Prebiotics and Metformin Influences Gut and Hormones in Type 2 Diabetes Youth (MIGHTY)
Phase: PHASE2
Conditions: Diabetes
Sponsor: National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)

Principal Investigator: Chung, Stephanie T.  
Last updated: February 9, 2021
Page 1
Clinical Research Protocol
National Institute of Diabetes and Digestive and Kidney Diseases
Protocol Number:
Protocol Version: 
[IND] [IDE] NUMBER:  N/A 
[IND] [IDE] NAME:  N/A 
[IND] [IDE] HOLDER:  N/A 
[IND] [IDE] MFG:  N/A 
Title: Prebiotics and Metformin Influences Gut and Hormones in Type 2 Diabetes Youth (MIGHTY-Fiber)
Short Title: MIGHTY - Fiber
Identifying Words: Gastrointestinal side effects, Metformin, Type 2 Diabetes, Youth
Principal Investigator:           Stephanie T. Chung, M.B.B.S. 
NIDDK
Estimated Duration of Study: 4 years
Start Date: Summer 2019
End Date: Summer 2023
Number and Type of Patients:  Accrual Ceiling: 50
Number
Sex
Age Range
Ethnic Group
Patients
50
Male/Female
10-25 years
All 
Project Uses Ionizing Radiation: 
_X__Research indicated:      RSC Approval Number:
2741
 Expiration Date: 06/27/2022
Project Uses “Durable Power of Attorney”: No                        Off-site Project: No
Multi-site enrollment: No
Multi-institutional project: No
Contents
Precis.......................................................................................................................................................................4
1.0
Background .................................................................................................................................................4
2.0
Specific Objectives/ Aims...........................................................................................................................6
2.1 Study Outcomes .................................................................................................................................................................7
3.0
Study Design................................................................................................................................................7
3.1 Study Design and Timeline................................................................................................................................................7
3.2 Study Protocol....................................................................................................................................................................8
3.2.1 Study Procedures ......................................................................................................................................................10
3.3 Inclusion and Exclusion Criteria......................................................................................................................................11
3.4 Randomization .................................................................................................................................................................12
3.5 Study Agents/ Intervention ..............................................................................................................................................13

Principal Investigator: Chung, Stephanie T.  
Last updated: February 9, 2021
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3.6 Follow-up and Post-Study Treatment ..............................................................................................................................15
4.0 Clinical and Laboratory Methods................................................................................................................15
4.1 Microbiome Analysis.......................................................................................................................................................15
4.2 Short Chain Fatty Acid Analysis .....................................................................................................................................16
5.0 Statistical and Power Analyses.....................................................................................................................16
5.1 Sample Size Justification .................................................................................................................................................16
5.2 Analysis of Primary and Secondary Outcomes................................................................................................................16
6.0 Collection and Storage of Human Specimens or Data ...............................................................................16
6.1 Research Use, Storage and Disposition of Human Subjects’ Samples............................................................................16
6.2 Materials Transfer Information........................................................................................................................................17
6.3 Genomic Data Sharing Plan.............................................................................................................................................17
7.0 Multiple-site Studies ......................................................................................................................................17
8.0 Human Subject Protection............................................................................................................................17
8.1 Investigators and Roles ....................................................................................................................................................17
8.2 Informed Consent/Assent Procedures..............................................................................................................................17
8.2.1 Language for Minors.....................................................................................................................................................17
8.2.2 Telephone consenting ...................................................................................................................................................18
8.3 Rationale for Subject Selection........................................................................................................................................18
8.4 Recruitment Strategies .....................................................................................................................................................19
8.5 Risks/Benefits Analysis including Considerations of Alternatives to Participation........................................................19
9.0 Adverse Event Reporting..............................................................................................................................21
9.1 Event Characterization and Reporting to the IRB ...........................................................................................................21
9.2 Data and Safety Monitoring Plan.....................................................................................................................................22
9.3 Monitoring Subjects and Criteria for Withdrawal of Subjects from the study ................................................................22
9.4 Protection of Participant’s Privacy and Confidentiality ..................................................................................................22
9.5 Investigational New Drug Determination........................................................................................................................23
10.0 Renumeration...............................................................................................................................................23
11.0 Conflict of Interest.......................................................................................................................................23
Appendix A: Sample GI Symptom Questionnaire in RedCap........................................................................24
Appendix B: Continuous glucose monitoring (CGM) and blood glucose management instructions ..........25
Appendix C: Stool Collections............................................................................................................................28
Appendix D: Quality of Life Questionnaire (PedsQL).....................................................................................29
Appendix E:  Keeping Food Records.................................................................................................................30
Appendix F: NIDDK IND Determination Checklist for Marketed Drugs.....................................................35
Appendix G: Non-nutritive Sweetener Avoidance Handout ...........................................................................37
References.............................................................................................................................................................39

Principal Investigator: Chung, Stephanie T.  
Last updated: February 9, 2021
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Precis
Metformin is the most widely prescribed anti-diabetes medication in the world and the first-line therapy for treating type 2 
diabetes (T2D) in youth and adults.  However, metformin’s glucose-lowering ability is variable in clinical practice, and 
efficacy is further limited by poor medication adherence because of metformin-associated adverse effects. Gastrointestinal 
(GI) symptoms such as bloating, abdominal discomfort, cramping, and diarrhea are the most common side effects 
associated with metformin use occurring in up to 80% of individuals at drug initiation and up to 30% in individuals on 
chronic treatment.  In youth with type 2 diabetes, the burden of metformin-associated side effects is high because 
metformin is the only oral FDA-approved for treatment and there are no other oral alternatives.  Therefore, identifying 
ways to mitigate these GI side effects, especially in youth with type 2 diabetes, is of high clinical significance. New data 
suggest that metformin-induced changes in the gut and/ or the microbiome may be related to both its beneficial (glucose-
lowering) and adverse effects. To address this clinical challenge, prebiotic fibers that are non-digestible food ingredients, 
may help to improve metformin tolerability by increasing beneficial bacteria and stool metabolites, such as short chain 
fatty acid (SCFA) stool concentrations. This pilot study will test the hypothesis that a prebiotic microbiome modulator 
(MM) – containing prebiotic fibers and polyphenols – will reduce GI side effects of metformin at time of initiation and 
change the stool metabolite profile in youth and young adults with T2D treated with metformin, age 10-25 years who are 
not on insulin therapy. The 9-week study will have 2 phases and 6 outpatient visits at the NIH Clinical Center. Phase 1 is a 
5-week randomized double blind cross-over trial with two 1-week intervention periods (metformin + prebiotic and 
metformin + placebo) during which subjects will eat a standardized diet.  Phase 2 will occur immediately following phase 
1 in which participants will start an open-label 4-week intervention with metformin and the prebiotic MM.  
1.0 Background
Metformin is the most widely prescribed anti-diabetic agent in the world and is first line for treatment of type 2 diabetes 
(T2D) in both adults and children [1, 2] . However, metformin non-responsiveness is an important clinical challenge, 
occurring in 20-50% of youth and adults [3], and reduced treatment adherence is a well-recognized and potentially 
modifiable risk factor [4]. Metformin related gastrointestinal (GI) side effects (bloating, diarrhea, cramping, nausea and 
vomiting) are a common barrier to maximal 
dose escalation and treatment optimization [5]. 
These side effects are also a frequently 
reported reason for non-adherence [5]. Side 
effects are observed in > 80% of individuals 
newly initiated on metformin and ~10-30% of 
patients on long-term therapy [6, 7], with 
estimates of 1 in 4 youth taking metformin 
having at least one GI side effect [8]. 
Challenges are magnified in youth with T2D, 
as metformin is the only oral medication that is 
approved for use in the 10-17-year age group. 
Yet, there is a paucity of studies examining the 
mechanisms of metformin’s GI side effects in 
vivo, and thus limited ability to mitigate these 
Figure 1Potential mechanisms of metformin-related changes in gut metabolites and lower 
GI side effects. H2S: hydrogen sulfide, GLP-1: glucagon-like peptide 1,FXR: farnesoid X 
receptor (References: 14-16)

Principal Investigator: Chung, Stephanie T.  
Last updated: February 9, 2021
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issues. This pilot study, a randomized double-blind crossover trial, examines the use of a prebiotic as an adjunct to 
improving metformin tolerability, facilitating short-term dose escalation, and also explores the underlying gut-based 
mechanisms of both metformin action and intolerance in youth and young adults with type 2 diabetes.
Metformin-induced changes in the microbiome are emerging as an important primary mechanistic target. Initial studies in 
adults suggest that short-term metformin therapy increases production of short chain fatty acids (SCFAs) and incretins 
(glucagon-like peptide-1 [GLP-1] and peptide YY [PYY]) [9, 10] by increasing populations of SCFA-producing bacteria, 
such as Akkermansia muciniphila and Bifidobacterium adolescentis [11-13]. However, other changes in the gut milieu 
may also contribute to the adverse GI side effects commonly seen with metformin use, but few studies have resolved the 
cellular mechanisms that cause of metformin’s GI side effects. Figure 1 illustrates three (3) hypothetical mechanisms of 
metformin-induced changes in the gut microbiome that may contribute to its GI symptoms. For example, metformin-
induced inhibition of colonic bacterial glycerophosphate dehydrogenase increases colonic lactate acidosis that may be 
toxic to the gut mucosa [14]. Alternatively, high concentrations of stool bile acids, secondary to direct farnesoid X 
receptor (FXR) inhibition by metformin, may cause osmotic diarrhea [15]. Additionally, metformin-treated patients have 
increased abundance of Escherichia sp that was associated with functional shifts with enrichment of virulence factors and 
gas metabolism genes, specifically for the production of hydrogen sulfide gas [16].  
While the precise mechanism of 
metformin-induced GI side effects 
remains to be elucidated, microbiome-
influencing supplements may benefit 
patients with diabetes. Even without other 
medications, prebiotic fiber has been 
shown to improve the metabolic profile in 
patients with and without diabetes  [17-
19]. Prebiotics are substrates for bacterial 
fermentation and SCFA production 
(Figure 2) and are defined as 
“nondigestible food ingredients that 
beneficially affect the host by selectively 
stimulating the growth and/or activity of one or a limited number of bacteria in the colon, thus improving host health” 
[20]. These supplements are effective in reducing the severity and duration of infectious diarrhea [21], as well as 
improving bowel regularity. Prebiotics may also improve intestinal permeability and gut inflammation – two factors 
which have been linked to obesity and metabolic syndrome [22, 23]. However, prebiotic supplements, when used in 
isolation, could increase flatulence by producing methane and hydrogen sulfide gas as byproducts via methanogen and 
sulfate-reducing bacteria, respectively (Figure 2) [24]. To decrease these unwanted side effects, preliminary studies have 
demonstrated that using polyphenols -- naturally occurring compounds that are metabolized by the SCFA-producing 
bacteria (acetogens)-- may be helpful in reducing flatulence (Figure 2) [25]. The presence of polyphenols, commonly 
found in fruits and vegetables, allows acetate-producing bacteria to thrive, while methane- or hydrogen sulfate-producing 
bacteria have less available hydrogen substrate to convert to their respective gases [25, 26].  In a pilot randomized trial of 
10 adults with T2D, a prebiotic microbiome modulator (MM) with inulin and beta glucan plus polyphenols from 
blueberry pomace improved metformin tolerability and fasting glycemia [27]. This same cocktail improved glucose 
profiles in healthy adults with overweight and obesity during an oral glucose tolerance test  [28].  However, the 
mechanisms by which prebiotics influence metformin-induced GI side effects are unknown. Importantly, it remains to be 
established whether using this supplement is feasible in youth and young adults with T2D, whose microbiome signatures 
may be different from adults [29]. This study is innovative and clinically significant because it uses a prebiotic 
intervention to evaluate gut-incretin pathways while identifying whether a relatively safe supplement could 
Figure 2- Proposed Mechanism of Prebiotic Microbiome Modulator, adapted from [25]

Principal Investigator: Chung, Stephanie T.  
Last updated: February 9, 2021
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improve quality of life and glycemia in youth and young adults with type 2 diabetes who are high-risk for diabetes-
related complications. 
Preliminary studies: 
Currently, we are exploring metformin’s mechanism of action in African American youth with T2D (NCT02960659), 
including changes in their microbiome. Our preliminary findings from 4 youth who were evaluated before and after 3-
months of metformin therapy showed a decreasing trend for HbA1c (-0.56±0.2%) and increasing trends for stool 
propionate and butyrate (P=0.1, Figure 3).  In 
collaboration with Dr. Hariom Yadav at Wake 
Forest, using 16S Ribosomal targeted sequencing 
and Qiime analysis of Operational Taxonomic 
Units (OTUs), we also observed modest changes in 
microbial diversity, a decreasing trend in the 
Shannon index (an index of complexity, P=0.15). 
This decreased diversity has been seen in other 
studies of metformin-induced changes in the gut 
microbiota [14].  Therefore, metformin may change gut 
microbial diversity in association with changes in SCFA stool 
concentrations in youth, but larger samples sizes are needed 
for confirmation. While metformin alone can increase SCFA, prebiotic MM may be able to augment this increase by 
shifting substrate metabolism away from gas (hydrogen sulfide and methane) to acetate production. Further investigations 
into these mechanisms will help us determine the feasibility of an oral supplement to mitigate side effects of metformin 
and improve quality of life, as well as glycemia, in youth with T2D.  
2.0 Specific Objectives/ Aims
The primary objective of this study is to determine the feasibility and efficacy of a prebiotic MM to improve metformin-
induced lower GI side effects in youth with T2D and the secondary objective is to measure the change in stool SCFA 
content after prebiotic MM intervention.
Aim 1. To compare lower-GI side effects and stool consistency (tolerability score) in youth and young adults with T2D at 
initiation of daily metformin therapy when used with a daily prebiotic microbiome modulator (MM) vs placebo over a 1-
week period. 
Hypothesis: A prebiotic MM will be associated with higher tolerability scores (a composite score of lower GI-related side 
effects and stool consistency) compared to placebo. 
Rationale: A prebiotic MM, containing both fiber and polyphenols, reduced metformin-related GI side effects in adults 
with T2D while retaining, and even augmenting, the hypoglycemic effects expected with metformin therapy in a small 
study (4). Due to the significant potential for clinical benefit, we propose to validate these results in youth, in whom 
metformin is the only FDA approved anti-diabetic therapy.
Aim 2. To compare the change in SCFA stool content in youth with T2D on metformin therapy when treated with 
prebiotic MM vs. placebo after 1 week and 4 weeks.  
Hypothesis: The prebiotic MM will be associated with higher SCFA stool content compared to placebo.
Rationale: SCFAs are microbial metabolites derived from fermentation of dietary fibers that are likely gut mediators of 
glucose metabolism. Metformin and prebiotic fiber individually have been demonstrated to increase stool SCFA [13, 16, 
30, 31]. We propose to further elucidate the mechanism of metformin and MM, both individually and when taken 
together.  
Figure 3. Stool short Chain Fatty Acid Concentrations in feces before 
and after 3 months of metformin therapy in youth with type 2 diabetes 
(n=4).

Principal Investigator: Chung, Stephanie T.  
Last updated: February 9, 2021
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Exploratory aims: To evaluate the relationship of change in tolerability score with change in stool SCFA, change in gut 
microbial diversity and SCFA-producing phyla, change in intestinal permeability markers, determine tolerability score for 
metformin and prebiotic MM over 1 month, and change in measures of glucose, insulin and incretin kinetics after 
treatment with placebo compared to 4 weeks of continuous prebiotic MM intervention.
2.1 Study Outcomes
Primary Outcome: A composite tolerability score based on 4 GI side effect profile categories over 1 week.
Secondary Outcome: Stool SCFA content (proprionate and butyrate levels) after 1 week and 4 weeks of treatment.
Exploratory Outcomes: Stool microbial diversity indices, stool metabolites (bile acid concentrations, lactic acid, 
hydrogen sulfide), serum markers of intestinal permeability; glucose, insulin, GLP-1 and PYY concentrations during a 
mixed meal test.
3.0 Study Design 
3.1 Study Design and Timeline
This 9-week pilot study will have 2 phases and 6 outpatient visits at the NIH Clinical Center (Figure 4). Phase 1 is a 5-
week randomized double-blind randomized cross-over trial with two 1-week intervention periods (metformin + prebiotic 
and metformin + placebo).  Phase 2 will occur immediately following phase 1 in which participants will start an open-
label 4-week intervention with metformin and the prebiotic MM.  
Phase 1: This is a randomized double-blind cross-over study of youth with T2D with two intervention periods (metformin 
+ prebiotic MM BiomeBliss® ®, and metformin + placebo). Participants will have 5 study visits over the 5-week period.  
After screening history and physical examination are performed at visit 1, participants will enter a 1-week run-in period in 
which metformin therapy will be discontinued and blood glucose will be monitored twice daily via finger stick or with a 
continuous glucose monitor. The order of intervention will be randomized at Visit 2; when initiating metformin, 
participants will have 1-3 days of dose escalation, then arrive at a final dose of 850mg twice daily. After 7-9 days of 
treatment, participants will be evaluated as described in Table 1, then enter a 2-week washout period of prebiotic/placebo 
and metformin, and then cross over to the opposite intervention for 1 week.
Figure 4. Study Design and Timeline

Principal Investigator: Chung, Stephanie T.  
Last updated: February 9, 2021
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Phase 2: This is an open-label 4-week extension of metformin + prebiotic MM. Participants will receive a 1-month supply 
of metformin and prebiotic at the end of Phase 1 (Visit 5) and be evaluated for measures of stool metabolite and glycemia 
at the end of the trial (Visit 6).
Every effort will be made to adhere to the proposed timelines, but some flexibility is required for scheduling of other 
studies, unanticipated equipment maintenance, etc. Scheduling variations will not be reported. 
3.2 Study Protocol
Table 1 illustrates study procedures and timing over the 2 study phases. All visits will take place at the NIH Hatfield 
Clinical Center.  Except for the screening visit, participants will be evaluated after an 8-10 hour overnight fast. Detailed 
descriptions of each procedure/ visit are given below.
Table 1 – Study Procedures and timeline
Subject Identification: potential participants will be referred by local providers responsible for clinical care. This 
includes the Children’s National Multidisciplinary Type-2 Diabetes clinic, as well as from other community providers to 
whom we will provide information about our study (see recruitment strategies, 8.4). Interested participants will be given 
our study information and will contact us via the study line or email. Participants will be phone screened to determine 

Principal Investigator: Chung, Stephanie T.  
Last updated: February 9, 2021
Page 8
baseline eligibility and additional study information.  If participants are eligible, we will arrange a screening visit at the 
NIH.
Screening visit (Visit 1): After initial screening for eligibility and informed consent process completed, participants will 
undergo history and physical examination, routine bloodwork, and urine test to determine eligibility. Nutrition staff will 
also obtain body circumference measures, such as neck, waist and hip circumference. These measurements will be done 
over minimal clothing with a non-stretch measuring tape. 
Participants will receive instructions on stool collection at home and stool collection kits - one to be mailed back to the 
study team for analysis prior to stopping their baseline metformin dose (unless they are able to provide a stool sample 
after consent while still at NIH), and one to collect a sample in the 24 hours prior to visit 2 if has a bowel movement. 
Patients will also be taught how to fill out daily surveys to assess GI symptoms (see details below). They will meet with a 
member of the nutrition team to select foods for their metabolic diet and to be educated on completing a 3-day food 
record. Participants will also be educated on foods we will ask them to avoid during the study, such as pre/pro-biotics 
(kombucha, kefir, yogurt, etc.) and foods containing non-nutritive sweeteners. 
Period 1 (Drug-free run-in, Visit 2 and 3)
Drug-free run-in period:  Participants will discontinue metformin for the 7-10 days preceding Visit 2, to assess their 
metabolic and gut microbiome response off anti-diabetic medications, as well as to wash out the immediate effect of 
metformin on the participants microbiota. The timing of this 1 week period was chosen because changes in gut 
microbiome have been observed in as little as 1 day and on average 1 week [33]. During this time, participants will 
complete daily GI symptom questionnaires and monitor their blood glucose and upload their readings daily while not 
taking metformin. Participants with persistently elevated fasting glucose readings and/or signs and symptoms of metabolic 
decompensation (e.g. vomiting, dehydration, lethargy, abdominal pain, ketonuria and acidosis) will be withdrawn from 
the study and restarted on his/her medication. During the drug-free run-in period, individuals who cannot either wear the 
continuous glucose monitor (CGM), or complete at least 2 finger stick blood glucose measurements daily will be 
withdrawn from the study. 
  
Visit 2: This visit will take place 7-10 days after discontinuing metformin. Study personnel will review dosing and the 
completeness of GI questionnaires, as well as glucose trends with the participants. Participants will leave with a study 
metformin supply from the NIH pharmacy, as well as bottles of either placebo or prebiotic for a 1 week +15% supply and 
be instructed to take metformin and the powder mixed with a shake provided by the study team at the same time twice 
daily, with the first dose given with dinner that evening. Participants will take metformin 850mg with dinner on day 1-
3and the dose will be increased to 850mg with breakfast and with dinner on day 3 and thereafter. Participants will fill out 
daily GI symptom questionnaires and will be instructed to contact the NIH study team if they are unable to tolerate the 
medication. Participants will be provided with 1 week of food to take home and consume on the week prior to Visit 3. The 
NIH Clinical Center Metabolic Kitchen will pack out meals for 7 days. Energy provided will be estimated using Mifflin St 
Jeor equation and an activity factor of 1.3 [34]. Participants will also leave with an accelerometer to be worn around the 
wrist 24 hours/day during the period between visits 2 and 3.
Visit 3: Participants will return to the NIH CC 7-9 days after visit 2. Stool and urine sample collection and bottle/ pill 
counts and assessment of uneaten food will be performed, and the accelerometer will be returned. Participants will 
undergo a MMT (see description below). Completeness of GI symptom questionnaires and glucose trends will be 
reviewed. 
Period 2 (Washout, Visit 4 and 5)

Principal Investigator: Chung, Stephanie T.  
Last updated: February 9, 2021
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Participants will be instructed to stop taking all metformin and MM/placebo powder during the washout period between 
visit 3 and 4; instructions for CGM and hyperglycemia precautions that were given at the screening visit will be reviewed 
with the family. The 2-3 week washout was chosen to minimize any carry-over effects and has been optimal for detecting 
gut microbiota after discontinuing therapy [9, 27]. 
Visit 4: Visit 4 will occur at the NIH CC 14-24 days after the beginning of the wash out period; The NIH Clinical Center 
Metabolic Kitchen will pack out frozen and non-perishable items to provide meals for 7 days. Participants will also be 
asked to wear the accelerometer on wrist until they return for visit 5. 
Visit 5: Participants will bring a stool sample and study medications and have identical assessments as described in visit 3 
above. They will also be reminded about keeping food records, which they will be asked to do prior to visit 6
Phase 2 (open-label study and Visit 6)
At the end of Visit 5, patients will leave with a 1-month supply of metformin and MM, to continue taking twice daily, as 
well as an accelerometer device daily for 4 weeks. Final study Visit 6 – Participants will undergo assessments as outlined 
in Table 1 and receive counseling on resuming their medication regimen from prior to the study, as well as follow up 
nutrition counseling on healthy diet and lifestyle practices. 
Rescue visit for GI intolerance
If participants are unable to tolerate 850mg twice daily during the study, they will be withdrawn from the study and have 
the option come in for a rescue visit, where they will provide a stool sample and obtain bloodwork. They will then be 
offered the opportunity to enter the open label arm using BiomeBliss® in combination with their highest tolerable 
metformin dose. 
3.2.1 Study Procedures

GI symptom questionnaires: A questionnaire derived from validated sets used for GI symptoms of irritable 
bowel syndrome [35] which previous investigators have used [27]. Assessments of stool consistency (not 
applicable, very hard, hard, formed, loose, watery), urgency to evacuate (no need to evacuate within 3 hours after 
dosing, need to evacuate within 3 hours, need to evacuate within 2 hours, need to evacuate within 1 hour), daily 
bowel movements (at least 1 movement every 3-4 days, at least 1 movement every 2 days, at least 1 movement 
per day, at least 2 movements per day), bloating sensation (not applicable, mild, moderate, severe), flatulence 
(less than normal, normal, moderately increased, greatly increased), and evacuation completeness (not applicable, 
incomplete, constipated), in addition to a King’s stool chart, will be included (see Appendix A) [27]. This survey 
will be available in an online platform designed for mobile device but may also be filled out in paper form. 

Blood glucose monitoring: Blood glucose will be monitored with either fingerstick blood glucose twice daily 
(fasting and bedtime) or by using an FDA- approved continuous glucose monitoring device (CGM) (for example, 
the Freestyle Libre). The device will be used to monitor glucose in real-time, approximately every 5 minutes.  The 
system consists of a small sensor, transmitter, and hand-held receiver. The small sensor, with a small needle 
attached, will be inserted subcutaneously. The transmitter, which is attached to the sensor, will send the measured 
glucose to the receiver. The sensor is changed at least once every two weeks. Participants will receive training on 
removing and replacing the sensor and education on the signs and symptoms of hyperglycemia (see Appendix B). 
Participants will be asked to upload their readings daily to be monitored by a member of the study team daily 
while participants are not taking metformin, and weekly while taking metformin. Participants will be asked to 
contact the study team immediately after confirming with a finger stick BG if their glucose is low (<50mg/dL), or 
if they have a fasting blood glucose reading of >200mg/dl, or a reading anytime of >300mg/dl. Participants with 
persistently elevated fasting glucose readings and/or signs and symptoms of metabolic decompensation (e.g. 
vomiting, dehydration, lethargy, abdominal pain, ketonuria and acidosis) will be withdrawn from the study and 
restarted on his/her medication. 

Principal Investigator: Chung, Stephanie T.  
Last updated: February 9, 2021
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
Activity and Sleep Monitoring: Free-living physical activity and sleep quality will be quantified using a small, 
non-invasive, portable watch accelerometer (GT3X+ by Actigraph Inc., Pensacola FL) worn on the participant’s 
wrist. Physical activity levels, sleep duration and efficiency, amount of time spent in sedentary, moderate, 
vigorous intensity categories and estimated activity-associated energy expenditures will be extracted using 
established predictive equations.  This is an FDA 510(k) cleared Class II medical device.

Urine sample collection: urine will be collected at every visit for storage; for females, this same sample will be 
used for a pregnancy test

Stool sample collection: Participants will bring stool samples from home collected in the previous 24 hours to be 
processed by the study team or stool samples will be collected during outpatient visits. The stool sample will be 
stored at 4C in a sterile plastic vial and processed same day (see Appendix C). 

Mixed Meal Test (MMT): One intravenous catheter will be placed in the arm for blood draws upon arrival on 
the morning of the MMT.A liquid meal to provide ~30% of the estimated daily calorie requirements for weight 
maintenance determined by the Mifflin St. Jeor equation with an activity factor of 1.3 [34]. Blood samples will be 
obtained at 0, 10, 20, 30, 40. 50, 60, 90, 120, 150 and 180 minutes to measure hormones and metabolites 
including glucose, lactate, free fatty acid, triglyceride, C-peptide, and insulin concentrations. Blood to measure 
markers of intestinal permeability will be obtained at time 0.

Quality of Life Questionnaire: Participants will fill out the PedsQL quality of life questionnaire [32] (see 
Appendix D).

Food Records: Participants will review their food records with a member of the nutrition team. If participants do 
not bring records to this appointment, the nutrition team will perform a 24-hour recall capturing foods consumed 
in the day prior to admission (see Appendix E). 

DXA scan – dual x-ray absorptiometry scan will be performed to evaluate body composition and determine fat 
mass and lean body mass.
3.3 Inclusion and Exclusion Criteria
3.3.1 Inclusion Criteria 
1.
Age 10-25 years
2.
Pubertal or post-pubertal: Girls – Tanner stage IV-V breast; Boys – Testicular volume >10cc
3.
Diagnosis of type 2 diabetes by ADA guidelines [36] or with established diagnosis previously treated with 
metformin.
4.
Negative test for diabetes-related autoantibodies (glutamic acid decarboxylase 65 and tyrosine phosphatase-
related islet antigen 2 (IA-2)) documented in NIH CRIS chart or via outside laboratory assessment within the last 
10 years. 
5.
Hemoglobin A1C <8% at study initiation
3.3.2 Exclusion Criteria
1.
Pregnancy or breastfeeding
2.
Allergy to study medications
3.
Allergy or self-reported intolerance to blueberry, pomegranate, or oats, soy, gluten or dairy products.
4.
Chronic insulin therapy or insulin use within the last 3 months
5.
Treatment with other medications which are known to affect the parameters under study, including antibiotics 
within the last month, immunosuppressants, proton-pump inhibitors, supraphysiologic systemic steroids, probiotic 
or prebiotic supplements
6.
Heavy yogurt consumption (2 or more servings of ≥6 oz per day)
7.
Chronic GI disease, gastric bypass surgery, cancer diagnosis or autoimmune disease

Principal Investigator: Chung, Stephanie T.  
Last updated: February 9, 2021
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8.
Metabolic derangement such as metabolic acidosis, severe hyperglycemia (fasting blood glucose ≥ 200mg/dL), 
and/or liver enzymes > three times the upper limit of normal. 
9.
Any other condition that, in the opinion of the investigators, will increase risk to the subject, or impede the 
accurate collection of study-related data.
10. Body weight ≥ 450lbs 
11. Body weight ≤58kg
12. Hemoglobin concentration <10g/dL
3.3.3 Rationale for Inclusion Criteria
Age Range 10-25 years and pubertal status
The age range of 10-25 years was chosen to maximize recruitment and includes a wide range of youth afflicted with type 
2 diabetes.  All participants will be pubertal or post-pubertal youth (defined as Tanner stage IV-V for girls and testicular 
size 11-25cc for boys) to minimize any potential differences in gut microbiota associated with insulin resistance of 
puberty.  We believe this approach is reasonable because we will have a well-defined cohort (by pubertal status).  In 
addition, the broad content of the microbiome is of similar structure after 3 years old until later adulthood [29]
Pubertal status will be defined based on breast examination (Tanner stage IV-V) in girls and testicular examination (11-
25cc) in boys.  Pubic hair and bone age will not be used to characterize pubertal stage.
Diabetes diagnosis criteria and HbA1c <8% 
Type 2 diabetes in youth will be diagnosed according to ADA criteria [36]and eligibility criteria were based on the 
Treatment Options for Type 2 Diabetes in Adolescents and Youth (TODAY) study design [37]. Youth with HbA1c ≥8% 
often have severe hyperglycemia and glucotoxicity which is associated with increased risk for dehydration and 
ketoacidosis and these conditions may be associated with. changes in the microbiome [38, 39].
3.3.4 Rationale for Exclusion Criteria
Pregnancy and Breastfeeding
Pregnancy and breastfeeding are associated with physiologic increases in insulin resistance, which would confound data 
interpretation.  In addition, the DXA scan cannot safely be performed in pregnant women.  Female participants of 
childbearing age who meet other eligibility requirements and wish to participate in the study will be informed of the 
potential risks to a pregnancy conceived while on any study medication. Participants who consent to participate will be 
asked to practice reliable birth control including systemic hormones and/or barrier methods.  Patients who are pregnant 
and/or sexually active and not using adequate birth control will be excluded from enrollment in the trial.  Pregnancy tests 
will be obtained from all female participants before inpatient visits and right before DXA scans. If a subject becomes 
pregnant while on study they will be removed from the study and will not be allowed to re-enroll on the study
Chronic insulin therapy
Participants who are currently taking insulin therapy or who are within 3 months of insulin treatment will be excluded 
from the study, as the run-in period would be riskier for these individuals.  
Chronic GI diseases or medications/supplements affecting the GI tract
Individuals who have a disease, such as known autoimmunity, inflammatory bowel disease or celiac, may have altered gut 
microbiota due to that underlying condition. Certain medications are known or theorized to influence the gut milieu, and 
so to avoid confounding in this small study, we will exclude these patients as well. Additionally, yogurt or other pre/pro-
biotic supplements will change the gut microbiome. 
Any condition which may increase risk to the subject or impede accurate data collection

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Examples include participants with a history of active thyroid disease, liver disease, pancreatitis, nephrotic syndrome or 
lupus.  
Weight ≥ 450lbs
Individuals with weight ≥ 450lbs will be excluded because the maximum weight for the dual energy absorptiometry 
(DXA) scan is 450lbs.
3.4 Randomization 
The NIH Clinical Center (CC) Pharmacy will perform randomization. The randomization schedules will be constructed by 
an independent statistician and shared with the NIH CC Pharmacy.  The PI and study staff will not view the 
randomization schedule a priori.  Subjects will be randomized as they enter the protocol as follows: The PI or study staff 
will notify the NIH CC pharmacy of the eligible enrolled participant and the pharmacy will assign the intervention arm 
according to the predetermined randomization schedule.  
3.5 Study Agents/ Intervention 
Three study agents will be used: metformin standard release 850mg oral tablet, BiomeBliss® powder, and placebo 
powder.  Metformin will be used within the approved dosing regimens. BiomeBliss will be used according to its labeled 
instructions, see package label (Appendix C)
Blinding of the investigators and participants will be maintained throughout until the end of the study with the exception 
of our research nurse who can communicate with participants about any problems with the powder they may be taking. 
Investigators will be unblinded in the case of a participant needing to be withdrawn due to metformin intolerance; if the 
intolerance is noted during treatment with placebo, participants will be offered the option to immediately enter the second 
phase. 
The following placebo was chosen to ensure appropriate blinding and similar sensory characteristics for the placebo and 
product: the placebo (Gatorade G2 (low sugar) powder, a mix of Glacier Freeze and Grape flavors The placebo will be 
formulated and packaged by NIH Clinical Center Pharmacy as outlined in the Table 2 below and based on the following 
justifications: 
1.
The non-fiber carbohydrate content and calories are comparable in the placebo and BiomeBliss.  Although the 
total carbohydrate content is different, the calories provided, and the non-fiber carbohydrate content are 
similar between placebo and Biomebliss.  It was important to ensure that there was no fiber in the proposed 
placebo because the fiber (beta glucan and inulin) is the active ingredient under study. We expect that the 
placebo should have no impact on stool consistency or frequency, so that we can best evaluate the effect that 
the study fiber supplement has on the stool.  
2.
Comparable non-nutritive sweetener (NNS) in placebo and BiomeBliss. The proposed placebo is sweetened 
with a non-nutritive sweetener rather than by sucrose alone to help keep the mass of each dose similar and 
isocaloric. We expect that stevia (a “natural” NNS) and the artificial NNSs, such as sucralose and acesulfame, 
will have comparable but negligible effects on glycemia in the short term [40].  However, we acknowledge 
that the effects of NNS on the microbiome is an emerging area of research. The data on the effects of non-
nutritive sweeteners and their potential alterations of gut microbiota are controversial. Over the last decade, 
there is an increasing number of rodent and in vivo models indicating modest changes in gut phyla with NNS 
doses that far exceed the maximum acceptable daily intake [41-45]. The clinical impact of these changes is 
still unclear; human studies are scarce and conflicting. A handful of clinical trials, using acesulfame do not 
agree on whether this sweetener exerts a change in gut microbiota [46-48]. To our knowledge, there are no 

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human trials rigorously evaluating gut microbiome with stevia and sucralose [49]. Additionally, we expect 
that during the phase 2 when we expect to see changes in the microbiome, our participants will be consuming 
NNS in possibly higher quantities in their usual diet. We will be asking them to avoid diet sodas and other 
items known to be sweetened principally with NNS (see appendix G), but even foods that are not advertised 
as “diet” or low-sugar frequently contain NNS, so they will be impossible to avoid in a real-world study. 
Table 2: Biome Bliss and G2 Placebo Nutrition Facts
BiomeBliss
Gatorade G2 Placebo Mixture
(4g Grape + 15g Glacier Freeze)
Calories per 
serving
60kcal  (27g)
63 kcal (19g)
Ingredients
Inulin, Blueberry Extract, 
Beta-glucan (oats), Soy 
Protein Isolate, 
Pomegranate Flavor, 
Xanthan Gum, Citric 
Acid, Stevia Extract
Glacier Freeze: sugar, citric 
acid, salt, sodium citrate, natural 
flavor, monopotassium 
phosphate, modified food 
starch, calcium 
silicate, sucralose, acesulfame 
potassium, blue 1
Grape: sugar, citricacid, salt, sodium 
citrate, monopotassium phosphate, 
natural and artificial flavor, cornstarch, 
calcium silicate, sucralose, acesulfame 
potassium, blue 1, red 40
Dietary Fiber
9g
None
Total 
Carbohydrate
22g
17.5g
Non-fiber 
Carbohydrate
13g
17.5g
The NIH CC Pharmacy and the prebiotic fiber will be packaged and dispensed in a similar container.  Each product will 
be mixed in a pre-specified shake, provided by the study team, immediately prior to consumption. Mixing the powders 
with the shake prior to consumption will assure that consistency is also similar. 
3.5.1 Dose titration for gastrointestinal intolerance
At the start of each period, participants will take metformin 850mg daily, regardless of their dose prior to beginning the 
study. Participants will be titrated up to 850mg twice daily within 1-3days of Period 1 and 2. For participants who 
experience intolerable gastrointestinal side effects, metformin dose will be decreased by 850mg.  Examples of such side 
effects include 3 or more diarrheal episodes or intense discomfort or cramping for more than 3 hours. If gastrointestinal 
symptoms resolve x 2 days, and at the discretion of the PI, the dose of metformin may be increased again to 850mg twice 
daily. Participants who are unable to tolerate 850mg daily will come for the rescue visit and be withdrawn from the study. 
3.5.2 Dispensing and Storing Study Medications
The NIH CC Pharmacy will acquire and store metformin and BiomeBliss® powder for the study and will formulate and 
compound the placebo powder.  The study medication will be coded and dispensed by the NIH CC Pharmacy.  The 

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pharmacy will dispense the study medication according to the predetermined randomization table.  The Randomization 
section contains additional details on the processes we will use to assign randomization.  The storage of medications will 
be according to the package insert for each study drug. Medication will be supplied in 7 day +15% supply at Visit 2 and 4.  
3.5.3 Compliance and Adherence
When potential participants are considering participation, the importance of compliance with the intervention assignment 
will be stressed.  The ability to adhere to the protocol of the study, and take medication twice daily, as instructed, is one of 
the inclusion criteria of the study.  Medication adherence will be determined by study personnel counting of pills and 
sachets that were dispensed at each medical visit and returned at the next medical visit (Visits 3 and 5).  The percent of 
pills and sachets taken over each intervention period will be assessed. We will employ the following strategies:
1.
Fifteen-percent extra of metformin tablets and powder sachets will be given to the participant to improve 
compliance, as the participants will not be told how many extra doses are sent (although they will be told that 
there are more than the exact number of pills so that we can be sure they are taking it).  Participants will return the 
remaining doses and the number of unused tablets/sachets will be recorded.
2.
Adherence to blood glucose monitoring will be assessed by reviewing CGM or glucometer downloads at every 
visit.
3.
The participants will be contacted frequently by text message to encourage and assess compliance. Text and 
email-based reminders are an important adherence tool which can be tailored to the needs of the individual youth 
and family.  Text messaging has been shown to improve outcomes in children with other chronic diseases, such as 
asthma [50] and sickle cell anemia [51] and in adults with type 2 diabetes [52]. 
3.6 Follow-up and Post-Study Treatment
The patient’s clinically relevant data, available at the end of the study, will be shared with their diabetes provider with the 
consent of the participant and the parent. At the family’s request, it will also be provided to his or her primary care 
provider.  
Post-Study Obligations
There are no anticipated post-study obligations.
The study drugs (metformin and BiomeBliss®) will only be provided to participants while participating in this protocol.  
After completion or withdrawal from the study, if the patient would like to continue the medication, their clinically 
relevant data may be shared with their diabetes provider (with the consent of the participant and the parent) for further 
management.
4.0 Clinical and Laboratory Methods
4.1 Microbiome Analysis
Fecal samples will be collected in sterile cryovials and stored at 4C for less than 24 hours.  The sample will be transferred 
to -80C and stored until further analysis for microbial diversity and phylla.  This analysis will be performed in 
collaboration with Dr. Hariom Yadav at Wakeforest University.  After thawing of fecal sample, fecal DNA will 
be isolated for microbial compositional analysis using PowerSoil® DNA Isolation Kit or comparable kit.  Amplicon 
sequencing using next generation technology (bTEFAP®) will be used to for microbial sequencing using Illumina MiSeq 
and HiSeq platforms technologies.  In brief, the 16S universal Eubacterial primers 27F AGAGTTTGATYMTGGCTCAG 
and 519R GTNTTACNGCGGCKGCTG will be utilized to evaluate the microbial ecology of each sample on the Illumina 
MiSeq with methods via the bTEFAP® DNA analysis service.  A single-step 30 cycle PCR using HotStarTaq Plus Master 
Mix Kit (Qiagen, Valencia, CA) will be used under the following conditions: 94C for 3 minutes, followed by 28 cycles of 

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94C for 30 seconds; 53C for 40 seconds and 72C for 1 minute; after which a final elongation step at 72C for 5 minutes 
will be performed.  Following PCR, all amplicon products from different samples will be mixed in equal concentrations 
and purified using Agencourt Ampure beads (Agencourt Bioscience Corporation, MA, USA).  Samples will be sequenced 
utilizing the Illumina MiSeq chemistry following manufacturer’s protocols.
The Q25 sequence data derived from the sequencing process will be processed using a pipeline (MR DNA, Shallowater, 
TX).  Sequences will be depleted of barcodes and primers then short sequences < 200bp are removed, sequences with 
ambiguous base calls removed, and sequences with homopolymer runs exceeding 6bp removed.  Sequences are then 
denoised and chimeras removed.  Operational taxonomic units (OTU) will be defined after removal of singleton 
sequences, clustering at 3% divergence (97% similarity)[53-56].  OTUs will be taxonomically classified using BLASTn 
against a curated GreenGenes/RDP/NCBI derived database[57] and will be compiled into each taxonomic level into both 
“counts” and “percentage” files.  Count files contain the actual number of sequences while the percent files contain the 
relative (proportion) percentage of sequences within each sample that map to the designated taxonomic classification.
4.2 Short Chain Fatty Acid Analysis
Fecal samples will be grounded with a pellet pestle motor, followed by being resuspended in 1 mL PBS buffer (0.1 M, pH 
7.4). After 4 h dissolution with 1 min vortex every 20 min, samples will be centrifuged (12, 000 g, 10 min) and passed 
through the 0.45 µm membrane filter.  Cell-free samples were used for determining the concentrations of SCFA (lactate, 
acetate, propionate and butyrate) using a high-performance liquid chromatography (Waters-2695 Alliance HPLC system, 
Waters Corporation, Milford, MA, USA) with DAD detector at 210 nm, equipped with a Aminex HPX-87H column (Bio-
Rad Laboratories, Hercules, CA). Sample (10 µL) will be injected each time and H2SO4 (0.005 N) will be used to elute the 
column with a flow rate of 0.6 mL/min at 35ºC.
5.0 Statistical and Power Analyses
5.1 Sample Size Justification 
Sample size estimates are based on the mean changes and standard deviations in GI tolerability score observed in the 
study of adult subjects taking the MM [27] including a mean tolerability score of 7 with standard deviation 4. Using alpha 
0.05 with 80% power, we will need 10 participants to complete the study. Assuming a 50% attrition rate for this crossover 
study, we expect to recruit approximately 20 individuals.  For the secondary outcome, our preliminary data reporting a 
mean change of 2.95±3.3 μmol/g of proprionate and 4.17±5.72 μmol/g of butyrate (Figure 3), 20 participants will achieve 
90% power, assuming an α=0.05, and correlation coefficient of 0.5.
5.2 Analysis of Primary and Secondary Outcomes
The primary outcome, the participant’s composite score of tolerability will be constructed using the principal component 
analysis (PCA) based on 4 GI side effect profile categories (stool consistency, urgency to evacuate, bloating sensation, 
and flatulence). PCA will be used to account for the expected high inter-patient variability. Comparison of mean 
tolerability score over 1 week will be with linear mixed models, adjusting for baseline score.  Pre-specified covariates are: 
treatment period and sequence effects from the cross-over design. 
The secondary outcome, change in mean stool SCFA over 1 and 4 weeks, will be analyzed using repeated measures of 
analysis variance (ANOVA) to consider correlation of observations within each participant. Participant and exploratory 
characteristics will be expressed as mean and standard deviation, median and interquartile range for continuous variables 
and frequency for categorical variables. Between group comparison of means will also be analyzed using repeated 
measures ANOVA. Spearman correlations will be used to compare relationships between continuous variables. A P-value 
of <0.05 will be used for statistical significance.

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6.0 Collection and Storage of Human Specimens or Data
6.1 Research Use, Storage and Disposition of Human Subjects’ Samples 
For future reference and potential use, we will store all samples collected in this protocol in our locked freezers for an 
unlimited period of time. Samples will be labeled with coded identifiers linked to patient identity only via a secured 
database. Research records and data with personal identifiers will be stored in our locked offices, the medical record 
department, and the electronic study database. This material will additionally be protected by medical record and 
computer access procedures.  Access to records and data associated with personal information will be restricted to the 
Principal Investigator, Co-Investigators, study support staff, study monitors and database support staff.
Subjects may request that unused samples be removed from our freezers and be destroyed.  If no such request is made, we 
will keep samples until they are completely used or no longer of scientific value, at which time they will be destroyed. We 
do not plan to destroy personal medical information or stored data. The Principal Investigator will report loss or 
destruction of data or samples to the IRB. 
6.2 Materials Transfer Information
Stored samples and/or data may be sent to outside collaborating laboratories, or shared with other NIH collaborating 
investigators, to study questions related to diabetes or its complications (including, for example: microbiome sequencing, 
stool metabolite analysis, diabetes, obesity, weight, and lipid metabolism). Samples may be sent to outside commercial 
laboratories for analysis.  Samples and data sent to outside laboratories and collaborators for analysis and/or testing will 
contain only coded numbers, without personal identifiers. Materials Transfer Agreements will be completed before the 
exchange of samples and/or data with outside collaborators.
6.3 Genomic Data Sharing Plan
This protocol involves the collection of non-human genomic DNA.  Fecal samples will be stored for analysis of microbial 
DNA.  The participants will consent for their data to be shared through publicly accessible databases over the internet, 
NCBI or NIH’s genebank.  Data will be submitted at the time of publication and will include the relevant genomic data 
and corresponding phenotypic data.  Users must agree to the conditions of use governing access to the public release data, 
including limitation of research to investigations consistent with the participants’ consent, restrictions against attempting 
to identify study participants, destruction of the data after analyses are completed, reporting responsibilities, restrictions 
on redistribution of the data to third parties and proper acknowledgment of the data resource. 
7.0 Multiple-site Studies
The primary site for study visits will be at the NIH Clinical Center.   Primary enrollment and informed consent and assent 
will only occur at the NIH Clinical Center.    
8.0 Human Subject Protection
8.1 Investigators and Roles
Please see Key Study Personnel document. Dr. Stephanie Chung (PI), Dr. Abby Meyers, Nurse Practitioner Michael 
Stagliano and Nurse Lilian Mabundo will obtain informed consent. 
8.2 Informed Consent/Assent Procedures
Informed consent/ assent will be obtained as consistent with the requirements of SOP 12 and 14D.  Written consent/assent 
will be obtained from each subject after detailed explanations of the planned procedures by the principal or a designated 

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Page 17
associate investigator. Prospective subjects will be given the research information in layman’s term and in language 
understandable to the subjects. The consent process will take place prior to any study procedures.  Subjects have the right 
to withdraw participation from this protocol at any time.
8.2.1 Language for Minors
Written informed consent and assent will be obtained from the minor and his/her parent/guardian or (LAR) prior to any 
screening visits, study procedures or interventions. The Principal Investigator or other designated qualified protocol 
investigators will explain the study in language understandable to the parent/guardian or (LAR)).  The investigator will 
also explain the study to the minor who is of a younger age and level of understanding.  Enough time and opportunity will 
be given for discussion of the research as well as to answer any questions they may have, taking care to minimize or 
eliminate the perception of coercion or undue influence.  In accordance with 45 CFR 46.408, [the parent/guardian or 
(LAR)], will sign the current IRB approved informed consent.  The minor ages 10-17yrs will provide written assent, as we 
expect that all of those enrolled will be capable of understanding and signing the assent document. The investigator will 
sign the assent as well. A copy of the consent and assent will be given to the minor and [his/her [parent/guardian or 
(LAR)] for future reference. The signed documents will be sent to the Medical Records Department for placement in the 
subject's permanent CC medical record. The consent process will additionally be documented in the electronic medical 
record (CRIS). 
For minors who reach the age of consent while enrolled in our protocol, the parent and the minor will be forewarned at the 
time of enrollment, that there will be a need to consent the minor on reaching the age of consent as consistent with the 
requirements of SOP-12 and 14D—Requirements for Informed Consent.  This is because the prior parental permission 
and child assent are not equivalent to legally effective informed consent for the now-adult subject (consistent with NIH 
HRPP SOP 14D.8). All individuals will be given the opportunity to ask questions. In these situations, reconsenting will be 
performed at the NIH CC or using the telephone consent process.
8.2.2 Telephone consenting
When executing the consent process by telephone, the subject will receive a copy of the protocol consent in the mail prior 
to being consented.  After they have had an opportunity to review the consent the investigator will contact the subject by 
telephone.  The investigator will review the investigational nature of the protocol with the subject and answer questions. If 
the subject chooses to participate, the subject will sign and date the consent.  The informed consent document will be 
mailed to the principal or associate investigator who led the discussion, who will sign and date and mail back a fully 
executed copy for the subject’s records.  The informed consent process will be documented on a progress note and a copy 
of the note and the original informed consent will be filed in the subject’s record.
8.3 Rationale for Subject Selection
Type 2 diabetes in youth is a rapidly progressive and aggressive disease yet metformin therapy, the only oral- approved 
anti-diabetic therapy, is associated with GI sides effects in up to 80% of newly initiated patients and up to 30% of patients 
on chronic therapy. The burden of metformin-related GI side effects is magnified in youth because it is a potentially 
modifiable reason for medication non-adherence and subsequent poor glycemic control.  Therefore, investigating a non-
pharmacological supplement that may improve metformin tolerance in youth and young adults is of high clinical 
significance. Volunteers eligible for this study include mature adolescents and young adults with type 2 diabetes.  Young 
adults will be included in the protocol to enhance recruitment and because the disease progression is similar to adolescents 
with type 2 diabetes.  We will not recruit pre-pubertal or peri-pubertal children with diabetes because this increases the 
risk that they have type 1 diabetes (a condition not relevant to the aims of the current study). 
8.3.1 Rationale for Exclusion of Vulnerable Populations

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This study will involve recruitment of children age 10-17 years as described above.  The study will not involve 
recruitment of other vulnerable populations including pregnant women, prisoners, adults who are or maybe unable to give 
consent because they do not meet the inclusion criteria for this study. We will protect participants as described in SOP 
14A.
8.4 Recruitment Strategies
Tools used for the recruitment of subjects will include the posting of the study description on the NIH CC website, the 
NIH Record, the Clinical Center News, Children’s National Medical Center (CNMC),newspaper advertisements and 
social media). Additionally, fliers will be placed on bulletin boards at different buildings throughout the NIH campus, the 
CNMC, George Washington University, the University of Maryland and other pediatrician or doctors’ office sites in the 
Washington DC Metropolitan area. Recruitment material will be IRB approved prior to dissemination. Also, potential 
participants may be referred by local providers responsible for clinical care. This includes the Children’s National 
Multidisciplinary Type-2 Diabetes clinic, as well as from other community providers to whom we will provide our study 
information and fliers. 
8.4.1 Strategies to address the challenges of clinical trials in youth with type 2 diabetes 
The research team is experienced in successfully recruiting youth with type 2 diabetes and familiar with the major 
challenges to pediatric research in type 2 diabetes in youth and young adults: (1) difficulty with recruiting eligible 
participants and (2) poor retention rates[58].  To maximize recruitment multiple techniques will be employed including: 
partnering with major organizations in the Washington DC metro area including CNMC, INOVA, Georgetown 
University, University of Maryland, Kaiser, and Johns Hopkins University, who we will meet with on a periodic basis and 
provide handouts for them to give to any of their patients who may be interested. 
To improve retention in this study we will employ the following strategies including: periodic telephone, e-mail and/ or 
text message communication, as well as strategizing with individual families regarding their unique socio-economic 
barriers and offer alternative and supportive strategies to encourage study adherence (e.g. finding creative solutions to 
transportation issues).
8.5 Risks/Benefits Analysis including Considerations of Alternatives to Participation
This study involves research that is greater than minimal risk but presenting the prospect of direct benefit to the 
individual subjects.  
8.5.1 Evaluation of Benefits
This research protocol is designed to elucidate mechanisms of the side effects caused by metformin and the ability of a 
supplemental product to improve those in patients who are already requiring metformin. Metformin is approved for the 
treatment of youth with type 2 diabetes 10 years or older and is standard of care.  Limited data (Burton et al 2015) 
demonstrates that there may be both an improvement in GI symptoms as well as in glycemia for patients with type 2 
diabetes, so there is a theoretical possibility that the MM will reduce GI side effects of metformin, and therefore quality of 
life, and improve blood sugars.  Participating in the protocol procedures may also be beneficial to the individual because it 
may provide specific information about the individual’s glycemic control and diabetes management, as well as more 
frequent interaction with the study team.
8.5.2 Evaluation of Risks
This research protocol will be associated with greater than minimal risk.  The specific risks and our approach to minimize 
these risks are outlined below.  

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1.
Blood Sampling:  Peripheral blood draws (venipuncture) performed during this study for research will not exceed 
10.5 mL/kg, or 550 mL (whichever is smaller) per 8-week period for adults.   For pediatric patients, blood draws 
will not exceed 5 mL/kg in a single day, or 9.5 mL/kg or 550 mL (whichever is smaller) per 8-week period.  The 
total blood volume for this study is 372 ml over the 9-week period.  Patients may experience some discomfort at 
the site of the needle entry, and there is a risk of bruising at the site.  There is a remote risk of fainting or local 
infection.  
2.
Intravenous Catheter Placement:  The placement of intravenous catheters can be uncomfortable and pose the 
potential risk of bleeding, bruising and infection.  All catheters will be placed under sterile conditions and 
universal precautions will be observed.  Should any complications occur, they will be addressed immediately. 
3.
Risk associated with metformin 
i.
Gastrointestinal upset:  Nausea, vomiting, diarrhea, flatulence, abdominal discomfort and indigestion are 
common side effects associated with metformin use. To be eligible for this study, participants will have 
an indication to start metformin as first line therapy for type 2 diabetes, if they are not already taking it 
prior to the start of the study. The following actions will be taken to minimize the risk for gastrointestinal 
disturbance:
a. Metformin dosing will be dose escalated over a 1-3-day time period
b. Gastrointestinal symptoms will be recorded daily by participants and communicated with the 
study team with each submission of the online questionnaire. 
c. Participants who experience severe and/or recurrent gastrointestinal symptoms, defined as ≥ 3 
diarrheal episodes or intense discomfort or cramping > 3 hours, will be withdrawn from the study. 
ii.
Boxed Warning – Lactic acidosis (rare): The risk of lactic acidosis with metformin use is increased in 
elderly individuals with renal dysfunction or congenital heart failure. Participants with chronic renal or 
heart failure, or other chronic metabolic illnesses will not be recruited for this study. 
iii.
Kidney problems: In individuals with kidney failure, diarrhea, nausea and vomiting may cause a loss of 
fluids and dehydration.  Subjects with impaired renal function at baseline evaluation will not be recruited 
for this study.
4.
Risk associated with drug-free periods
i.
Severe Hyperglycemia with metabolic decompensation: During the 7-10-day period between Visit 1 and 
Visit 2, and again in the 21-day period between Visit 3 and 4, youth may be at increased risk for severe 
hyperglycemia since the youth will be asked to stop their anti-diabetic medication.  This risk is reduced 
because we will only enroll individuals who are in relatively good glycemic control (HbA1c <8%) who 
will be at lower risk for decompensation.  Subjects will receive counseling on diet and lifestyle treatment 
to minimize this risk, as this is in keeping with current standard of care clinical guidelines as an important 
treatment modality for youth with type 2 diabetes [59].  
The following steps will be taken to minimize the risks of severe hyperglycemia: 
a.Participants at higher risk for severe hyperglycemia defined as those with HbA1c ≥8% will not be 
eligible to participate.
b.All participants will receive up-to-date education on diet and lifestyle management by the PI and 
study staff.
c.During the run-in drug free period all individuals will monitor glucose levels by CGM.  Those 
who are unwilling to wear CGM will be required to check 2 fingerstick glucoses per day. Study 
staff will be in daily contact with participants to review trend uploads. 

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d.If CGM or glucometer readings show >200mg/dl fasting, or 300mg/dl bedtime is noted during the 
run-in period, they will notify the study PI immediately.  Individuals with persistently elevated 
glucose levels and signs and symptoms of metabolic decompensation (e.g. vomiting, dehydration, 
lethargy, abdominal pain) will be withdrawn from the study and restarted on his/ her medication. 
e.If vomiting, abdominal pain or dehydration is noted, participant will be evaluated by a medical 
professional or PI immediately for metabolic evaluation of acidosis and ketosis. If metabolic 
decompensation (acidosis or ketosis) is noted, participants will be promptly treated and 
withdrawn from the study. 
5.
Stool sampling: Stool sampling is not associated with any health risk but may be uncomfortable for some 
participants.
6.
Radiation Exposure: Dual energy absorptiometry (DXA) scan. The DXA scan is a reliable and reproducible 
method to measure body composition, specifically body fat and lean body mass.  The patient lies on a flat table 
with the x-ray source below the table and the detector above. Each scan takes about 5-7minutes. For the DXA 
scan the effective dose of radiation is 0.00006 rem, which is below the guideline of 5 rem (or 0.5 rem in children) 
per year allowed for research subjects by the NIH Radiation Safety Committee.  The average person in the United 
States receives a radiation exposure of 0.3 rem per year from natural sources, such as the sun, outer space, and the 
earth’s air and soil.  If participants want to learn more about radiation, they will be given the pamphlet: An 
Introduction to Radiation for NIH Research Subjects. 
7.
Activity and Sleep monitoring: There are no risk associated with these monitors, but participants may find them 
inconvenient to wear. 
8.
Continuous glucose monitoring system: According to the device manufacturer, there is minimal risk associated 
with the device. Possible side effects include but are not limited to local infection, inflammation, pain or 
discomfort, bleeding at the insertion site, bruising, itching. Because this protocol represents the first use of this 
device by our research team, we do not know the frequency of such complications of the device. A medical 
provider will be available should any of these problems occur. 
9.
Risk associated with prebiotic MM: The MM Biome Bliss is a fiber supplement that is available and marketed in 
the USA.  In the dose prescribed for this study, the beta-glucan supplement will be equivalent to ~3cups of 
cooked oatmeal per day and provide ~ 40% of recommended daily fiber intake.  This supplement can 
uncommonly be associated with minor gastrointestinal upset (stomach bloating, loose stools) and has not been 
associated with any serious adverse effects. 
8.5.3 Alternatives to participation
Participation in clinical trials is completely voluntary.  Refusal to participate will not affect a subject's ability to participate 
in other studies at NIH or elsewhere.
9.0 Adverse Event Reporting 
9.1 Event Characterization and Reporting to the IRB 
Unanticipated problems, non-compliance, and other reportable events will be reported to the NIH IRB as per NIH HRPP 
SOP “Reporting Research Events,” Policy 801. All adverse events occurring during the study, including those observed 

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Last updated: February 9, 2021
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by or reported to the research team, will be recorded.  All actual or suspected non-compliance, major deviations, 
unanticipated problems (UP), new information that might affect the willingness of a participant to enroll or remain in the 
study, or suspension or termination of research activities placed by the NIH or IC leadership will be reported to the NIH 
IRB as soon as possible but not more than 7 days after the PI first learns of the event.  
Deaths will be reported to the Clinical Director and IRB within 24 hours after the PI first learns of the event.
At the time of CR, a high-level summary (not a line item listing) of events will be submitted including: major and minor 
protocol deviations; noncompliance reported to the IRB that is not related to a protocol deviation; Adverse Events and 
Serious Adverse Events that do not meet the definition of an UP; and UPs reported to the IRB
9.2 Data and Safety Monitoring Plan
Study procedures will be subject to audits and/or monitoring visits to ensure compliance with the protocol and applicable 
regulatory requirements consistent with the NIDDK quality assurance program plan.  Audit and/or monitoring visit results 
will be reported to the Principal Investigator for further reporting as appropriate.   Study documents and pertinent hospital 
or clinical records will be reviewed to verify that the conduct of the study is consistent with the protocol plan.
The collection, monitoring and analysis of adverse events will be the responsibility of the Principal Investigator and the 
investigative team.  
As required by FDA 21 CFR 312.50, trial procedures will be subject to review to ensure compliance with the protocol and 
applicable regulatory requirements.  Results will be reported to the Principal Investigator/Sponsor for further reporting to 
the FDA consistent with applicable regulations.   The specific monitoring plan will be developed with the Principal 
Investigator and frequency of monitoring visits determined by such factors as study enrollment, data collection status and 
regulatory obligations.  Monitoring visit results will be reported to the Principal Investigator for further reporting as 
appropriate.  Study documents and pertinent hospital or clinical records will be reviewed to verify that the conduct of the 
study is consistent with the protocol plan.
9.3 Monitoring Subjects and Criteria for Withdrawal of Subjects from the study
9.3.1 Withdrawal Criteria
1.
Withdrawal of consent.  A subject wishes to withdraw from the study as stated in the informed consent (all 
subjects reserve the right to withdraw from the study without prejudice).
2.
Adverse event.  A subject experiences an adverse event that in the investigator’s opinion necessitates withdrawal 
from the study.  Specific examples of adverse events that would result in withdrawal are:
a)
Severe gastrointestinal intolerance to study drug (metformin therapy or prebiotic supplement) defined as 
≥ 3 diarrheal episodes or intense discomfort or cramping > 3 hours.
b)
Diabetic ketoacidosis or severe hyperglycemia requiring insulin replacement.
c)
Severe hypoglycemia defined as: 1) severe symptomatic hypoglycemia with seizures or unconsciousness 
and blood glucose <50mg/dl or 2) fasting hypoglycemia <30mg/dl on two consecutive days with or 
without symptoms.
3.
Abnormal screening blood tests. Subjects who have abnormal blood tests and who meet exclusion criteria will 
be withdrawn from the study by the investigator. 
4.
Investigator decision.  An investigator feels it is in the subject's best interest to terminate participation. The 
detailed reasoning behind this decision will be documented.
5.
Protocol deviation.  Includes subject noncompliance with CGM during the run-in period, pregnancy, study entry 
criterion deviation, or start of a concomitant medication that would impede accurate study analysis.  If a female 
participant’s urine pregnancy test is positive, we will ask the girl’s permission to inform her family so that she can 
get optimal medical care.  During the drug-free run-in period, individuals who cannot complete at least 2 

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fingerstick blood glucose measures, wear CGM or who cannot be reached for >3 days during this period will be 
withdrawn from the study.
9.4 Protection of Participant’s Privacy and Confidentiality 
At the time of enrollment each subject is given a study-specific code name. The coding system is available only to study 
staff and kept in a secure, password protected database accessible only to study staff. The actual names of the subjects will 
not be provided or ever made available in any publication.
9.5 Investigational New Drug Determination
This dietary supplement will be purchased from the original manufacturer and dispensed by the research 
pharmacy. It is not being researched to market as a drug nor is it being investigated under the regulatory 
definition of a drug (cure, treat, diagnose, mitigate, or prevent a disease). The supplement is being used to 
decrease the side effects of metformin.  The treatment of side effects is not considered a disease, and therefore 
per FDA part 312 does not meet criteria for requirement of an IND (see Appendix F). 
10.0 Renumeration
All participants will receive financial compensation for their time per NIH Clinical Center guidelines for on-site visits 
(which occur at NIH), and additional compensation will be provided for specific procedures based on inconvenience units, 
as outlined in Table 3. 
Table 3:  Compensation per Visit/ Procedure 
Compensation Estimated repeats
Total
Visit
$ 40
6
$ 240
MMT
$ 50
3
$ 150
DXA
$ 30
1
$   30
Stool collection
$ 20
6
$ 120
GI Symptom Questionnaire
$   1
30
$   30
Completion Bonus
$ 250
1
$ 250
Total 
$ 820
If a participant were to drop out, the participant will only receive payment for whatever visits and procedures they 
completed up to that point. If a participant does not complete daily GI symptoms questionnaires, he/she will be 
compensated for each that is completed. Compensation will be given in two installments: after Visit 5 and at the end of 
the study. Total compensation for guardians accompanying minors for the entire study is $50 per visit x 6 visits = 
$300. The guardians will only receive payments for whatever visits were completed and will be paid as a lump sum at the 
completion of the study.  
11.0 Conflict of Interest 
1.
The National Institutes of Health reviews NIH employees at least yearly for conflicts of interest. The following link 
contains details on this process: http://ethics.od.nih.gov/forms/Protocol-Reveiw-Guide.pdf. 
2.
This protocol has investigators who are not NIH employees. They are expected to comply with their Institutions’ 
conflict of interest policies. 

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Principal Investigator: Chung, Stephanie T.  
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