event within 24 hours of first awareness of the SAE and document the time of 
his/her first awareness of the SAE. For all SAEs, the Investigator will pursue and provide 
information to the Sponsor or designee in accordance with the timeframes for reporting 
specified above.  This will generally include a description of the AE in sufficient detail to allow for 
a complete medical assessment of the case and independent determination of possible 
causality.  Information on other possible causes of the event, such as concomitant medications 
and illnesses must be provided.  All observed or volunteered AEs regardless of treatment group 
or suspected causal relationship to the investigational product(s) will be reported.  
 
Participant Notification of New Information:  
 
Participant will be notified via email, text message and/or phone call.  
10.4. Safety Monitoring 
Investigators will assess subjects at each visit and over the phone or via text (or email) each week of the 
study at days 0, 7±2, 14±2, 21±2, and 28±2 as well as day 7±2 post-intervention. If AEs as defined in this 
protocol are identified (a 2 level change in one or more of the symptoms as compared to baseline as 
assessed by questions 2 (abdominal pain), 6 (abdominal discomfort), 8 (constipation), 16 (diarrhea), 22 
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Gastrointestinal Symptom Scale). The treatment will be first be reduced to one 7.5g sachet per day 
(taken either at once or divided between two time periods if the participant has been participating for > 
10 days. The treatment will be discontinued if the participant is in the first 10 days of participation, and 
the AE will be recorded. Also, participants who report severe side effects (interfering significantly with 
subject’s usual function) will be withdrawn from the study immediately. If 5 or more of the first 20 
subjects enrolled experience a 2 level change in the most reported side effects (bloating, flatulence, 
watery stools, abdominal pain as assessed by questions 2 (abdominal pain), 6 (abdominal discomfort), 8 
(constipation), 16 (diarrhea), 22 (abdominal distention), 26 (abdominal bloating), 31 (nausea), or 33 
(vomiting) on the PROMIS Gastrointestinal Symptom Scale compared to baseline or higher adverse 
event leading to subject discontinuation, we will stop the study temporarily and request the medical 
monitor to do an unblinded assessment of the data. 
 
10.5. Study Suspension / Early Termination of the Study 
Reports of severe adverse events interfering significantly with daily function in >20% of participants will 
lead to early termination of the study. Premature termination of this study may also occur because of 
unexpected, significant or unacceptable risk to participants, incomplete or unevaluable data, 
determination of futility, a regulatory authority decision, change in opinion of the IRB, or intervention 
safety problems. All investigators in the study will be notified of events that may warrant early 
termination of the study.   
 
11. Supporting Documentation and Operational 
Considerations 
11.1. Regulatory, Ethical, and Study Oversight Considerations 
11.1.1. 
Informed Consent Process 
11.1.1.1. Consent/Assent and Other Informational Documents Provided 
to Participants 
Consent forms describing in detail the study intervention, study procedures, and risks will be provided to 
the participant virtually and written documentation of informed consent is required prior to starting 
intervention/administering study intervention.  The following consent materials are submitted with this 
protocol: 
 
Adult Consent Form 
Consent Addendum for Unencrypted Communication  
11.1.1.2. Consent Procedures and Documentation 
Informed consent is a process initiated prior to the individual’s agreeing to participate in the study and 
continues throughout the individual’s study participation. Consent forms will be approved by the 
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consent process will occur virtually over zoom. The investigator will explain the research study to the 
participant and answer any questions that may arise. A verbal explanation will be provided suited to the 
participant’s comprehension of the purposes, procedures, and potential risks of the study and of their 
rights as research participants.  Participants will be able to carefully review the written consent form and 
ask questions before signing. The participants should have the opportunity to discuss the study with 
their family or surrogates or think about it prior to agreeing to participate. The participant will sign the 
informed consent document electronically prior to any procedures being done specifically for the study. 
Participants must be informed that participation is voluntary and that they may withdraw from the 
study at any time without prejudice. The participants will be sent an electronic copy and given a printed 
copy of the informed consent document for their records. The informed consent process will be 
conducted and documented in the source document (including the date), and the form signed before 
the participant undergoes any study-specific procedures.  
 
 
11.2. Study Discontinuation and Closure 
This study may be suspended or prematurely terminated if there is sufficient reasonable cause.  Written 
notification, documenting the reason for study suspension or termination, will be provided by the 
suspending, or terminating party to study participants, investigator, funding agency, and regulatory 
authorities.  If the study is prematurely terminated or suspended, the Principal Investigator (PI) will 
promptly inform study participants, the Institutional Review Board (IRB), and sponsor and will provide 
the reason(s) for the termination or suspension.  Study participants will be contacted, as applicable, and 
be informed of changes to study visit schedule. 
  
Circumstances that may warrant termination or suspension include, but are not limited to: 
• 
Determination of unexpected, significant, or unacceptable risk to participants 
• 
Demonstration of efficacy that would warrant stopping    
• 
Insufficient compliance to protocol requirements 
• 
Data that are not sufficiently complete and/or evaluable 
• 
Determination that the primary endpoint has been met 
• 
Determination of futility 
 
Study may resume once concerns about safety, protocol compliance, and data quality are addressed, 
and satisfy the sponsor, and the IRB. 
 
11.3. Confidentiality and Privacy 
Participant confidentiality and privacy is strictly held in trust by the participating investigators, their 
staff, and the sponsor. This confidentiality is extended to cover testing of biological samples in addition 
to the clinical information relating to participants. Therefore, the study protocol, documentation, data, 
and all other information generated will be held in strict confidence. No information concerning the 
study or the data will be released to any unauthorized third party without the prior written approval of 
the sponsor.  
 
All research activities will be conducted in as private a setting as possible. 
 

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The study monitor, other authorized representatives of the sponsor, representatives of the Institutional 
Review Board (IRB), regulatory agencies, or pharmaceutical companies supplying the study product may 
inspect all documents and records required to be maintained by the investigator. The clinical study site 
will permit access to such records. 
 
The study participant’s contact information will be securely stored at the site for internal use during the 
study. At the end of the study, all records will continue to be kept in a secure location for as long a 
period as dictated by the reviewing IRB, Institutional policies, or sponsor requirements. 
 
Study participant research data, which is for purposes of statistical analysis and scientific reporting, will 
be transmitted to and stored in RedCap. This will not include the participant’s contact or identifying 
information. Rather, individual participants and their research data will be identified by a unique study 
identification number. The study data entry and study management systems used by clinical sites and by 
research staff will be secured and password protected. At the end of the study, all study databases will 
be de-identified and archived. 
 
Certificate of Confidentiality (if applicable) 
To further protect the privacy of study participants, a Certificate of Confidentiality will be issued by the 
National Institutes of Health (NIH).  This certificate protects identifiable research information from 
forced disclosure. It allows the investigator and others who have access to research records to refuse to 
disclose identifying information on research participation in any civil, criminal, administrative, legislative, 
or other proceeding, whether at the federal, state, or local level. By protecting researchers and 
institutions from being compelled to disclose information that would identify research participants, 
Certificates of Confidentiality help achieve the research objectives and promote participation in studies 
by helping assure confidentiality and privacy to participants. 
11.4. Future Use of Stored Specimens and Data 
Data collected for this study will be analyzed by the investigators of this study and stored in RedCap. 
After the study is completed, de-identified, sequencing microbiome data will be submitted to the 
Sequence Read Archive (SRA) at the National Center for Biotechnology Information (NCBI), for use by 
other researchers including those outside of the study. Permission to transmit data will be included in 
the informed consent.  
 
With the participant’s approval and as approved by local Institutional Review Board (IRB), de-identified 
biological samples will be stored at the UNC Microbiome Core. These samples could be used to research 
the gut microbiome and intestinal permeability, and other conditions for which older adult individuals 
are at increased risk.  
 
During the study, an individual participant can choose to withdraw consent to have biological specimens 
stored for future research. However, withdrawal of consent with regard to biosample storage may not 
be possible after the study is completed.  
 
11.5. Key Roles and Study Governance 
Principal Investigator 

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Sylvia Becker Dreps, MD, MPH 
UNC – Chapel Hill  
590 Manning Drive, CB # 7595 
Chapel Hill, NC 27599-7595 
(984)-974-4888; (919) 943-7445 
sbd@email.unc.edu 
 
Overall structure of the study team  
Dr. Sylvia Becker Dreps, MD, MPH is the Principal Investigator in the proposed studies. 
Dr. Andrea Azcarate-Peril, PhD is a collaborator in the proposed studies and will assist with data analysis 
of de-identified data.  
Dr. Vivan Lee MD (Co-investigator) is a clinical gastroenterologist with experience in patients with a 
broad spectrum of gastrointestinal conditions including colon polyps, gastrointestinal bleeding, 
gastrointestinal infections, heartburn, abdominal pain, dyspepsia, IBD, constipation and diarrhea. She 
will serve as a medical monitor if needed during the study. 
Ms. Kelli Hammond (Clinical research coordinator) will assist with day to day study activities, consent 
process, participant contact, and data entry. 
\ 
Dr. Roach, Ph.D. (Co-investigator, Bioinformatician) and Dr. Kia, Ph.D. (Co-investigator, Bioinformatics, 
and biostatistics specialist) will provide support on data analysis and interpretation. 
 
 
11.6. Safety Oversight 
 
Safety oversight will be under the direction of the study’s principal investigator.. 
 
11.7. Clinical Monitoring 
Clinical site monitoring is conducted to ensure that the rights and well-being of trial participants are 
protected, that the reported trial data are accurate, complete, and verifiable, and that the conduct of 
the trial follows the currently approved protocol/amendment(s), with International Conference on 
Harmonization Good Clinical Practice (ICH GCP), and with applicable regulatory requirement(s).  
 
• 
The Medical Monitor or designee will perform monitoring of the study in cooperation with the 
Investigator, including review of AE reports.  
• 
When additional information is required, the Medical Monitor will contact the Investigator or 
designee. 
 

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11.8. Quality Assurance and Quality Control  
The site will perform internal quality management of study conduct, data and biological specimen 
collection, documentation, and completion.  A quality management plan will be developed to describe a 
site’s quality management. 
 
Quality control (QC) procedures will be implemented beginning with the data entry system and data QC 
checks that will be run on the database will be generated. Any missing data or data anomalies will be 
communicated for clarification/resolution. 
 
Following written Standard Operating Procedures (SOPs), the PI will verify that the clinical trial is 
conducted and data are generated, and biological specimens are collected, documented (recorded), and 
reported in compliance with the protocol, International Conference on Harmonization Good Clinical 
Practice (ICH GCP), and applicable regulatory requirements (e.g., Good Laboratory Practices (GLP), Good 
Manufacturing Practices (GMP)).  
 
The investigational site will provide direct access to all trial related sites, source data/documents, and 
reports for the purpose of monitoring and auditing by the sponsor, and inspection by local and 
regulatory authorities. 
 
11.9. Data Handling and Record Keeping 
11.9.1. 
Data Collection and Management Responsibilities 
Data collection is the responsibility of the clinical trial staff at the site under the supervision of the site 
investigator. The investigator is responsible for ensuring the accuracy, completeness, legibility, and 
timeliness of the data reported. 
 
All source documents should be completed in a neat, legible manner to ensure accurate interpretation 
of data.   
 
Hardcopies of the study visit worksheets will be provided for use as source document worksheets for 
recording data for each participant enrolled in the study.  Data recorded in the electronic case report 
form (eCRF) derived from source documents should be consistent with the data recorded on the source 
documents.  
 
Clinical data (including adverse events (AEs), concomitant medications, and expected adverse reactions 
data) and clinical laboratory data will be entered into RedCap a 21 CFR Part 11-compliant data capture 
system provided by UNC Chapel Hill. The data system includes password protection and internal quality 
checks, such as automatic range checks, to identify inconsistent, incomplete, or inaccurate data. Clinical 
data will be entered directly from the source documents. 
 
 

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11.9.2. 
Study Records Retention 
Study documents should be retained for a minimum of 2 years after the last approval of a marketing 
application in an International Conference on Harmonization (ICH) region and until there are no pending 
or contemplated marketing applications in an ICH region or until at least 2 years have elapsed since the 
formal discontinuation of clinical development of the study intervention. These documents should be 
retained for a longer period, however, if required by local regulations. No records will be destroyed 
without the written consent of the sponsor, if applicable. It is the responsibility of the sponsor to inform 
the investigator when these documents no longer need to be retained. 
 
11.10. Protocol Deviations 
A protocol deviation is any noncompliance with the clinical trial protocol, International Conference on 
Harmonization Good Clinical Practice (ICH GCP), or Manual of Procedures (MOP) requirements. The 
noncompliance may be either on the part of the participant, the investigator, or the study site staff. As a 
result of deviations, corrective actions are to be developed by the site and implemented promptly.  
These practices are consistent with ICH GCP:  
• 
4.5 Compliance with Protocol, sections 4.5.1, 4.5.2, and 4.5.3  
• 
5.1 Quality Assurance and Quality Control, section 5.1.1  
• 
5.20 Noncompliance, sections 5.20.1, and 5.20.2.  
It will be the responsibility of the site investigator to use continuous vigilance to identify and report 
deviations within 2 working days of identification of the protocol deviation, or within 2 working days of 
the scheduled protocol-required activity. Protocol deviations will be sent to the reviewing Institutional 
Review Board (IRB) per their policies. The site investigator will be responsible for knowing and adhering 
to the reviewing IRB requirements. Further details about the handling of protocol deviations will be 
included in the MOP.  
11.11. Publication and Data Sharing Policy  
This study will be conducted in accordance with the following publication and data sharing policies and 
regulations: 
National Institutes of Health (NIH) Public Access Policy, which ensures that the public has access to the 
published results of NIH funded research. It requires scientists to submit final peer-reviewed journal 
manuscripts that arise from NIH funds to the digital archive PubMed Central upon acceptance for 
publication. 
This study will comply with the NIH Data Sharing Policy and Policy on the Dissemination of NIH-Funded 
Clinical Trial Information and the Clinical Trials Registration and Results Information Submission rule. As 
such, this trial will be registered at ClinicalTrials.gov, and results information from this trial will be 
submitted to ClinicalTrials.gov. In addition, every attempt will be made to publish results in peer-
reviewed journals.  Data from this study may be requested from other researchers 10 years after the 
completion of the primary endpoint by contacting Dr. Becker-Dreps. 
In addition, this study will comply with the NIH Genomic Data Sharing Policy, which applies to all NIH-
funded research that generates large-scale human or non-human genomic data, as well as the use of 
these data for subsequent research.  

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11.12. Conflict of Interest Policy 
The independence of this study from any actual or perceived influence, such as by the pharmaceutical 
industry, is critical.  Therefore, any actual conflict of interest of persons who have a role in the design, 
conduct, analysis, publication, or any aspect of this trial will be disclosed and managed. Furthermore, 
persons who have a perceived conflict of interest will be required to have such conflicts managed in a 
way that is appropriate to their participation in the design and conduct of this trial.  The study 
leadership in conjunction with the North Carolina Translational and Clinical Sciences Institute and UNC 
Chapel Hill has established policies and procedures for all study group members to disclose all conflicts 
of interest and will establish a mechanism for the management of all reported dualities of interest. 
12. Additional Considerations 
This section should include a description of any additional considerations not currently covered in this 
protocol template, such as particular institutional or IRB-related requirements.  
 
<Insert text> 
 
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supplementation in prefrail older and healthy adults increased faecal bifidobacteria, but did not impact 
immune function and oxidative stress. Clin Nutr, 2021. 40(5): p. 3019-3031. 
 
 
 
14. List of Appendices 
This section should include a listing of any tables, questionnaires, investigator brochure, device manual, 
subject ‘handouts’, etc that should accompany the study protocol.   
 
Appendix 1: 38-point Gastrointestinal Symptom and Severity Checklist (GSSC) 
 
Appendix 2: Food frequency questionnaire. 
 
Appendix 3: [include version number and date, and a short description.]  

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