Acute Effect of HP-211 (Axulin) on Blood Glucose and Serum Insulin Responses in Healthy Lean and Overweight Humans
Phase: PHASE1
Conditions: Insulin Resistance, Diabetes
Sponsor: Housey Healthcare ULC

 
Date: 22 December, 2016 
 
Page 1 
 
 
 
 
 
 
Protocol 
GIL-1665 
Acute effect of Axulin on Blood Glucose and Serum Insulin 
Responses in Healthy Lean and Overweight Humans. 
 
 
Principal Investigator: 
Thomas MS Wolever, BM, BCh, PhD, DM 
Glycemic Index Laboratories, Inc. 
20 Victoria St., Suite 300, 
Toronto, Ontario M5C 2N8 
B: 416-978-5556;  e-mail: thomas.wolever@utoronto.ca 
 
 
Co-Investigator: 
Alexandra Jenkins, RD, PhD 
B: 416-861-0506 ext. 200; E-mail: alexandrajenkins@gilabs.com 
 
Study Coordinator: 
Janice Campbell, MSc, CCRP 
 
Sponsor: 
Housey Pharmaceutical Research Laboratories 
16800 West 12 Mile Road, Suite 201 
Southfield, MI 48076 
USA 
 
Sponsor Contact: 
Gerard M Housey, MD, PhD 
President and CEO 
tel: 248-663-7000 ext. 227 
FAX: 248-663-7007 
E-mail: ghousey@housey.com 
 
20 Victoria Street, Suite 300, 
Toronto, Ontario Canada M5C 2N8 
Tel 416-861-0506 Fax 416-861-0649 
www.gilabs.com

Protocol: GIL-1665 
 
 
 
Date: 22 December, 2016 
 
Page 2 
INTRODUCTION 
After consumption of a meal, pancreatic secretions of various digestive enzymes results in the 
breakdown of carbohydrates into monosaccharides including glucose.1 These sugars are 
subsequently absorbed through the intestinal lumen, resulting in an increased plasma glucose 
concentration.1 In response to high glucose levels, pancreatic beta-cells are stimulated to release 
the hormone insulin which circulates through the bloodstream and binds to insulin-responsive 
cells including adipocytes (fat tissue), myocytes (muscle tissue), and hepatocytes (liver).1  The 
resulting insulin-mediated signaling cascade initiates intracellular glucose uptake within 
peripheral tissues leading to a corresponding decrease in circulating plasma glucose.1 
In insulin responsive cells glucose uptake stimulation begins after the binding of insulin to 
Insulin Receptors (IR), which are found on the membrane surface of cells in insulin responsive 
tissues such as fat, muscle and liver.  The IR consists of an extracellular domain which binds to 
insulin, and an intracellular domain that has a protein tyrosine kinase activity.2  The binding of 
Insulin to the IR initiates a series of auto-phosphorylation events within the protein kinase 
domain that permit interaction and phosphorylation of downstream signaling proteins in the cell 
that mediate the cellular response to insulin.3-4 The resulting signaling complex includes proteins 
in the Insulin Receptor Substrate  (IRS) family known as IRS-1 and IRS-2. 5-8 These key targets 
of the insulin signaling pathway link IR activation to downstream signaling cascades that 
mediate intracellular processes including GLUT4 mediated glucose uptake. 9-16 
Pre-diabetes and Type II diabetes involve an impaired post-receptor response to insulin that 
hinders the glucose uptake response after meal consumption. 17-19 Chronic hyperglycemia and 
the resulting compensatory hyperinsulinemia promote a cohort of acute and chronic sequelae 
including cardiovascular disease, liver complications, central nervous system degeneration and 
hyperglycemic osmotic stress. 20-23 Axulin is a natural health product consisting of a mixture of 
extracts from herbs and vegetables present in normal diets which was identified by screening 
more than 100,000 compounds and  extracts in a patented cell-culture based assay system 
targeting the IRS proteins.  In vitro, Axulin has marked effects on the IRS-2 branch of the insulin 
signaling cascade to enhance downstream insulin signaling.  Axulin has been shown in animal 
models to increase glucose uptake in peripheral tissues and decrease  circulating blood glucose 
and triglyceride concentrations. Regular supplementation of the diet with Axulin would be 
expected to reduce the incidence of associated pre-diabetic and diabetic complications, resulting 
in an increased quality of life for patients without resorting to current anti-diabetic prescription 
drugs such as metformin and others that may have substantial unwanted side effects in patients. 
 
HYPOTHESES 
Axulin will reduce postprandial glucose and insulin responses in a dose-dependent fashion in 
healthy subjects without diabetes.  The reduction in glucose and insulin will be relatively greater 
in insulin-resistant than insulin-sensitive subjects. 
 

Protocol: GIL-1665 
 
 
 
Date: 22 December, 2016 
 
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OBJECTIVES 
The primary objective of this study is to determine the dose of Axulin which will reduce the 
incremental area under the blood glucose response curve (AUC) elicited by 75g oral glucose in 
healthy subjects by ≥33%. 
The secondary objectives are to determine the dose-response effects of Axulin on: 
- blood glucose and serum insulin AUC 
- % reduction of insulin AUC 
- the blood glucose and serum insulin increments at 15, 30, 45, 60, 90 and 120min 
- insulinogen index 
- Matsuda insulin sensitivity index 
- ISSI-2 index of beta-cell function 
- incremental blood lipid response at 120min 
- plasma triglyceride response 
- plasma axulin response 
The other objectives are to determine whether the effects of Axulin on glucose, insulin and 
triglyceride responses differ in lean vs overweight subjects and to determine whether the effects 
of Axulin on postprandial responses are similar when administered in capsule versus tablet form. 
 
SUBJECTS 
Inclusion criteria 
Subjects will be males or non-pregnant, non-lactating females aged 18-60 years who have no 
history of diabetes and are in good health.  Lean subjects will have a BMI >18.5 and <25.0kg/m², 
overweight subjects will have a BMI ≥25.0 and <35.0kg/m².  Subjects taking stable doses (for at 
least 4 weeks of signing the consent form) of the birth control pill, thyroxin replacement therapy, 
statins, fibrates, cholesterol absorption inhibitors, anti-hypertensive medications, asprin, non-
steroidal anti-inflammatories and/or mild anxiolytics or sedatives can be included. 
Female subjects of child-bearing potential must, for the duration of the study, be using one of the 
following birth control methods: total abstinence, a hormonal birth control method (oral, 
injectable, transdermal, or intra-vaginal), intrauterine device, or confirmed successful vasectomy 
of partner. 
 
Exclusion criteria 
- Fasting serum glucose >6.9mmol/L (124mg/dL) 
- HbA1c >6.4% 
- Fasting serum triglycerides >4.5 mmol/L (399 mg/dL) 
- Fasting LDL cholesterol >4.99 mmol/L (192 mg/dL) 
- Blood pressure >149 systolic or >89 diastolic 
- Serum creatinine, or aspartate- or alanine transaminases >1.2 times upper limit of normal 
- White cell count, red blood cell count, hemoglobin or hematocrit outside normal range 
- Hospitalization for surgery or a medical condition within 3 months of signing the consent form 

Protocol: GIL-1665 
 
 
 
Date: 22 December, 2016 
 
Page 4 
- Use of any drug to treat diabetes 
- Use of medications other than those listed above or the presence of any condition which might, 
in the opinion of Dr. Wolever, either: 1) make participation dangerous to the subject or to 
others, or 2) affect the results 
- Allergy or sensitivity to tarragon, chromium, escarole, lettuce, microcrystalline cellulose, 
inulin, food colouring (FD&C Yellow#5 and Blue#1) or vegetable-based capsules (silicon 
dioxide, titanium dioxide, hydroxypropylmethylcellulose) 
 
Number of subjects 
The primary endpoint is the % reduction in 2-hour incremental AUC for glucose.  Using the t-
distribution with 2-tailed p<0.05 and assuming an average CV of within-individual variation of 
2-hour incremental AUC values of 25%, n=20 subjects (10 lean and 10 overweight) has 80% 
power to detect a 17% difference in AUC, 90% power to detect a difference of 19% and 99% 
power to detect a difference of 26%.  Within each subject group (n=10) there is 80% power to 
detect a difference in AUC of 25%, and 80% power to detect a 33% difference between the 
responses of lean and overweight subjects.  To allow for 10% dropout, we will recruit n=11 lean 
and n=11 overweight subjects. 
 
Subject Recruitment 
Participants will be recruited from the pool of participants at GI Laboratories who have indicated 
that they are willing to be contacted and asked if they wish to participate in new studies.  If an 
insufficient number of subjects can be recruited, then a newspaper advertisement will be used to 
recruit new subjects. 
 
Selection of participants 
Participants willing to be considered will be invited to come to the research centre to have the 
study procedures explained to them and be given a copy of the consent form which they may 
either sign then, take away to sign at a later date, or decline to participate.  Participants will be 
encouraged to ask any questions they may have and not to sign the consent form until all of their 
questions have been answered to their satisfaction.  Those who consent to participate will come 
to the research centre for a pre-selection visit when subject eligibility will be determined.  During 
this visit, the 10-14hr overnight fasted participants will complete a questionnaire (Appendix 1) to 
assess medical history, and lifestyle factors, have height, weight, waist circumference and blood 
pressure measured, and have a blood sample drawn for measurement of glucose, HbA1c, lipids, 
creatinine, AST, ALT and complete blood count.  Females will have a pregnancy test. 
 
PROCEDURES 
The study will have a randomized block design with repeated measures.  One block, which will 
be double-blinded, will test the effect of Axulin capsules; the other block, which will be open 
label, will test the effect of Axulin tablets. Within each participant, the order of the blocks will be 
randomized and within each block the order of the treatments will be randomized. 

Protocol: GIL-1665 
 
 
 
Date: 22 December, 2016 
 
Page 5 
Eligible participants will be instructed to maintain stable dietary and exercise habits and for the 
duration of the study, to avoid vigorous physical activity for 24hr before each visit and to and 
consume their normal dinner the night before each visit.  At each visit current use of medications 
and natural health products participants will be reviewed with each participant and any changes 
will be discussed with the investigator.  Female participants will be advised that if they become 
pregnant during the study they are to contact the investigators and discontinue their participation 
immediately. 
Each subject will be studied on 6 separate days with at least a 3-day interval between tests.  On 
each test day, subjects will come to GI Labs (20 Victoria Street, Toronto) in the morning after a 
10-14h overnight fast.  On each test occasion the subject will be weighed, and give fasting 
finger-prick and venous blood samples.  Blood from a forearm vein will be collected into a 6ml 
EDTA tube (Vacutainer, BD Canada, Mississauga, ON).  Finger-prick blood samples will be 
collected into 2 separate vials: one (2-3 drops blood into a fluoro-oxalate tube) for glucose 
analysis and the other (6-8 drops of blood into an 0.3ml Microvette with clot activator, Sarsted, 
Germany) for insulin.  After the fasting sample the subject will take 12 capsules or 2 tablets with 
a drink of 250ml water.  Subjects will rest for 3-10min and have their blood pressure measured; 
40min after swallowing the first capsule or tablet a finger-prick blood sample (2-3 drops for 
glucose and 6-8 drops for insulin analysis) will be collected and then subjects will consume 
82.5g dextrose (containing 75g anhydrous glucose) dissolved in 300ml water within 5min plus a 
drink of 150ml water within 10min of starting the glucose drink.  Further finger-prick blood 
samples for glucose (2-3 drops) and insulin (6-8 drops) will be obtained at 15, 30, 45, 60, 90 and 
120 min after starting to drink the glucose solution.  Venous blood samples (7ml) will be 
collected after the 60 and 120 min finger-prick.  During the test subjects will remain seated 
quietly except for trips to the washroom if necessary.  After the last blood sample subjects will 
be offered a snack and be free to leave. 
Treatments 
The 6 treatments administered will consist of the following: 
 
- 12 placebo capsules wit 250ml water (0g Axulin) 
 
- 250ml water alone (0g Axulin 
 
 
 
- 4 placebo and 8 Axulin capsules (2g Axulin) 
 
- 16 Axulin capsules (4g Axulin) 
 
- 2 Axulin tablets (2g Axulin) 
 
- 4 Axulin tablets (4g Axulin) 
Each placebo capsule contains 250mg inulin, each identical-looking Axulin capsule contains 
250mg Axulin.  It is anticipated that Axulin will be sold in tablets because 1g can be compacted 
into a tablet without loss of biological activity, but, because Axulin is a low-density powder, only 
400mg can be placed in a capsule of similar size.  We will use capsules in this study because it is 
more economical to produce the relatively small number of identical-looking placebo capsules 
required as compared to producing placebo tablets.  To obtain doses in 1g increments, we will 
use smaller capsules containing 250mg each.  The rationale for including tablets in the study is 
that Axulin in tablet form might be more slowly bioavailable than when encapsulated; thus, it is 

Protocol: GIL-1665 
 
 
 
Date: 22 December, 2016 
 
Page 6 
important to determine whether the effects of Axulin tablets on postprandial responses is similar 
to the same dose taken in capsules. 
Blinding 
The number of Placebo and Active Axulin capsules and tablets required for the study will be 
provided to GI Labs.  A person not involved in any other aspect of the study will make up 6 
small zip-lock plastic bags for each subject labelled with a subject number and visit number and 
containing the treatment for vists 1 through 6 (the bag for the water alone treatment will be 
empty).  The treatments will be randomized as shown in Table 1.  The order of the blocks and 
the order of the treatments within each block were randomized using the @rand() function of 
Excel with 2 blocks of 6 created for lean subjects and 2 blocks for overweight subjects; the 2 
extra orders are for use in case of drop-outs so that at least n=20 subjects complete the study (see 
Table 1).  The person labeling and filling the bags with the treatments will assign the treatments 
to the codes and place the treatment assignments in a sealed opaque envelope kept in an 
accessible location.  The envelope will be opened only after the study has been completed and 
the database locked, or in case of emergency if required for participant safety. 
 
Table 1: Randomization Scheme 
 
ID 
Group 
Visit 1 
Visit 2 
Visit 3 
Visit 4 
Visit 5 
Visit 6 
1 
Lean 
A 
B 
C 
E 
D 
F 
2 
Lean 
B 
C 
A 
E 
F 
D 
3 
Lean 
F 
D 
E 
C 
A 
B 
4 
Lean 
B 
A 
C 
F 
E 
D 
5 
Lean 
D 
E 
F 
A 
C 
B 
6 
Lean 
D 
F 
E 
C 
B 
A 
7 
Lean 
F 
D 
E 
A 
C 
B 
8 
Lean 
C 
B 
A 
D 
E 
F 
9 
Lean 
D 
F 
E 
B 
C 
A 
10 
Lean 
C 
A 
B 
E 
D 
F 
11 
Lean 
B 
A 
C 
E 
F 
D 
12 
Lean 
F 
E 
D 
A 
B 
C 
21 
Overweight 
D 
E 
F 
C 
B 
A 
22 
Overweight 
F 
D 
E 
B 
A 
C 
23 
Overweight 
A 
B 
C 
E 
D 
F 
24 
Overweight 
A 
C 
B 
F 
E 
D 
25 
Overweight 
B 
C 
A 
E 
F 
D 
26 
Overweight 
C 
A 
B 
D 
F 
E 

Protocol: GIL-1665 
 
 
 
Date: 22 December, 2016 
 
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27 
Overweight 
B 
C 
A 
F 
D 
E 
28 
Overweight 
D 
F 
E 
B 
A 
C 
29 
Overweight 
D 
E 
F 
C 
B 
A 
30 
Overweight 
F 
E 
D 
A 
B 
C 
31 
Overweight 
A 
C 
B 
E 
F 
D 
32 
Overweight 
E 
D 
F 
C 
A 
B 
Instructions: please assign capsules to codes  A, B or C, and tablets or empty bag to codes D, E or F.  
Place treatment assignments into a sealed opaque envelope and keep the envelope in an accessible 
location.  Treaments are as follows: 
 
BLOCK 1 (A, B or C) 
 
- Placebo 1; 16 Placebo capsules 
 
- 2g Axulin caps: 4 Placebo capsules plus 8 Axulin capsules 
 
- 4g Axulin caps: 16 Axulin capsules 
 
BLOCK 2 (D, E or F) 
 
- empty bag 
 
- 2g Axulin tablets: 2 Axulin tablets 
 
- 4g Axulin tablets: 4 Axulin tablets 
Biochemical Analysis 
Finger-prick blood samples for glucose analysis will be placed in the refrigerator immediately 
after being obtained and at the end of the test placed in a -20˚C freezer until analysis which will 
be performed within 5 days. Glucose analysis is done using an YSI model 2300 STAT analyzer 
(Yellow Springs, OH).  The microvette tubes used to collect blood for insulin analysis will be 
centrifuged at 3622×g for 2 min and the serum transferred to labeled polypropylene tubes and 
stored at -20˚C prior to analysis of insulin using the Human Insulin EIA Kit (Alpco Diagnostics).  
Venous blood samples will be stored at 4ºC until the end of the study after which they will be 
centrifuged at ~1400×g for 10 min and the plasma removed and separated into 2×1ml aliquots; 1 
for analysis of triglycerides at St. Michael’s Hospital biochemistry laboratory and the other for 
measurement of the active ingredient in Axulin by liquid chromatrography/mass spectrometry. 
 
Statistical Analysis 
Incremental areas under the blood glucose, insulin and triglyceride response curves (AUC), 
ignoring area below fasting, will be calculated.  Blood glucose and insulin concentrations and 
AUC values will be analyzed using repeated measures ANOVA examining for the main effects 
of dose, form (tablets vs capsules) and subject group (lean vs overweight) and their interactions.  
After demonstration of significant heterogeneity, differences between individual means will be 
assessed using Tukey’s test to adjust for multiple comparisons. 
 
Informed Consent 
The GI Testing protocol has been approved by the Western Institutional Review Board® which 
meets all requirements of the US Food and Drug Administration (FDA), the Department of 
Health and Human Services (DHHS), the Canadian Health Protection Branch (HPB), Canadian 

Protocol: GIL-1665 
 
 
 
Date: 22 December, 2016 
 
Page 8 
Institutes for Health Research (CIHR) and the European Community Guidelines.  Informed 
consent is obtained from all subjects for each series of tests they participate in. 
 
Participant Withdrawal Criteria 
The Investigator may elect to withdraw the participant from the study or the participant may 
withdraw from the study at any time.  Reasons for withdrawing include, but are not limited to: 
 Development of an exclusion criterion during the course of the study 
 Study product never consumed 
 Participant was lost to follow-up 
 Participant did not follow study procedures  
 Participant elected to withdraw from the study for any reason 
 Investigator elected to remove participant from the study for any reason 
 Subject takes longer than the specified time to complete the study 
 
In the case of premature withdrawal, all planned assessments for the end of the study visit will be 
completed and the sponsor will be informed as soon as possible. The subject’s Case Report Form 
(“CRF”) will be completed including the final assessment. The date and main reason for the 
premature withdrawal will be clearly documented in the subject’s file and written down on the 
CRF 
 
ADVERSE EVENT REPORTING 
An adverse event (AE) is any symptom, injury, illness, or medical/surgical procedure whether 
planned or not or whether associated with the study foods or not.  AEs will be assessed at each 
visit and recorded in the Case Report Forms. AEs will be evaluated for duration, severity, 
seriousness, and causal relationship to the food items provided in the study by the study 
physician who will decide if any action is required and provide referral, treatment, and/or follow-
up as appropriate until resolved, judged to no longer be clinically significant, or if a chronic 
condition, until fully characterized.  Research staff will record the final outcome and resolution 
date of the event wherever possible. The action taken and the outcome will be recorded in the 
Case Report Form. 
 
It is anticipated that participants enrolled in the study will be clinically managed without the 
need to add medications or to change the dosage(s) assigned to their current medications.  In the 
event that additional medication is required, or an increase in their medication dosage is 
warranted, these changes will be recorded as adverse events.   
 
Investigators are required to report adverse events that fit the following criteria within 10 
working days of the time the investigator becomes aware of them: 
 
 Event is Unexpected (in terms of nature, severity, or frequency) given (a) the 
research procedures that are described in the protocol-related documents, such as 
the IRB-approved research protocol and informed consent document, or the 

Protocol: GIL-1665 
 
 
 
Date: 22 December, 2016 
 
Page 9 
Investigator Brochure; and (b) the characteristics of the subject population being 
studied. 
 Related or possibly related to participation in the research (possibly related 
means there is a reasonable possibility that the incident, experience, or outcome 
may have been caused by the procedures involved in the research); and 
 Suggests that the research places subjects or others at a greater risk of harm 
(including physical, psychological, economic, or social harm) than was previously 
known or recognized. 
 
If the adverse event is clearly not related to the study drug, device, procedures, or 
washout process, it would not represent a risk to other subjects in the research or a 
“problem” for the study and, therefore, does not have to be reported to WIRB. 
 
The Principal Investigator, Dr. Thomas Wolever, will be informed within 48 hrs of the nature of 
the reportable AE and the treatment allocation of the participant in whom it occurred by sending 
a copy of the AE report via email (thomas.wolever@utoronto.ca).  Dr. Wolever will track all 
reportable AEs and notify the sponsors if they occur. 
 
In addition to the above, all adverse events will be reported to Health Canada.  All serious 
adverse events (any adverse event that results in death, is life-threatening, requires in-patient 
hospitalization or prolongation of existing hospitalization, results in persistent or significant 
disability/incapacity or is a congenital anomaly/birth defect) will be reported to Health Canada, 
the Sponsor and the IRB within 24hr of their ascertainment. 
 
Risks 
Healthy volunteers have been found to be able to tolerate doses of up to 60g of resistant 
maltodextrins with few symptoms (Storey et al. 2007).  Since the test meals used in this study 
contain a maximum of 12g resistant starch, we do not expect subjects to experience any GI 
symptoms, or if symptoms do occur, they would be very mild (eg. mild flatulence). However, 
there may be rare occurrences of study product intolerance, as manifested by severe 
gastrointestinal symptoms (such as crampy gas or watery stools), eczema, rashes, or other signs 
of allergy, or infection.   
 
Dispensing 
It is the responsibility of the Investigator or his designee to ensure that study product is 
only provided to study participants.   
All study products will be consumed during test sessions at Glycemic Index Laboratories 
and will not be dispensed to subjects to consume outside of the facility.  The specific serving size 
for each product will be provided to the Investigator along with instructions for administration of 
each product. 
 
Source documents and archiving 
 
All documents relating to the study will be kept by the investigator: the original informed 
consent form, source documents, copy of the CRF, copy of the accountability of products 

Protocol: GIL-1665 
 
 
 
Date: 22 December, 2016 
 
Page 10 
administered, copy of the WIRB (ethics committee) approval and correspondence with the 
sponsor.  The investigator will authorize direct access to the source documents during 
monitoring.  As required by Canadian law, documents will be kept by the investigator for at least 
25 years after the closure of the study. 
 
BUDGET 
 
Expense Category 
Amount ($CDN)
Screening 
8,450 
Hospital Lab fees 
3,475 
WIRB submission 
4,375 
Subject payments 
9,840 
Supplies 
14,370 
Staff 
 
      Study coordinators: 144hr @ $45 
6,480 
      Junior Staff: 138hr @ $30 
4,140 
      Lab technicians: 439hr @ $30 
13,170 
Data analysis 
4,000 
Protocol/Report /Manuscript 
5,800 
Space utilization 
31,400 
 
TOTAL 
105,500 
 

Protocol: GIL-1665 
 
 
 
Date: 22 December, 2016 
 
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