Colonic Delivery of Butyrate to Improve Insulin Sensitivity and Lower Triglycerides in Type 1 Diabetes Subjects
Phase: PHASE1
Conditions: Type1diabetes
Sponsor: Mayo Clinic

Study to Evaluate the Effects of BKR-017 on 
Insulin Sensitivity and Triglycerides in Type 1 
Diabetes Subjects 
NCT03999853 
13July2023 

 
CONFIDENTIAL 
 
 
CL-301 
Study to Evaluate the Effects of BKR-017 on Insulin Sensitivity and  
Triglycerides in Type 1 Diabetes Subjects 
 
PROTOCOL DATE:   
 July 13, 2023Amendment 5 
Protocol Version 6 
 
 
 

 
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Revision History 
Protocol 
Date 
Version Number 
Original Protocol 
March 1, 2020 
1.0 
Amendment 1 
May 20, 2022 
2.0 
Amendment 2 
July 18, 2022 
3.0 
Amendment 3 
December 5, 2022 
4.0 
Amendment 4 
April 25, 2023 
5.0 
 
Section 
Change Summary 
Title Page 
Protocol Date changed to July 13, 2023 
TOC & Page 
Numbers 
Appropriate page number changes 
 
Footer and 
synopsis page 
Changed Ver.  5 to 6where appropriate 
Signature Page 
Protocol Date changed to  July 13, 2023D, added version 
 
 
Baseline Visit: 
Screen (Day -
28) 
 In addition, study CGM sensors will be dispensed with subject instructions 
on use of Dexcom CLARITY app to capture glucose data throughout study 
participation. 
Diary cards will be mailed/emailed to the study team every 7 to10 days 
throughout the study. Subjects using an insulin pump, will be advised to 
mail/email a copy of their pump settings and insulin doses every 7 to10 days 
or upload their pump data to a prespecified, linked online account 
compatible with their pump.  
Height will be measured at screen visit. 
 
Urine pregnancy test added. 
An eight-week supply of investigational product (BKR-017) will be 
dispensed. Subjects will not start dosing until Day 0. 
Females of childbearing potential and able to become pregnant, must agree to 
use one of the birth control methods listed in the consent form, for the duration 
of the study.  
Treatment 
Period: Day 0 
to 48 
Study Diary cards will be mailed/emailed to the study team every 7 to10 
days throughout the study.  
. 
 
 
 
Visit 2: Final 
Visit (Day 48) 
Height will be measured. 
 
 
 
 

 
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TABLE OF CONTENTS 
 
LIST OF TABLES 
 
 
 
 
 
 
 
 
 
       vii 
ABBREVIATIONS  
 
 
 
 
 
 
 
 
       vii 
SIGNATURE PAGE                                                                                                                      viii 
SYNOPSIS                                                                                                                                      ix 
  
1. 
INTRODUCTION 
1 
2. 
STUDY PURPOSE AND OBJECTIVES 
6 
3. 
INVESTIGATIONAL PLAN SUMMARY 
7 
4. 
STUDY POPULATION 
7 
4.1 
Number of Subjects 
7 
4.2 
Method of Assigning Subject Numbers 
7 
4.3 
Inclusion/Exclusion Criteria 
7 
4.4 
Excluded Therapies 
8 
5. 
INVESTIGATIONAL PRODUCT 
9 
5.1 
Investigational Product Description and Packaging 
9 
5.2 
Investigational Product Storage, Dispensing, and Return 
10 
5.3 
Investigational Product Administration 
10 
5.4 
Per Protocol Population 
11 
6. 
STUDY PROCEDURES 
12 
6.1 
Screening Period 
13 
6.1.1 
Recruitment and Prequalification 
13 
6.1.2 
Visit 1 (Screening) 
13 
6.2 
Study Periods 
13 
6.2.1 
Baseline Period (Day -28 to Day 0, Visit 1) 
13 
6.2.2 
Termination of Baseline Period, Initiation of Test Period 
15 
6.2.3 
Test Period (Day 0  to Day 48 , Visit 2 ) 
Error! Bookmark not defined. 
7. 
SAFETY AND EFFICACY EVALUATIONS 
16 
7.1 
Vital Sign Measurements 
16 
7.2 
Tests for Safety Assessments 
16 
7.3 
Tests for Efficacy Assessments 
17 
7.3.1 
Primary Efficacy Assessments 
17 
7.3.2 
Secondary and Exploratory Efficacy Assessments 
17 
7.4 
Adverse Events 
18 
8. 
PROVISIONS TO MONITOR DATA TO ENSURE SUBJECT SAFETY 
19 
9. 
DISCONTINUATION OF A STUDY PARTICIPANT 
19 
9.1 
Screen Failures 
19 
9.2 
Reasons for Discontinuation 
19 
10. DATA REVIEW AND DATABASE MANAGEMENT 
20 
10.1 
Data Collection 
20 
10.2 
Database Management and Quality Control 
21 
10.3 
Subject Confidentiality 
21 
11. DATA ANALYSIS 
21 
11.1 
Objectives and Endpoints 
21 

 
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11.1.1 
Analysis Objectives 
21 
11.1.2 
Primary Efficacy Endpoint 
22 
11.1.3 
Secondary Efficacy Endpoints 
22 
11.1.4 
Safety Endpoints 
22 
11.1.5 
Exploratory Endpoints 
22 
11.2 
Sample Size 
22 
12. ANALYSIS CONVENTIONS 
23 
12.1 
Analysis Sets 
23 
12.2 
Missing Data 
23 
12.3 
Pharmacy Records 
23 
12.4 
Demographics and Baseline Characteristics 
23 
12.5 
Subject Disposition 
23 
12.6 
Efficacy Analysis 
23 
12.6.1 
Primary Efficacy Analysis 
24 
12.6.2 
Secondary Efficacy Analysis 
24 
12.6.3 
Exploratory Efficacy Analysis 
24 
12.7 
Safety Analysis 
24 
12.7.1 
Adverse Events 
24 
12.7.2 
Laboratory Data 
`24 
12.7.3 
Vital Signs 
24 
13. ETHICAL CONSIDERATIONS 
25 
13.1 
Regulatory and Ethical Compliance 
25 
13.2 
Informed Consent Procedures 
25 
13.3 
Responsibilities of the Investigator and IRB 
25 
13.4 
Publication of Study Protocol and Results 
25 
13.5 
Protocol Adherence 
25 
13.6 
Protocol Amendments 
26 
14. APPENDICES 
27 
14.1 
APPENDIX 1:  Previous Human Experience with Butyrate 
28 
14.2 
APPENDIX 2:  Tables 
29 
15. REFERENCES 
31 

 
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 LIST OF TABLES 
 
Table 1: Time & Events Schedule ................................................................................................ 12 
Table 2: Previous human experience with butyrate (selected studies) ......................................... 28 
Table 3: Safety Hematology Laboratory Tests ............................................................................. 29 
Table 4: Safety and Efficacy Chemistry Laboratory Tests ........................................................... 30 
 
ABBREVIATIONS 
AE 
ADG 
ALP 
ALT 
API 
AST 
BUN 
CBC 
CGM 
CPR 
eCRF 
eGDR 
GLP-1 
HbA1c 
HDPE 
HIPAA 
HOMA-IR 
hs-CRP 
HWR 
HTN 
ICF 
ITT 
IRB 
OPC 
PI 
SAP 
T1D 
USP 
1,5-AG 
 
Adverse Event 
Average Daily Glucose 
Alkaline Phosphatase 
Alanine Aminotransferase 
Active Pharmaceutical Ingredient 
Aspartate Aminotransferase 
Blood Urea Nitrogen 
Complete Blood Count 
Continuous Glucose Monitoring  
C-Peptide 
Electronic Case Report Form 
Estimated Glucose Disposal Rate 
Glucagon-Like Peptide-1 
Hemoglobin A1c 
High Density Polyethylene 
Health Insurance Portability and Accountability Act  
Homeostatic Model Assessment and Insulin Resistance 
High-sensitivity C-Reactive Protein Hip-to-Waist Ratio 
Hip-to-Waist Ratio 
Hypertension 
Informed Consent Form 
Intent-To-Treat  
Institutional Review Board 
Out-Patient Clinic  
Principle Investigator 
Statistical Analysis Plan 
Type 1 Diabetes 
United States Pharmacopeia 
1,5-Anhydroglucitol  

 
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SIGNATURE PAGE 
Study Title: 
Study to Evaluate the Effects of BKR-017 on Insulin Sensitivity and  
Triglycerides in Type 1 Diabetes Subjects 
Protocol Date 
and Version: 
 
 July 13, 2023Amendment5, Version6  
Protocol 
Number: 
CL-301 
Investigator: 
Adrian Vella, MD 
Mayo Clinic 
200 1st St SW 
Rochester, MN 55905 
Institutional  
Review Board: 
Mayo Clinic Institutional Review Board 
 
200 First Street SW 
Rochester, Minnesota 55905 
 
We, the undersigned, have carefully read this protocol and agree that it contains all the necessary 
information required to conduct the trial and that it complies with principles of Declaration of 
Helsinki. 
 
Investigator:  
 
Adrian Vella, MD 
 
 
 
__________________________________ 
Signature 
_______________________ 
Date (month/dd/year) 
 
 

 
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CLINICAL PROTOCOL SYNOPSIS 
Protocol No.:  CL-301 
Sponsor: Mayo Clinic 
Investigator: Adrian Vella, MD 
Title of Study:  Study to Evaluate the Effects of BKR-017 on Insulin Sensitivity and Triglycerides in Type 1 
Diabetes Subjects 
Study Center:  Mayo Clinic, Rochester, MN 
Country: US 
Objectives:  To evaluate effect of colon-targeted butyrate tablets (BKR-017) on insulin sensitivity and 
triglycerides in type 1 diabetes subjects.  
 
Number of Tablets 
Dose 
Test 
Group  
Test 
Product 
No. of 
Subjects 
Dose  
Regimen 
Dosing 
Duration 
(days) 
Each dose 
(tablets) 
Daily 
(tablets) 
Single 
Dose  
Total Daily 
Dose  
1 
Active 
16 
BID 
48  
3 
6 
1.5 g 
3.0 g 
Test Product: BKR-017 is an oral tablet formulation designed to target delivery of butyrate to the colon via a 
colon-targeting technology. 
 
 
 
 
 
The tablets will be supplied by BioKier in 150-ml high density polyethylene (HDPE) white bottles.  
Design:  This study will test effect of BKR-017 (colon-targeted 500 mg butyrate tablets) as adjuvant therapy in 
type 1 diabetes (TID) Subjects on metabolic control in this population. Insulin sensitivity, insulin usage, glucose 
control (variability), and triglycerides will be measured after 7 weeks of treatment and compared to Day -28. 
Number of Subjects:  Assuming a 30% (approximate) early discontinuation rate, 16 Subjects will be consented 
and enrolled to ensure that 13 Subjects complete the 11-week study (4-week run in and 7 -week treatment 
period). 
Diagnosis and Main Criteria for Inclusion:  Criteria for Subject recruitment will be: 20–80 years of age; type 
1 diabetes with C-Peptide (CPR) less than 0.5 ng/mL; HbA1c level of 6.4–8.9%; at week -4. The study design 
will be a single-arm, open-label study. Subjects will be expected to use the Dexcom G6 Continuous Glucose 
Monitor (CGM) system and be willing to share their data via the CLARITY app with the study team.  
Duration of Subject Enrollment:  Eleven (11) weeks; baseline period 28 days (4 weeks), treatment period:  48 
days (7 weeks). Two visits will be required: one screen visit and one visit after 48 days of treatment.  
STUDY PERIOD (See Time and Events Schedule, Section 6) 
Baseline Period (Day -28): 
Visit 1 (Day -28):  Following web- and phone-based pre-screening, subjects who remain eligible will be 
scheduled for a screening visit. Subjects will be asked to arrive for the screening visit (Visit 1) in a fasted state 
(10-hour fast). Subjects will be consented prior to obtaining any biological samples or performing any study 
procedures. Subject eligibility will be assessed, demographics, medical history, and concomitant medications 
will be obtained along with- height, weight, waist and hip circumference to calculate Hip-to-Waist Ratio (HWR), 
and an abbreviated physical exam will be performed (including signs for edema). Fasting samples will be 
obtained for glucose and triglycerides. Additional blood samples will be obtained for HbA1c, routine 
hematology, and routine chemistry assessments. Hypertension (HTN) will be determined from medical history 
and concomitant medications. Blood pressure will be measured and used, along with HbA1c, to calculate the 
eGDR (estimated Glucose Disposal Rate). A urine pregnancy test will be obtained for females of childbearing 
potential.  

 
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CLINICAL PROTOCOL SYNOPSIS 
Protocol No.:  CL-301 
Sponsor: Mayo Clinic 
Investigator: Adrian Vella, MD 
After signing consent and meeting all inclusion criteria, subjects will be instructed on, and supplied with a study 
provided Dexcom G6 sensors , to collect glucose data. Subjects will be instructed to keep their Dexcom 
CLARITY app active, so that glucose monitoring data can be accessed by the study team throughout the study. 
Diary cards will be provided for the subject to record insulin doses, changes to concomitant medications and 
adverse events. Diary cards will be mailed/emailed to the study team every 7 to10 days throughout the study. 
Subjects using an insulin pump, will be advised to mail/email a copy of their pump settings and insulin doses 
every 7 to10 days or upload their pump data to a prespecified, linked online account compatible with their pump 
(i.e. Medtronic CareLink, Tandem T Connect or Glooko), whereby the relevant data can be abstracted by the 
study team.  
An eight-week supply of investigational product (BKR-017) will be provided to subjects. Subjects will not start 
dosing until Day 0. 
During the entire study, subjects will be asked to maintain their daily routines, including diet and exercise. 
Females of childbearing potential and able to become pregnant, must agree to use one of the birth control 
methods listed in the consent form, for the duration of the study.  
Treatment Period:  Day 0 to Day 48: 
 
Day 0 will occur approximately 28 days after the screen visit (agreed upon with the subject). The study team 
will communicate with the subject via a phone call or virtual visit on this day. After ensuring that the CGM data 
collected is acceptable, subjects will be instructed to commence taking the investigational product (BKR-017) 
as instructed.   
Subjects will be instructed to keep their Dexcom CLARITY app active, so that glucose monitoring data can be 
accessed by the study team. Subjects will continue to fill out diary cards, to record insulin doses, changes to 
concomitant medications and adverse events. Diary cards will be mailed/emailed to the study team every 7 to 
10 days.  Subjects using an insulin pump, will be advised to mail/email a copy of their pump settings and insulin 
doses every 7 to 10 days or upload their pump data to a prespecified, linked online account compatible with their 
pump (i.e., Medtronic CareLink, Tandem T Connect or Glooko), whereby the relevant data can be abstracted by 
the study team. 
Subjects will be contacted by the study team on Days 18 and 28, to assess study compliance, review concomitant 
meds and adverse events. 
  
 
Final Visit (Day 48): 
At the final study visit, Visit 2 (Day 48), fasting samples (10-hour fast) will be obtained for glucose and 
triglycerides.  Additional blood samples will be obtained for HbA1c, routine hematology, routine chemistry 
assessments, CPR, and the exploratory tests 1,5 AG and hs-CRP.  Blood pressure, height, weight, waist and hip 
circumference to calculate HWR, be obtained and an abbreviated physical exam will be performed.  A urine 

 
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CLINICAL PROTOCOL SYNOPSIS 
Protocol No.:  CL-301 
Sponsor: Mayo Clinic 
Investigator: Adrian Vella, MD 
pregnancy test will be obtained for females of childbearing potential. The CGM sensor will be removed, and 
data downloaded.  
Subjects will be required to return the study CGM sensor, all unused test product in the original container along 
with any diary cards at this visit.  The diary card and returned product will be reconciled by the study staff, and 
medication changes and adverse events recorded on the diary will be reviewed. 
Subjects with the following conditions will be excluded: History of bariatric or intestinal surgery, currently 
pregnant, nursing, or trying to become pregnant, type 2 diabetes, active gastrointestinal disease (e.g., irritable 
bowel syndrome, inflammatory bowel disease (e.g., ulcerative colitis, Crohn’s Disease, diverticulitis, 
gastroparesis, chronic/frequent diarrhea or chronic/frequent constipation), active and clinically significant  
hepatic, renal, or pancreatic disease, history of congestive heart failure, prior MI or significant cardiovascular 
disease, hepatic, renal, or pancreatic disease, history of congestive heart failure, prior MI or significant 
cardiovascular disease, uncontrolled hypertension uncontrolled hyper- or hypothyroid disease, active infection 
or history of trolled hypertension uncontrolled hyper- or hypothyroid disease, active infection or history of  
hepatic, renal, or pancreatic disease, history of congestive heart failure, prior MI or significant cardiovascular 
disease, uncontrolled hypertension uncontrolled hyper- or hypothyroid disease, active infection or history of  
chronic infection, subjects reporting an anticipated change in exercise or diet, subjects on a high fiber diet, 
subjects with pitting edema, and subjects who, in the investigator’s judgment, are not suitable for the study for 
any other reason, or subjects who cannot commit to the requirements of the study. 
 
Subjects currently taking the following therapies are excluded: GLP-1 receptor agonists (e.g., Byetta®, 
Bydureon®, Adlyxin®, Victoza®, Saxenda®, Trulicity®, Tanzeum®, Ozempic®), Amylin analogs (e.g., 
Symlin®), Thiazolidinediones (e.g., Actos®, Avandia®) and any combination of these medications (e.g., 
Avandamet®, Avandaryl®, Duetact®, Actoplus Met®, PioMet®), Acarbose (Glucobay®), Miglitol  (Glyset®).  
Additionally, subjects taking oral and injectable corticosteroids (e.g., prednisone), oral and injectable antibiotics, 
opioids and probiotics or other medications that alter gastrointestinal motility, chemotherapy, radiation, 
cytotoxic therapy, immune suppressants, antiepileptic drugs, colesevelam (Welchol®), and other supplements 
containing butyrate are also excluded from the study. 
Efficacy and Safety Evaluations 
Primary  
Insulin sensitivity, as measured by the estimated glucose disposal rate (eGDR), a validated 
clinical tool for estimating insulin sensitivity in T1D 
Secondary  
The following secondary evaluations will be performed: 
1. Glucose variability 
2. Triglycerides 
3. Insulin usage, as reported on diary card 
Safety 
Blood pressure, physical exam, chemistry, hematology and treatment-emergent adverse 
events 
Exploratory 
1,5 AG and hs-CRP 

 
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CLINICAL PROTOCOL SYNOPSIS 
Protocol No.:  CL-301 
Sponsor: Mayo Clinic 
Investigator: Adrian Vella, MD 
Statistical 
Analysis of 
Primary 
Endpoint:  
Primary and Secondary Efficacy Data:  The primary endpoint is the change from 
baseline (Day 0) to Day 48 in insulin sensitivity as measured by eGDR.  
The secondary endpoint of glucose variability (as measured from CGM data) is also the 
change from baseline (Day 0) to Day 24 and Day 48.  However, the secondary endpoint 
of lowering of triglycerides measurements will be calculated from the baseline at Day -
28. 
 
Safety Data: Assessment of safety will be made using the change from baseline in vital 
signs, physical exam, chemistry, hematology, and treatment-emergent adverse events.  
The safety analysis set will include all Subjects that received any amount of study 
treatment. The safety analysis set will include all Subjects that received any amount of 
study treatment. 
 
Sample Size Estimates: Sample size estimates are based on practical considerations. 
Synopsis Date: July 18, 2022, Amendment 2, Version 3.0 

 
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1. 
INTRODUCTION 
 
Summary: BioKier, Inc. is developing colon-targeted, oral formulations of natural gut hormone 
secretagogues as non-prescription medical foods or supplements for nutritional use for 
improvement of insulin sensitivity in type 1 (T1D) and type 2 (T2D) diabetes. The natural 
compounds under investigation by BioKier are butyrate and L-glutamine. An earlier clinical study 
(CL-201), conducted at Pennington Biomedical Research Center, demonstrated that treatment for 
4 weeks with the L-glutamine formulation (BKR-013) improved insulin sensitivity in a significant 
number of T2D subjects.  BioKier now plans to investigate the effects of an oral butyrate tablet 
formulation (BKR-017) on insulin sensitivity and other metabolic parameters in diabetes patients; 
this study will be in T1D subjects. The long-term anti-diabetes effects of treatments that increase 
the presence of butyrate in the colon are well documented, which suggests that a colonic 
formulation of butyrate is a viable option for chronic use in both T1D and T2D. Along with L-
glutamine, butyrate is one of the most active gut hormone secretagogues among natural 
compounds. Butyrate, a short-chain fatty acid, is the active ingredient of BKR-017 to be tested in 
the current clinical protocol (CL-301). The findings from the pilot study CL-201 with the L-
glutamine capsule BKR-013 showed that insulin resistance is a relevant efficacy endpoint and 
therefore has been identified as the primary endpoint in this study. 
 
Gut hormones, including glucagon-like peptide-1 (GLP-1), secreted by L-cells in the lower gut in 
response to nutrient stimulation, are important in the body’s regulation of energy balance 1.  They 
signal the arrival of food in the gut to regulatory centers controlling glucose metabolism, appetite, 
and lipid metabolism. As well as regulating glucose homeostasis and satiety, an important effect 
of GLP-1 secreted from the gut is inhibition of fat absorption, which results in lower levels of free 
fatty acids and triglycerides in the circulation. Impaired secretion of gut hormones by L-cells in 
response to meals has been reported in obesity 2;3, T2D 3, and T1D 4, although in these conditions 
the L-cells retain their ability to respond to secretagogues.  Also, available evidence indicates that 
GLP-1 receptor agonists improve glycemic control when used in conjunction with insulin in 
patients with T1D 5, suggesting that augmented GLP-1 secretion can be beneficial in T1D.  
Butyrate is one of the most active natural L-cell agonists, as shown in in vitro 6-8 and has been 
shown to be an active secretagogue of GLP-1 in in vivo studies 8.  There is a preponderance of 
human data demonstrating that the beneficial effect on diabetes of delivery of non-digestible 
carbohydrates to the colon are due to the actions of butyrate which is a product of fermentation of 
carbohydrates by colonic bacteria. Furthermore, the improvement in metabolic control after 
fermentation of ingested carbohydrates in the colon of rats 9 and humans 10 has been shown to be 
due to increased GLP-1 secretion.  However, as effective as these approaches can be, they have 
not achieved wide therapeutic acceptance because the fermentation process causes serious GI side 
effects including diarrhea, bloating, and flatulence, which were not tolerated in clinical trials 11.  
Alternate methods of delivering butyrate to the colon to achieve the same effects, but avoiding 
fermentation, were patented by BioKier. 

 
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CL-301 Ver.4  
 
 
 
In order to affect the metabolic conditions that result from impaired gut hormone secretion, 
BioKier has formulated the nutrient butyrate into a colon-targeted tablet formulation (BKR-017) 
intended to stimulate secretion of GLP-1 from L-cells in the lower gut. Butyrate delivered to the 
colon in tablet form will not cause the side-effects seen with formation of butyrate by fermentation. 
 
It is well recognized in the scientific and medical community that stimulation of GLP-1 secretion 
by activation of L-cells in the lower gut is an area of expected dynamic development 12-14.  BioKier 
is utilizing the GLP-1 secretagogue properties of butyrate to develop this safe, natural compound 
as a medical food. While a number of metabolic conditions result from GLP-1 dysregulation, 
BioKier is evaluating the effect of BKR-017 on insulin sensitivity in T1D patients. In this 
population, BKR-017 is not intended to treat T1D, and as such would not replace insulin therapy, 
but rather BKR-017 will provide a nutritional support to any currently prescribed T1D therapy, 
which may involve prescription medication, or diet and exercise, or both. Specifically, we expect 
that use of BKR-017 will afford better glucose control including reduced variability and reduced 
frequency of hypoglycemic episodes. This would be due to improved insulin sensitivity which is 
expected to lead to lower insulin use in those patients and would cause weight loss in patients with 
metabolic obesity. Another benefit of lowering insulin resistance would be to lower cardiovascular 
risk as there is plenty of evidence associating insulin resistance with increased CV risk 15. Elevated 
triglycerides are one of the validated risk factors for CV disease in both type 1 and type 2 diabetes 
patients 16, and one of the beneficial effects of metabolic surgery and colonic butyrate produced 
by fermentation is significant lowering of triglycerides and therefore reduction in CV risk 17;18.  
Pertinently, GLP-1 receptor agonists 19 and DPP-4 inhibitors 20 that elevate endogenous GLP-1 
have been shown to decrease cardiovascular risk and inflammatory markers including 
triglycerides, free fatty acids, VLDL-C, apoB-48, and ApoB (reviewed in 21). 
 
Butyrate, formulated as BKR-017 to target the colon, is being developed by BioKier as a medical 
food. Regulations require that medical foods be consumed or administered enterally under the 
supervision of a physician and that they are intended for the specific dietary management of a 
condition for which distinctive nutritional requirements, based on recognized scientific principles, 
are established by medical evaluation such as appropriate laboratory and clinical science. An 
Investigational New Drug (IND) application is not required to conduct clinical investigations of a 
medical food, and a New Drug Application is not required for marketing.  
 
Previous studies conducted by BioKier, in which butyrate and L-glutamine was administered 
directly via infusion to the colon of T2D subjects, showed that single 1g doses of either butyrate 
or L-glutamine improved biomarker responses to an oral glucose challenge. Specifically, colonic 
administration of butyrate and L-glutamine both improved secretion of the glucoregulatory gut 
hormone GLP-1 and the subsequent insulin response. As follow-on to these effects observed on 
biomarkers, clinical outcome data is required to support marketing claims for BKR-017, the oral 

 
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formulation of butyrate, as a medical food, which would satisfy the FDA requirement of clinical 
data based on recognized scientific principles necessary for labeling a medical food. Note, in this 
study in T1D, it is not anticipated that improved GLP-1 secretion will necessarily increase insulin 
secretion but will improve insulin sensitivity, thus reducing insulin dose and glucose variability. 
 
BioKier’s Approach:  While several nutrients have gut hormone secretagogue activity, BioKier 
has selected the nutrients butyrate and L-glutamine to formulate as candidates for medical foods 
to advance through clinical development. Although both have potent acute GLP-1-secretagogue 
activity, butyrate has well documented chronic effects on glucose regulation, including the reports 
of non-digestible carbohydrates improving metabolic control over the long term, discussed above. 
There is large body of scientific and clinical evidence supporting the role of butyrate in improving 
metabolic control in diabetes:  
1. α-Glucosidase inhibitors, such as acarbose and miglitol, help control patients’ glucose by 
shuttling saccharides to the colon where they are processed to butyrate and increase GLP-
1 secretion 22;23.  That the increase in magnitude of improvement is in proportion to the 
increase in adverse gastrointestinal effects provides additional evidence that fermentation 
of saccharides to butyrate is the mechanism by which those drugs work and also why those 
drugs are not used, because of the adverse side-effects. An increase in colonic butyrate and 
augmented GLP-1 secretion were demonstrated in patients treated with those drugs. 
2. Tagatose, which was in clinical trials 11, produces butyrate in the colon by fermentation. 
Major outcomes after 1 year of tagatose dosing in this study were: 
a. Mean HbA1c fell from 10.6% to 9.6%  
b. Body weight declined from 108.4 to 103.3 kg  
c. HDL cholesterol progressively rose from 30.5 to 41.7 mg/dL 
However, Tagatose, which was in development as anti-diabetes drug by Spherix, was not 
commercialized due to side-effects of fermentation. 
3. Oligofructose supplementation, which produces butyrate in the colon by fermentation, 
resulted in weight loss and better glucose control 24. Also, insulin decreased in the 
oligofructose group and increased in the control group between initial and final tests (P < 
0.05), indicating that insulin sensitivity improved with treatment. 
BioKier has developed the colon-targeted tablet formulation, BKR-017, to deliver butyrate to the 
site of action in the colon. The dose of butyrate delivered is at least as large as the amounts of 
butyrate that are very effective for diabetes patients when produced from products that deliver 
saccharides to the colon.  BKR-017 is designed to protect butyrate from digestion and absorption 
in the stomach and upper digestive system and deliver it to the colon where it can activate the 
secretion of gut hormones from L-cells. A safety advantage of targeting the luminal surface of the 
gut epithelium is that use of a compound such as butyrate, with local activity and very short 
systemic half-life, will result in negligible change to systemic levels and negligible off-target 

 
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CL-301 Ver.4  
 
 
effects. Accordingly, there should not be interference or interactions with other medications that 
are released in the small intestine or that circulate in plasma. 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
  
 
BioKier’s Previous Studies - Pre-Clinical: BioKier has completed acute and chronic exploratory 
pre-clinical studies. The studies provided proof of the concept that butyrate was able to restore oral 
glucose-induced GLP-1 secretion when administered to the colon of diabetic rats and, when dosed 
chronically in an oral, colon-targeted, sustained-release formulation, prevented the development 
of diabetes in a rat diabetes model. Effects noted were a reduction in basal glucose levels and an 
improvement in insulin sensitivity 8. 
 
BioKier’s Previous Studies – Clinical: BioKier has conducted acute studies in T2D patients in 
which single doses of either butyrate or L-glutamine were administered to the colon of subjects in 
an enema solution. In diabetes drug-naïve patients, both butyrate and L-glutamine were effective 
in restoring oral glucose-induced secretion of GLP-1 and insulin to levels approaching those seen 
in non-diabetic controls 8. 
 
BioKier has also conducted a 4-week clinical study (CL-201 at Pennington BioMedical Research 
Center) in T2D subjects to determine effect of an oral formulation of L-glutamine on glucose levels 
and insulin sensitivity. The exploratory clinical investigation employed a two-way crossover 
design, in which a BID dose of 1.5g L-glutamine in colon-targeted capsules (BRK-013), or 
placebo, were administered orally to T2D subjects. After 4 weeks of treatment with colon-targeted 
L-glutamine, which has the same mechanism of action as butyrate, there was an improvement in 
insulin sensitivity and lowering both fasting glucose and insulin in a significant number of patients. 
Significantly, in six of nine subjects there was a marked improvement in insulin sensitivity -- 
HOMA IR changed from 2.86 to 1.67 (n=6) in the responders, which compares favorably to 2.75 
to 1.27 (n=10) reduction for bariatric surgery patients 26.  These data supported the concept that 
chronic delivery of a natural gut hormone secretagogue to the colon of diabetes patients has 

 
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potential as a therapeutic in T1D, as well as T2D, because metabolic surgery and colonic 
fermentation of carbohydrates is beneficial in both T1D and T2D. 
 
Safety: Butyrate is a simple chemical entity, stable and readily available in United States 
Pharmacopeia (USP) grade and Active Pharmaceutical Ingredient grade (i.e., suitable for human 
use).  
 
BioKier has conducted IRB-approved clinical studies of butyrate in T2D patients; notably, three 
single-dose infusion studies under Western IRB approval. Furthermore, a study done at the Mayo 
Clinic in 1993 investigated the effects of butyric acid and glutamine delivered directly to the colon 
of pouchitis patients by suppository 27.  Butyric acid (4.4g bid and glutamine 1g bid) were given 
to patients for three weeks. The study reported positive effects on pouchitis but, significantly, 
found no safety issues of doses higher than those proposed by BioKier. In the combined clinical 
studies conducted by BioKier, there were 30 subjects exposed to 1g butyrate. Additionally, in a 
BioKier-sponsored study at the Mayo Clinic in which Dr. Vella was the Principal Investigator, 4 
patients were treated with butyrate suppositories at 3g a day for 4 weeks with no adverse effects 
observed. Butyrate is also used as an enema in doses up to 8g for treatment of colon inflammatory 
conditions. Furthermore, the safety of butyrate supplementation of enteral or parenteral nutrition 
has been widely studied in chronic conditions (solid tumors) at doses of 14-28g/d. For Crohn’s 
disease it was determined that 4 g/day butyrate as enteric-coated tablets for 8 weeks 28 was safe 
and well tolerated. Butyrate toxicity has been studied in humans and it was determined that dose-
limiting toxicity was not identified 29. The escalation was halted at 200 mg/kg (of butyrin, a 
butyrate prodrug) three times daily (40 g per day) as further increases were felt to be not practical 
due to the large number of capsules which would need to be administered. In another study 27 
butyric acid (4.4g BID) was given to patients for three weeks without any adverse effects. See 
Appendix 1 for human use summary. 
 
A safety advantage of BKR-017 over administration of large oral or parental doses of butyrate is 
that it will lead to minimal systemic exposure to butyrate because it is released slowly, acts locally 
in the colon, and is metabolized quickly.  
 
Rationale for the current study design: Butyrate was selected as a candidate compound based 
on clinical data from studies with non-digestible saccharides (tagatose, lactitol, and oligofructose), 
and -glucosidase inhibitors; both approaches result in fermentation of saccharides by butyrogenic 
bacteria in the colon, producing butyrate and augmenting GLP-1 secretion. Long-term treatment 
with these products has been shown to be beneficial for diabetes patients, both T1D and T2D.  
 
A BID dosing regimen was selected because the tablet core is designed to release butyrate in the 
ascending colon over a period of eight or more hours. This timing of the sustained-release profile 
suggests that BID dosing will be necessary to achieve optimal effects.  

 
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The 1.5g dose of BKR-017 was selected based on several factors including: safety data from 
BioKier studies and other relevant studies; practical considerations (three 500mg tablets was 
determined to be the highest number of tablets that could practicably be administered as a single 
dose); BioKier’s efficacy data from single intracolonic dosing in which 1.0g was effective in 
increasing GLP-1, in diabetes drug-naïve patients; and on the relative activities of nutrients and 
gastrointestinal compounds reported in published studies.  Considerations included: 
 
1. Fiber producing ~1g of butyrate in the colon was effective in improving glucose regulation 
9;9 (BioKier anecdotal data). 
2. Comparison of concentration of butyrate produced in the colon after use of maximal dose 
of acarbose and amount of butyrate release from BKR-01730; 
3. The amount of colon-infused bile acids effective in induction of GLP-1 secretion 31; and 
4. The amount of bile acids shuttled to the colon by Welchol 32. 
 
The proposed dose for this study has been increased from the 1.0-gram dose used in the single-
dose studies to 1.5g to account for the lower concentrations of butyrate that will result from the 
sustained release profile of the BKR-017 tablets. Even at 1.5g BID (3.0g total daily dose), this 
dose is within the practical range for repeated dosing and well within dose and duration ranges 
safely administered in clinical studies and currently used in clinical practice (see Appendix 1: 
Previous human experience with butyrate [selected studies]). 
 
Considering that BKR-017 will require chronic dosing to supplement a T2D patient’s prescribed 
diabetes treatment in order to maintain adequate insulin sensitivity, longer-term safety should be 
taken into consideration. A seven -week duration of dosing was selected based on the safety data 
from reported clinical investigations (see Appendix 1) and this is within the duration of dosing of 
the butyrate in reported clinical trials. Furthermore, this duration of dosing was selected in order 
to cross reference the “placebo effect” observed in T2D patients. This placebo effect, which is 
known to affect daily glucose levels, has been shown to diminish by approximately 3 weeks 33.  
Therefore, in consideration of safety data and practical considerations a seven -week dosing 
regimen was selected. 
 
2. 
STUDY PURPOSE AND OBJECTIVES 
 
This study will test the hypothesis that delivery of the butyrate-containing BKR-017 tablet to the 
colon of T1D subjects will improve insulin sensitivity. Additionally, the effect of BKR-017 on 
triglycerides and glucose variability will be assessed. The safety and tolerability of repeat dosing 
(up to 48 days of administration) of BKR-017 will be determined. By exploration of the effects of 
BKR-017 on various metabolic biomarkers, the data generated from this study will be used to 
support continued evaluations of BKR-017 as non-prescription product for T1D patients requiring 

 
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nutritional support to improve cardiometabolic health. Furthermore, these data will support 
continued evaluation of BKR-017 for the regulation of weight and hypertriglyceridemia. 
 
3. 
INVESTIGATIONAL PLAN SUMMARY 
 
This study is an open-label study that will be conducted at Mayo Clinic. The total duration of 
subject involvement is approximately 11 weeks; 4-week Baseline Period which is followed by a 7 
-week Test Period. During the Test Period, subjects will self-administer three tablets of 
investigational product (BKR-017) two times daily: before breakfast and before bedtime.  
 
4. 
STUDY POPULATION 
 
4.1 
Number of Subjects 
 
Approximately 28 subjects with T1D will be screened to determine eligibility. Sixteen eligible 
subjects will be enrolled into the study with the expectation that 13 will successfully complete the 
study. 
 
4.2 
Method of Assigning Subject Numbers  
 
All subjects who consent to participate in the study will be assigned a unique three-digit number 
(assigned sequentially, in chronological order beginning with number 301). The subject number 
will be assigned at Visit 1 (Day -28) and will identify the subject for the remainder of the study.  
 
4.3 
Inclusion/Exclusion Criteria 
 
Inclusion Criteria: 
1. Males and females between the ages of 20 and 80 years at the time of screening, inclusive 
2. Diagnosed with T1D and under the care of a healthcare professional for its management 
3. HbA1c 6.4-8.9%, inclusive 
4. Has given written informed consent to participate in this study 
5. Willing to complete 28-day baseline period and 48 -day test period 
6. Willing to maintain current diet and exercise routine for the duration of the study 
7. Current use of a Dexcom Continuous Glucose Monitor (CGM) 
 
Exclusion Criteria: 
1. History of bariatric or intestinal surgery 
2. Active gastrointestinal disease including but not limited to irritable bowel syndrome, 
inflammatory bowel disease (e.g., Crohn’s disease and ulcerative colitis), diverticulitis, 
gastroparesis, or chronic/frequent diarrhea or chronic/frequent constipation 

 
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3. Active and clinically significant hepatic, pancreatic disease, or renal disease as determined by 
the investigator 
4. History of significant heart disease, including congestive heart failure, prior MI, chronic atrial 
or ventricular fibrillation, coronary artery disease, cerebral vascular disease, or other 
cardiovascular disease, that in the opinion of the investigator should exclude the subject from 
the study 
5. Severely uncontrolled hypertension at screening defined as a systolic blood pressure 
> 180 mmHg or a diastolic blood pressure > 110 mmHg on the average of two seated 
measurements after being at rest for at least 5 minutes 
6. Uncontrolled hyperthyroidism or hypothyroidism, or other significant thyroid disease 
7. Active significant infection as determined by the investigator  
8. Known allergy to butyrate or any of the components of the tablets  
9. Participation in a clinical trial and/or treatment with an investigational drug during the 30 days 
before screening, or within 5 half-lives of receipt of an investigational drug or twice the 
duration of the biological effect of any investigational drug (whichever is longer) 
10. Pregnant, nursing, or trying to become pregnant  
11. Presence of pitting edema on physical exam 
12. High fiber diet 
13. In the investigator’s judgment, the subject is not suitable for the study for any other reason or 
cannot commit to the requirements of the study. 
14. Subject is taking one or more of the excluded therapies. See list of excluded therapies in section 
4.4. 
 
4.4 
Excluded Therapies 
 
Subjects should not be enrolled in the study if they are currently (defined as 2 weeks prior to and 
at the screening visit) taking any of the following agents listed below: 
 
1. 
GLP-1 receptor agonists (e.g., Byetta®, Bydureon®, Adlyxin®, Victoza®, Saxenda®, 
Trulicity®, Tanzeum®, Ozempic®) 
2. 
Amylin analogs (e.g., Symlin®) 
3. 
Thiazolidinediones (e.g., Actos®, Avandia®) 
4. 
Any combination medications that contain 1-4 above (e.g., Avandamet®, Avandaryl®, 
Duetact®, Actoplus Met®, PioMet®)  
5. 
Oral and injectable antibiotics 
6. 
Oral or injectable corticosteroids (e.g., prednisone) 
7. 
Chemotherapy 
8. 
Opioids 
9. 
Probiotics 
10. Radiation 
11. Cytotoxic therapy 

 
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12. Immune suppressants 
13. Antiepileptic drugs 
14. Colesevelam (Welchol®) 
15. Acarbose (Glucobay®)  
16. Miglitol (Glyset®) 
17. Over-the-counter products that contain ephedrine 
18. Other supplements containing butyrate  
19. Medications that alter gastrointestinal motility including but not limited to metoclopramide 
(Reglan®), Metamucil® or other bulk fiber agents, emollient agents like ducosate, stimulants 
like Dulcolax® or castor oil, magnesium hydroxide (Milk of Magnesia®), hyperosmolar 
preparations (lactulose, sorbitol, MiraLAX®), opioids (e.g., Vicodin®, codeine, Percocet®), 
Adsorbents (Immodium®, Kaopectate®, Pepto-Bismol®), or anti-emetics (e.g., Zofran®, 
Kytril®, Compazine®, Phenergan®) 
 
If a subject requires any of the above agents during the study, he/she should contact the study team 
to determine if there is an alternative available.  
 
5. 
INVESTIGATIONAL PRODUCT 
 
5.1 
Investigational Product Description and Packaging 
 
BKR-017 is a proprietary oral tablet formulation designed to target delivery of butyrate to the 
colon and provide sustained-release as the tablet passes through the colon. 
 
 
 
 
 
 
 
BioKier will arrange for the shipment of tablets directly from the manufacturer to the Mayo Clinic 
pharmacy in 150-ml HDPE white bottles (54 tablets per bottle). Each bottle contains enough test 
product for one week of dosing (plus 2 extra days). Each bottle will be labeled with the following 
information: 
 
 
 
 
 

 
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Study ID: 
CL-301 
Investigator: 
Adrian Vella, MD 
Mayo Clinic, Rochester, MN 
 
Take 3 tablets before breakfast and 3 tablets before bedtime daily 
 
Keep at room temperature 
 
Investigational use only 
Bottle contains 54 tablets of BKR-017 
 
Subject Number:  ________ Bottle Number:  ________ 
 
 
 
 
5.2 
Investigational Product Storage, Dispensing, and Return 
 
Bottles containing test product (BKR-017 tablets) will be stored at room temperature at the Mayo 
Clinic Research Pharmacy. 
 
The Research Pharmacist will keep detailed records of dispensation and return.  
 
During Test Period 
 
At Visit 1 (Day -28), eligible subjects will be given an 8-week supply of test product. Subjects will 
be instructed to begin dosing on Day 0. Additionally, Subjects will be instructed to complete the 
diary card daily and to return the diary card (with insulin usage, dosing information, changes in 
medications, and any adverse events) every 7 to 10 days by mail/email to the study team.  
 
.   
 
 
At Visit 2 (Day 48), the study CGM sensor, will be returned as well as all unused tablets in the 
original container(s). Diary cards will be reviewed and reconciled with the number of tablets 
returned. Subjects will be questioned about any missed doses based on the number of tablets 
returned, and the information supplied will be documented in the subject’s study records.  
 
5.3 
Investigational Product Administration 
 
The tablets will be self-administered orally twice daily; the first daily dose before breakfast, and 
the second daily dose before bedtime.  
 

 
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If the first daily dose is not administered before breakfast, it can be administered up to 12:00, noon. 
However, if the first daily dose is not taken by 12:00, noon it will be considered a missed dose; 
the bedtime dose should be taken as scheduled even if the morning dose was taken late or missed. 
If an evening dose is not taken by midnight, it will be considered a missed dose. All missed doses 
should be recorded on the diary card and returned in the original container at the next visit.  
 
The study staff will counsel subjects who repeatedly miss doses about the importance of being 
compliant with the dosing regimen. However, if the investigator determines that a subject is not 
willing or able to reasonably comply with the dosing regimen, the subject should be discontinued. 
 
5.4 
Per Protocol Population  
 
If a subject misses 15% of the required 96 doses (i.e., 14 or more doses) during the test period, 
then data from that subject will be excluded from the per-protocol efficacy assessment and 
included only in the intent-to-treat (ITT) analysis. 
 

 
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6. 
STUDY PROCEDURES 
Table 1: Time & Events Schedule 
 
 
 
Pre-
Scre
en 
Baseline Period 
Test Period 
Visit 1 
 
 
 
 
 
Visit 2  
D -28 
 
D 0  
D 18 
D 28 
 
D 48  
Review of Eligibility & Study Requirements  
X 
 
 
 
 
 
 
 
 
Written Informed Consent  
 
X 
 
 
 
 
 
 
 
Review of Inclusion / Exclusion criteria  
 
X 
 
 
 
 
 
 
 
Medical History & Demographics 
 
X 
 
 
 
 
 
 
 
Blood Pressure (seated position at the start of visit)  
 
X 
 
 
 
 
 
 
X 
Body Weight and Waist-to-Hip-Ratio 
 
X 
 
 
 
 
 
 
X 
Hematology and Chemistry (including CPR) 
 
X 
 
 
 
 
 
 
X 
Abbreviated Physical Exam 
 
X 
 
 
 
 
 
 
X 
Urine Pregnancy Test (women of childbearing 
potential) 
 
X 
 
 
 
 
 
 
X 
Dispense and/or Review Diaries for Compliance 
(Insulin and treatment) 
 
X 
 
X 
 
X 
X 
 
X 
Subject number assigned 
 
X 
 
 
 
 
 
 
 
CGM sensors dispensed 
 
X 
 
 
 
 
 
 
 
CGM sensor collected 
 
 
 
 
 
 
 
 
X 
 
Treatment Dispensed  
 
X 
 
 
 
 
 
 
 
Fasting Glucose and Triglycerides  
 
X 
 
 
 
 
 
 
X 
HbA1c  
 
X 
 
 
 
 
 
 
X 
Exploratory Tests hs-CRP and 1,5AG 
 
X 
 
 
 
 
 
 
X 
Concomitant Medication Review 
 
X 
 
X 
 
X 
X 
 
X 
Adverse Event Review 
 
 
 
X 
 
X 
X 
 
X 

 
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6.1 
Screening Period 
 
6.1.1 Recruitment and Prequalification 
 
Subjects will be recruited through advertisements and other materials approved by the Mayo Clinic 
IRB. Potential study subjects interested in participating may also be referred to the research team.  
 
Pre-screening will be performed to determine basic study eligibility information including: 
• Age 
• Type and age of onset of diabetes; confirmation of T1D diagnosis 
• Understanding and willingness to commit to the study requirements 
• Concomitant medications, including the need to refrain from use during the study of drugs that 
alter gastrointestinal motility 
 
Subjects who meet the initial criteria and are willing to participate will be scheduled for a 
Screening Visit (Visit 1). Subjects will be required to fast (i.e., no food or beverages other than 
water) for at least 10 hours prior to the screening visit.  
 
6.1.2 
Visit 1 (Screening) 
 
The screening visit will take place at the Outpatient Clinic (OPC), as described in Section 6.2.1 
below, and include: 
 
• Review of inclusion/exclusion criteria and eligibility 
• Subject consent will be obtained; documented with an Informed Consent Form (ICF) signed 
by subject 
 
6.2 
Study Periods 
 
The timing and evaluations for all visits to occur within the Baseline and Treatment Periods are 
defined in this section. Every attempt should be made to have all visits occurred at the required 
visit day. If, however, the subject is unable to adhere to the required visit day, a visit window of -
/+ 2 days is allowed (i.e., 2 days prior or 2 days after the required visit day). 
 
6.2.1 
Baseline Period (Day -28 to Day 0, Visit 1) 
 
The screening visit will take place on Day -28 before midday at the Outpatient Clinic (OPC). The 
following will be performed: 
1. Review of inclusion/exclusion criteria and eligibility 
2. Subject consent will be obtained; documented with an Informed Consent Form (ICF) signed 
by subject. Written informed consent must be obtained before any study procedures are 

 
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performed and a record of the subject’s consent (i.e., an original, signed and dated consent 
form) is to be maintained in the study files and a copy provided to the subject. 
Subjects whose HbA1c is within the range of 6.4-8.9% and meet all other preliminary eligibility 
criteria will continue with all other screening procedures, as follows:  
 
1. Urine pregnancy test for females of childbearing potential (exclude if positive and do not 
continue with other screening assessments). 
2. Confirm that the subject is in a fasted state. 
3. Obtain blood samples for routine hematology and chemistry tests, including CPR for safety 
evaluations, plus fasting glucose and triglycerides. See Tables 3 and 4, Appendix 2, for safety 
laboratory evaluations. 
4. Demographics (date of birth, gender, race, and ethnicity). 
5. Confirm medical history, including HTN determination, and including diabetes classification, 
onset/diagnosis date of diabetes, diabetes therapy(s), and therapy duration(s). 
6. Obtain concomitant therapies (generic name, dose, route, frequency, date started, and reason 
for use). 
7. Obtain vital signs (heart rate and blood pressure) in a seated position. 
8. Obtain subject’s height and weight 
9. Obtain waist and hip circumference to calculate the WHR. 
10. Perform standard physical exam including an exam to detect any signs of edema. Any amount 
of pitting edema will exclude a subject. 
11. After signing consent and meeting all inclusion criteria, subjects will be instructed on, and 
supplied with a study provided Dexcom G6 sensors, to collect glucose data. Subjects will be 
instructed to keep their Dexcom CLARITY app active, so that glucose monitoring data can be 
accessed by the study team.  
12. Diary cards will be provided for the subject to record insulin doses, changes to concomitant 
medications and adverse events. Diary cards will be mailed/emailed to the study team every 7 
to 10 days. Subjects using an insulin pump, will be advised to mail/email a copy of their pump 
settings and insulin doses every 7 to 10 days or upload their pump data to a prespecified, linked 
online account compatible with their pump (i.e. Medtronic CareLink, Tandem T Connect or 
Glooko), whereby the relevant data can be abstracted by the study team.  
13. Subjects will receive an 8-week supply of test product (BKR-017) 
14. Subjects will also be instructed not to make any modifications to their diet or exercise routine.  
 
 
 

 
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On Day 0:  
 
1. Day 0 will occur approximately 28 days after the screen visit. The study team will 
communicate with the subject via a phone call or virtual visit on this day.  
2. Study staff will review the subject’s diary and CGM data, in order to confirm compliance. 
3. Subjects will be questioned about any changes in health status/adverse events and any changes 
in therapy or medications. 
 
4.   
 
6.2.2 
Termination of Baseline Period, Initiation of Test Period 
 
 Day 0, the following will be performed: 
1. Subjects will be instructed to administer the first dose of test product prior to their mid-day 
meal and the second daily dose just prior to bedtime on Day 0. 
2. Subjects will be instructed to keep their Dexcom CLARITY app active, so that glucose 
monitoring data can be accessed by the study team.  
3. Subjects will be reminded to mail/email diary cards to the study team every 7 to 10 days. 
Subjects using an insulin pump, will be advised to mail/email a copy of their pump settings 
and insulin doses every 7 to 10 days or upload their pump data to a prespecified, linked online 
account compatible with their pump (i.e. Medtronic CareLink, Tandem T Connect or Glooko), 
whereby the relevant data can be abstracted by the study team.  
 
4. Subjects will also be instructed not to make any modifications to their diet or exercise routine.  
5. Subjects will be scheduled to return to the OPC for Visit 2 and instructed to return all unused 
tablets in the original container(s) along with any diary cards.  
 
 
 
On Day 48 (Visit 2, Final Visit): 
1. Subjects will report to the OPC in the morning, before 12:00 noon. If a subject is not able to 
comply with the Day 0 requirement, a variance of -2 days (or Day 46 or 50) is allowed. 
2. Urine pregnancy test will be obtained for females of childbearing potential.  
3. Subjects will return all unused tablets in the original container(s) along with any diaries. 
4. Blood pressure, height, weight, and waist and hip circumference will be obtained to calculate 
the WHR. Fasting samples will be obtained for glucose and triglycerides. Additional blood 
samples will be obtained for HbA1c, routine hematology, routine chemistry assessments CPR, 
and for exploratory biomarkers hs-CRP and 1,5AG.  
5. An abbreviated physical exam will be performed. 
6. Study staff will review the subject’s diary in order to reconcile tablets returned with any doses 
missed. 

 
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7. Subjects will be questioned about any changes in health status/adverse events and any changes 
in therapy or medications 
8. Study personnel will remove the CGM sensor from the subject’s abdomen and download the 
data to the Dexcom CLARITY data storage location.  
 
 
7. 
SAFETY AND EFFICACY EVALUATIONS 
 
7.1 
Vital Sign Measurements 
 
Single measurements of blood pressure and heart rate will be measured, with subjects in a seated 
position, at the start of every visit. 
 
7.2 
Tests for Safety Assessments 
 
Hematology (CBC), and chemistry testing and CPR, will be performed at Screening (Visit 1) to 
determine eligibility and at the completion of the Test Period (Visit 2) to evaluate changes in lab 
values. The complete list of hematology and chemistry laboratory safety parameters to be collected 
is provided in Appendix 2; See Table 3 and Table 4. 
 
A urine pregnancy test will be performed on women of child-bearing potential at screening to 
determine eligibility (pregnant women are not eligible) and at all subsequent Visits. Women who 
become pregnant at any time during the study should be discontinued at the investigator’s 
discretion and all pregnancies need to be followed to document the outcome of the pregnancy. 
 
In addition to the results of the urine pregnancy tests performed on women of child-bearing 
potential, study personnel will enter all laboratory data into the Electronic Case Report Form 
(eCRF).  
 
Blood and urine samples will be collected, processed, and stored according to Mayo Clinic’s 
established procedures.  
 
All laboratory testing, except the 1,5-Anhydroglucitol (1,5-AG) samples will be tested at Mayo 
Clinic.  The 1,5-AG samples will be tested by LabCorp.  
 
All Mayo Clinic analytical testing will be performed by validated/established Mayo Clinic test 
methods or by methods routinely performed in the Mayo Clinic Core Laboratory.  
 
Analytical testing performed at LabCorp will be performed by validated/established LabCorp test 
method. 
 

 
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7.3 
Tests for Efficacy Assessments 
 
7.3.1 
Primary Efficacy Assessments 
 
For the primary efficacy endpoint, HWR (Hip-to-Waist Ratio) and blood pressure will be 
measured at Day -28, Day 48 and used, along with HbA1c, to calculate the eGDR (estimated 
Glucose Disposal Rate) (WilliamsKV-ref 34).  
 
7.3.2 
Secondary and Exploratory Efficacy Assessments 
 
Secondary and exploratory evaluations will also be performed. The specific evaluations and 
sample collection and analysis time points are listed in Table 3, Appendix 2. 
 
Secondary efficacy evaluations require measurement of glucose variability, insulin usage, and 
triglycerides. Study personnel will enter the results into the eCRF. 
 
For the secondary endpoint of glucose variability, Dexcom CGM sensors will be supplied by 
BioKier and used to record continuous glucose levels every 5 minutes during the study.  
 
The CGM sensor is water-resistant and must be applied to the subject’s abdomen. The CGM 
sensor is held in place with a self-adhesive pad and requires no subject interaction with the device 
or the need for calibration.  
 
The CGM continuously measures glucose in interstitial fluid through a small (5mm long, 0.4mm 
wide) filament that is inserted just under the skin. It records glucose levels every 5 minutes, 
capturing up to 2880 glucose results for up to 10 days. Subjects will be instructed to keep their 
Dexcom CLARITY app active, so that glucose monitoring data can be accessed by the study team 
throughout the study. When the subject returns to the OPC at Visit 2, the CGM sensor will be 
removed by study staff and the data stored on the sensor will be retrieved by the reader and 
downloaded to the Dexcom CLARITY data storage location. The data will then be accessed by 
Mayo personnel and authorized BioKier personnel, including the statistician. 
 
The other secondary efficacy endpoint, triglycerides, will be measured in blood samples from 
Visits 1, and 2 by the Mayo Clinic Laboratory. 
 
Exploratory evaluations require analysis of the laboratory tests for hsCRP and 1,5-AG, from 
samples obtained Visits 1 and 2.  
 
All blood samples for exploratory evaluations will be collected and will be processed according to 
Mayo Clinic’s established procedures. All analytical testing will be performed on the secondary 

 
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and exploratory endpoints by validated/established Mayo Clinic test methods or by methods 
routinely performed in the Mayo Clinic Core Laboratory. 
 
Study personnel will enter the exploratory test results into the eCRF. 
 
7.4 
Adverse Events 
 
An adverse event is defined as any untoward medical occurrence that occurs during a clinical trial, 
whether or not considered related to the participation in the study or the test product received. 
Collection of adverse events in this study will begin at the time of administration of a subject’s 
first dose of test product. Any untoward events that may occur between the screening visit and the 
first dose of test product should be recorded as medical history. 
 
An adverse event or suspected adverse reaction is considered "serious" if, in the view of either the 
investigator or sponsor, it results in any of the following outcomes:  
 
• 
Death  
• 
Life-threatening  
• 
Inpatient hospitalization or prolongation of existing hospitalization  
• 
Persistent or significant incapacity or substantial disruption of the ability to conduct normal 
life functions, or 
• 
Congenital anomaly/birth defect 
 
Important medical events that may not result in death, be life-threatening, or require hospitalization 
may be considered serious when, based upon appropriate medical judgment, they may jeopardize 
the subject and may require medical or surgical intervention to prevent one of the outcomes listed 
in this definition. Examples of such medical events include allergic bronchospasm requiring 
intensive treatment in an emergency room or at home, blood dyscrasias or convulsions that do not 
result in inpatient hospitalization, or the development of drug dependency or drug abuse. 
 
An adverse event or suspected adverse reaction is considered "life-threatening" if, in the view of 
either the investigator or sponsor, its occurrence places the subject at immediate risk of death. It 
does not include an adverse event or suspected adverse reaction that, had it occurred in a more 
severe form, might have caused death. 
 
Suspected adverse reaction means any adverse event for which there is a reasonable possibility 
that the test product caused the adverse event. For the purposes of expedited safety reporting, 
"reasonable possibility" means there is evidence to suggest a causal relationship between the test 
product and the adverse event.  
 

 
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Subjects will be observed and questioned at every visit for the possible occurrence of adverse 
experiences; diaries will be reviewed to determine if any adverse experiences occurred between 
visits. Subjects should be asked to contact the investigator or study staff on any day on which an 
untoward event is experienced, whether the subject feels it is related to the test product or not.  
 
All adverse experiences, whether observed by the investigator or study staff, or reported by the 
subject, will be recorded in the subject’s study file and entered into the clinical database and will 
include the date of onset, outcome, date of resolution (if applicable), maximum severity, action 
taken, and relationship to the test product.  Whenever possible, a diagnosis will be assigned rather 
than the constellation of symptoms. 
 
Medical history/current medical conditions and adverse events will be coded using the medical 
dictionary (MedDRA) terminology. 
 
8. 
PROVISIONS TO MONITOR DATA TO ENSURE SUBJECT SAFETY 
 
The study protocol defines eligibility criteria designed to exclude patients for whom participation 
could pose unacceptable risks. Numerous medical assessments and laboratory tests throughout the 
study allow for continuous monitoring of safety. The Mayo Clinic Principal Investigator (PI) will 
review all lab work and adverse events. All adverse events will be reported to BioKier and the 
Mayo Clinic IRB in accordance with the Mayo Clinic IRB requirements.  
 
9. 
DISCONTINUATION OF A STUDY PARTICIPANT  
 
9.1 
Screen Failures 
 
A subject is considered a screen failure if he/she does not meet the inclusion/exclusion criteria or 
does not go on to receive test product for any reason.  
 
9.2 
Reasons for Discontinuation 
 
A subject will be discontinued from the study for any of the following reasons:  
1. Withdrawal of consent   
2. Hospitalization 
3. Treatment of an adverse event requires that the investigator be informed of the test product to 
which the subject was assigned 
4. Death 
5. Lost to follow-up 
6. Sponsor or investigator requests discontinuation of a subject for safety reasons  
7. Subject requires or has taken a prohibited medication for an extended period of time 

 
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8. Significant and/or persistent vomiting or diarrhea for an extended period of time, and 
vomiting/diarrhea can impact dosing/gut motility  
9. Subject is non-compliant with any of the other requirements of the study 
 
The reason for discontinuation will be documented in the subject’s study record and relevant page 
of the eCRF. 
 
For all subjects discontinuing before completing all study procedures and test visits, study 
personnel should perform a termination visit. At this visit subjects should return all unused test 
product and diary cards, vital signs should be obtained, and chemistry and hematology samples 
collected and tested. Additionally, if the subject is a woman of child-bearing potential, a urine 
pregnancy test should be performed. 
 
If the subject is unable or unwilling to come to the clinical site for a termination visit, study 
personnel should conduct a follow-up telephone call to assess the reason for the subject’s 
withdrawal from the study and to arrange for the return of unused test product. 
 
For subjects lost to follow up, every effort should be made, including sending a certified letter, to 
contact the subject to come to the clinical site for termination visit procedures and to return test 
product.  
 
10. 
DATA REVIEW AND DATABASE MANAGEMENT 
 
10.1 Data Collection 
 
All data should be recorded, handled, and stored at Mayo Clinic in a way that allows its accurate 
reporting and interpretation. 
 
Only specified study data and laboratory tests performed by Mayo Clinic will be entered into an 
eCRF. All data captured for this study must have an external originating source (either written or 
electronic); the eCRF is not considered as source. 
 
The investigator must maintain source documents for each study subject, which consist of ICFs, 
case and visit notes (e.g., clinic medical records) containing demographic and medical information, 
laboratory data, diary card data, and the results of any other tests or assessments. All data must be 
traceable to these source documents. The investigator must also keep the original ICF signed and 
dated by the subject (a signed copy is given to the subject). 
 
CGM data will be collected using the Dexcom G6 device. Data will be downloaded from the 
Dexcom G6 sensor to the Dexcom CLARITY data storage location in a manner that will allow 
accurate reporting and interpretation by the study team.  

 
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10.2 Database Management and Quality Control 
 
The study database consists of data from the eCRF database, and data retrieved from the CGM 
sensors used during the study.  
  
Study staff will enter required data into the eCRF on an ongoing basis as data are collected 
throughout the study. Automatic validation programs check for data discrepancies and generate 
appropriate error messages. Additionally, manual data queries will require resolution by the study 
personnel before query close-out. The eCRF will be accessible to authorized personnel only, and 
the system will be secured to prevent unauthorized access to the data and the system. This includes 
the requirement for a user ID and password to enter or change data. The level of access to the 
eCRF system (e.g., data entry, view rights only) will be dependent on each person’s role in the 
study. The investigator will be required to certify (via electronic signature) that the data entered in 
the eCRF are complete and accurate.  
 
Mayo Clinic personnel will review all data (eCRF) for completeness and accuracy and issue data 
queries about any inconsistent, incorrect or missing data. Once all queries have been resolved and 
the database is declared to be complete and accurate, it will be locked. After database lock, BioKier 
will provide the investigator with electronic media containing of all clinical study data for 
archiving. 
 
10.3 Subject Confidentiality 
 
In order to maintain subject confidentiality, only a subject number will be used to identify study 
subjects. Initials should not be used or referred to in any written communications.  
 
The investigator is expected to comply with all applicable privacy regulations such as the Health 
Insurance Portability and Accountability Act (HIPAA) of 1996.  
 
11. 
DATA ANALYSIS 
 
Analysis of the data will be conducted under the direction of BioKier personnel in compliance 
with the Statistical Analysis Plan (SAP) and internal guidance documents and industry standards.  
 
11.1 Objectives and Endpoints 
 
11.1.1 Analysis Objectives 
 
To evaluate the effects of BKR-017 tablets (active test product) when administered orally to T1D 
patients twice daily. Active test product will be administered for a 7 -week period. 

 
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11.1.2  Primary Efficacy Endpoint 
 
The primary efficacy endpoint is the comparison of insulin sensitivity, calculated as the eGDR 34 
on Days -28, and 48.  
 
11.1.3 Secondary Efficacy Endpoints 
 
Secondary endpoints include: 
1. Glucose variability (from CGM data) 
2. Fasting triglycerides 
3.  Insulin usage (from diary card) 
 
11.1.4 Safety Endpoints 
 
A descriptive assessment of safety will be made using vital signs, and selected chemistry 
assessments including bilirubin, alkaline phosphatase (ALP), asparate aminotransferase (AST), 
alanine aminotransferase (ALT), blood urea nitrogen (BUN), and creatinine. 
 
Additionally, clinically significant changes in the Visit 1 and 2 laboratory values obtained from 
routine hematology and chemistry (including CPR) testing will be reported in the eCRF along with 
adverse events reported during the test product administration period.  
 
Adverse events reported during the study will be attributed to the test product administered at the 
time the event occurred.  
 
11.1.5  Exploratory Endpoints 
 
The exploratory endpoints include changes in measurements of hsCRP and 1,5-AG. 
 
11.2 Sample Size 
 
 
Differences in eGDR (mg/kg/min) between Days -28, and 48 will be assessed by calculating eGDR 
from HWR (Hip-to-Waist Ratio), blood pressure, and HbA1c, off treatment and on treatment.  for 
each subject. Statistical significance will be assessed by comparing eGDR at each of Day -28and 
Day 48 to eGDR at Day -28 (i.e., baseline) with a paired t-test.  
 
This study of 12 subjects is considered large enough to draw some preliminary clinical 
conclusions. 

 
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12. 
ANALYSIS CONVENTIONS 
 
12.1 Analysis Sets 
 
An analysis database of all efficacy data collected will be generated (ITT Population). In addition, 
an analysis set of all subjects without major protocol violations (per protocol population) will 
consist of all subjects who took at least 85% of the 96 required doses (i.e., 82 or more doses).  
 
The safety analysis set will include all subjects who received any amount of test product.  
 
Analysis plans are detailed further in a Statistical Analysis Plan document (SAP).  
 
12.2 Missing Data 
 
All missing data will remain missing. No values will be imputed. 
 
12.3 Pharmacy Records 
 
Mayo Clinic pharmacy study records will be made available to BioKier since BioKier is supplying 
the test product. 
 
Inventory or dispensing records maintained by the Mayo Clinic pharmacy that identify the quantity 
of active test product that has been dispensed to a given subject will be reviewed. 
 
12.4 Demographics and Baseline Characteristics 
 
All data for background and demographic variables will be listed and summarized by subject. 
Relevant medical history, current medical conditions (including hypertension category (Y/N), 
results of laboratory screens, and any other relevant information will be listed. No statistical 
inference will be performed on these data.  
 
12.5  Subject Disposition 
 
The number and percentage of subjects in each analysis set who complete the study or discontinue 
early and the reasons for discontinuation will be summarized. No statistical testing will be 
performed. 
 
12.6  Efficacy Analysis 
 
All efficacy analyses will be repeated for both the ITT Population and the Per-Protocol Population. 
 

 
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12.6.1 Primary Efficacy Analysis 
 
 
12.6.2 Secondary Efficacy Analysis 
 
Blood glucose variability will be calculated for each subject, for each 24-hour day (midnight to 
midnight) during which a CGM device recording is available for the entire 24-hour period. Results 
will be presented graphically for each subject by study day.  
 
Triglyceride changes at Day -28 and Day 48 will be summarized as change from baseline where 
Day -28 is considered baseline. 
 
12.6.3 Exploratory Efficacy Analysis 
 
1,5 AG values at Day 48 will be compared to baseline (Day -28) with paired t-tests. 
 
hs-CRP values at Day 48 will be compared to baseline (Day -28) with paired t-tests. 
 
12.7 Safety Analysis 
 
12.7.1 Adverse Events 
 
The number and percentage of subjects experiencing adverse events reported during the test 
product administration period will be summarized by body system and preferred term for all 
subjects in the safety analysis set. Statistical testing will not be performed. 
 
12.7.2 Laboratory Data 
 
Selected laboratory tests will be summarized using an eight-number summary (sample size, mean, 
median, standard deviation, 25th quartile, 75th quartile, minimum, and maximum values) by visit. 
In addition, reported, graded laboratory abnormities will be summarized for each laboratory test. 
Statistical testing will not be performed. 
 
12.7.3 Vital Signs 
 
Vital signs will be summarized by visit using an eight-number summary (sample size, mean, 
median, standard deviation, 25th quartile, 75th quartile, minimum, and maximum values). Statistical 
testing will not be performed. 
Differences in eGDR (mg/kg/min) between Days -28, and 48 will be assessed by calculating eGDR 
on Days -28, and 48 for each subject. Statistical significance will be assessed by separate within-
subject comparisons of eGDR at each of Day -28, and Day 56 to eGDR at Day -28 (i.e., baseline) 
with paired t-tests. 

 
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13. 
ETHICAL CONSIDERATIONS 
 
13.1 Regulatory and Ethical Compliance 
 
This clinical study was designed and will be implemented in accordance with the ethical principles 
established in the Declaration of Helsinki. 
 
13.2 Informed Consent Procedures 
 
Eligible subjects may only be included in the study after providing written (witnessed, if required) 
consent using an Institutional Review Board (IRB)-approved ICF. Informed consent must be 
obtained before conducting any study-specific procedures. The process of obtaining informed 
consent should be documented in the subject’s medical record. Information should be given in 
both oral and written form and subjects must be given ample opportunity to inquire about details 
of the study.  
 
13.3 Responsibilities of the Investigator and IRB 
 
The protocol and the proposed informed consent form must be reviewed and approved by a 
properly constituted IRB before subjects can be enrolled in the study. For this study, Mayo Clinic 
IRB will be responsible for protecting the rights and welfare of human research subjects involved 
in the research activities and Mayo Clinic policies and procedures will be followed.  
 
A signed and dated statement that the protocol and informed consent have been approved by the 
IRB must be provided to BioKier before study initiation. Prior to study start, the investigator is 
required to sign a protocol signature page confirming agreement to conduct the study in accordance 
with the protocol and to give access to all relevant data and records to BioKier representatives 
including study monitors, and Mayo Clinic IRB, as required. 
 
13.4 Publication of Study Protocol and Results 
 
Upon study completion and finalization of a study report, the results of this trial may be submitted 
for publication in accordance with the publication terms defined in the Clinical Study Agreement.  
 
13.5 Protocol Adherence 
 
Deviations from the protocol should be avoided. All protocol deviations will be documented, 
recorded in the eCRF, and reported in the study report. Deviations will be reported to the Clinic 
IRB in accordance with their requirements. No waivers will be granted by the study sponsor.  
 

 
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13.6 Protocol Amendments 
 
Any change or addition to the protocol can only be made in a written protocol amendment that 
must be approved the Mayo Clinic IRB. Only amendments that are required for subject safety may 
be implemented prior to Mayo Clinic IRB approval. Notwithstanding the need for approval of 
formal protocol amendments, the investigator is expected to take any immediate action required 
for the safety of any subject included in this study, even if this action represents a deviation from 
the protocol. In such cases, the Mayo Clinic IRB will be informed in conformance with the IRB 
requirements. 
 
 

 
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14. 
APPENDICES 
 

 
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14.1 APPENDIX 1:  Previous Human Experience with Butyrate 
Table 2: Previous human experience with butyrate (selected studies) 
Patient Type 
Stage 
Dose/ 
Route 
of 
Administration 
Duration 
Reference 
Oral and Colon-Targeted Formulations Type 2 Diabetes 
Type 2 diabetes 
Efficacy, safety 
1g/ rectal infusion 
Single dose 
BioKier Study 8 
Type 2 diabetes 
Efficacy, safety 
3g/day butyrate 
suppositories  
4 weeks 
BioKier Study (data on file) 
Type 2 diabetes 
Efficacy 
6 x 100mg sodium butyrate 
capsules, orally 
45 days 
35;36 
Oral and Colon-Targeted Formulations in Other Patient Populations 
Cancer, advanced 
solid tumors 
Phase 1 
50-400 mg/kg/d, oral 
2 x 3 weeks 
37 
Cancer, advanced 
solid tumors 
Pilot efficacy, safety 150-200 mg/kg TID, oral 
 
29 
Crohn’s disease 
Pilot efficacy, safety 4g/d, oral – enteric-coated 
tablets 
8 weeks 
28 
Chronic pouchitis 
Pilot efficacy, safety 4.4g/d, suppository  
3 w 
27 

 
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14.2 APPENDIX 2:  Tables 
Table 3: Safety Hematology Laboratory Tests 
 
Screening 
Test Period 
Visit  
1 
2  
Day 
- 28 
48  
White Blood Cell Count (WBC) 
X 
X 
Red Blood Cell Count (RBC) 
X 
X 
Platelet 
X 
X 
Hemoglobin 
X 
X 
Hematocrit 
X 
X 
Mean Corpuscular Volume (MCV) 
X 
X 
Mean Corpuscular Hemoglobin (MCH) 
X 
X 
Mean Corpuscular Hemoglobin Concentration (MCHC) 
X 
X 
 
 
 

 
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Table 4: Safety and Efficacy Chemistry Laboratory Tests 
 Screening 
Test Period  
ASSESSMENTS 
Visit 
1 
 
 
 
 
 
2  
Day 
-28 
 
 
 
 
 
48  
 
Blood Urea Nitrogen (BUN) 
X 
 
 
 
 
 
X 
Creatinine 
X 
 
 
 
 
 
X 
Sodium, Calcium, Chloride, and Potassium 
X 
 
 
 
 
 
X 
Carbon Dioxide 
X 
 
 
 
 
 
X 
Total Protein 
X 
 
 
 
 
 
X 
Albumin 
X 
 
 
 
 
 
X 
Bilirubin (total)  
X 
 
 
 
 
 
X 
Aspartate Aminotransferase (AST) 
X 
 
 
 
 
 
X 
Alanine Aminotransferase (ALT) 
X 
 
 
 
 
 
X 
Alkaline Phosphatase (ALP)  
X 
 
 
 
 
 
X 
Cholesterol 
X 
 
 
 
 
 
X 
Triglycerides 
X 
 
 
 
 
 
X 
HDL 
X 
 
 
 
 
 
X 
LDL 
X 
 
 
 
 
 
X 
Glucose 
X 
 
 
 
 
 
X 
CRP 
X 
 
 
 
 
 
X 
HbA1C 
X 
 
 
 
 
 
X 
Urine pregnancy test 
X 
 
 
 
 
 
X 
Exploratory  
 
High-sensitivity C-reactive protein (hsCRP)  
X 
 
 
 
 
 
X 
1,5-Anhydroglucitol (1,5-AG)  
X 
 
 
 
 
 
X 
 
 
 

 
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