Phase 1, Open Label Dose-Finding Study of Intranasal Insulin in Healthy Participants
Phase: PHASE1
Conditions: Out-Of-Hospital Cardiac Arrest, Healthy
Sponsor: Robert Silbergleit

Phase 1, Open Label Dose-Finding Study of 
Intranasal Insulin in Healthy Participants 
 
NCT05062785 
 
Date of Actual IRB Approval: July 8, 2021 

 
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Phase 1, Open Label Dose-Finding Study of Intranasal 
Insulin in Healthy Participants 
 
 
National Clinical Trial (NCT) Identified Number: pending 
Principal Investigator: Robert Silbergleit, MD 
IND Sponsor: Robert Silbergleit, MD, University of Michigan 
IND Number: 155144 
 
Funded by: American Heart Association (AHA) 
 
 
Version 3 
 
15 April 2021 
 
 

 
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Table of Contents 
1 Protocol Summary 
7 
1.1 Synopsis 
7 
1.2 Schema 
9 
1.3 Schedule of Activities (SoA) 
10 
2 Introduction 
11 
2.1 Study Rationale 
11 
2.2 Background 
11 
2.3 Risk/Benefit Assessment 
12 
2.3.1 Known Potential Risks 
12 
2.3.2 Known Potential Benefits 
12 
2.3.3 Assessment of Potential Risks and Benefits 
12 
3 Objectives and Endpoints 
14 
4 Study Design 
15 
4.1 Overall Design 
15 
4.2 Scientific Rationale for Study Design 
16 
4.3 Justification for Dose 
17 
4.4 End of Study Definition 
17 
5 Study Population 
18 
5.1 Inclusion Criteria 
18 
5.2 Exclusion Criteria 
18 
5.3 Lifestyle Considerations 
18 
5.4 Screen Failures 
19 
5.5 Strategies for Recruitment and Retention 
19 
5.6 Considerations related to COVID-19 
19 
6 Study Intervention 
20 
6.1 Study Intervention(s) Administration 
20 
6.1.2 Dosing and Administration 
20 
6.2 Preparation/Handling/Storage/Accountability 
22 
6.2.1 Acquisition and accountability 
22 

 
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6.2.2 Formulation, Appearance, Packaging, and Labeling 
22 
6.2.3 Product Storage and Stability 
22 
6.3 Measures to Minimize Bias: Randomization and Blinding 
22 
6.4 Study Intervention Compliance 
22 
6.5 Concomitant Therapy 
23 
6.5.1 Rescue Medicine 
23 
7 Study Intervention Discontinuation and Participant Discontinuation/Withdrawal 
24 
7.1 Discontinuation of Study Intervention 
24 
7.2 Participant Discontinuation/Withdrawal from the Study 
25 
7.3 Lost to Follow-Up 
25 
8 Assessments 
27 
8.1 Efficacy Assessments 
27 
8.2 Safety and Other Assessments 
28 
8.3 Adverse Events and Serious Adverse Events 
28 
8.3.1 Definition of Adverse Events (AE) 
28 
8.3.2 Definition of Serious Adverse Events (SAE) 
28 
8.3.3 Classification of an Adverse Event 
29 
8.3.3.1 Severity of Event 
29 
The severity of adverse events will be graded using the Common Terminology Criteria for 
Adverse Events (CTCAE) – NIH/NCI 
29 
8.3.3.2 Relationship to Study Intervention 
29 
8.3.3.3 Expectedness 
29 
8.3.4 Time Period and Frequency for Event Assessment and Follow-Up 
29 
8.3.5 Adverse Event Reporting 
29 
8.3.6 Serious Adverse Event Reporting 
30 
8.3.7 Reporting Events to Participants 
30 
8.3.8 Events of Special Interest 
30 
8.3.9 Reporting of Pregnancy 
30 
8.3.10  Definitions of Selected or Anticipated Adverse Events 
31 
8.4 Unanticipated Problems 
31 

 
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8.4.1 Definition of Unanticipated Problems (UP) 
31 
8.4.2 Unanticipated Problem Reporting 
31 
8.4.3 Reporting Unanticipated Problems to Participants 
32 
9 Statistical Considerations 
33 
9.1 Statistical Hypotheses 
33 
9.2 Sample Size Determination 
33 
9.3 Populations for Analyses 
34 
9.4 Statistical Analyses 
34 
9.4.1 General Approach  
34 
9.4.2 Analysis of the Primary Efficacy Endpoint(s) 
35 
9.4.3 Analysis of the Secondary Endpoint(s) 
35 
9.4.4 Safety Analyses 
35 
9.4.5 Baseline Descriptive Statistics 
35 
9.4.6 Planned Interim Analyses 
35 
9.4.7 Sub-Group Analyses 
35 
9.4.8 Tabulation of Individual Participant Data 
35 
9.4.9 Exploratory Analyses 
35 
10 Supporting Documentation and Operational Considerations 
36 
10.1 Regulatory, Ethical, and Study Oversight Considerations 
36 
10.1.1 Informed Consent Process 
36 
10.1.1.1 Consent/assent and Other Informational Documents Provided to participants 
36 
10.1.1.2 Consent Procedures and Documentation 
36 
10.1.2 Study Discontinuation and Closure 
36 
10.1.3 Confidentiality and Privacy 
36 
10.1.4 Future Use of Stored Specimens and Data 
36 
10.1.5 Key Roles and Study Governance 
37 
10.1.6 Safety Oversight 
37 
10.1.7 Clinical Monitoring 
37 
10.1.8 Quality Assurance and Quality Control 
37 
10.1.9.1 Data Collection and Management Responsibilities 
37 

 
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10.1.9.2 Study Records Retention 
38 
10.1.10 Protocol Deviations 
38 
10.1.11 Publication and Data Sharing Policy 
38 
10.1.12 Conflict of Interest Policy 
38 
10.2 Additional Considerations 
38 
10.3 Abbreviations 
39 
10.4 Protocol Amendment History 
40 
11 References 
41 
 

 
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Statement of Compliance 
The trial will be conducted in accordance with applicable United States (US) Code of Federal 
Regulations (CFR). The Principal Investigator will assure that no deviation from, or changes to the 
protocol will take place without prior agreement from the funding agency and documented approval 
from the Institutional Review Board (IRB), and the Investigational New Drug (IND) sponsor, if 
applicable, except where necessary to eliminate an immediate hazard(s) to the trial participants. All 
personnel involved in the conduct of this study have completed Human Subjects Protection Training. 
The protocol, informed consent form(s), recruitment materials, and all participant materials will be 
submitted to the IRB for review and approval. Approval of both the protocol and the consent 
form(s) must be obtained before any participant is consented. Any amendment to the protocol will 
require review and approval by the IRB before the changes are implemented to the study. All 
changes to the consent form(s) will be IRB approved; a determination will be made regarding 
whether a new consent needs to be obtained from participants who provided consent, using a 
previously approved consent form. 
 

 
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1 PROTOCOL SUMMARY 
1.1 SYNOPSIS 
Title: 
Phase 1, Open Label Dose-Finding Study of Intranasal Insulin in Healthy 
Participants  
Study Description: 
The current study will evaluate the tolerability of intranasal insulin in 24 healthy 
adult volunteers after being serially exposed to escalating doses over 5 visits with 
a 1-week washout period in between each visit.  Regular insulin (Humulin R U500, 
Eli Lilly, Indianapolis IN) at a concentration of 500 U/mL will be prepared in doses 
of up to 2 mL containing between 0 – 1000 U. The ≤2 mL doses will be 
administered intranasally using a 5 mL syringe divided into ≤1 mL per nostril. 
Intranasal administration will be performed with the participants supine with the 
neck extended to mimic the position of comatose out-of-hospital cardiac arrest 
(OHCA) patients.   
  
A total of 11 possible doses will be tested, ranging from 0 to 1000 U intranasal 
insulin. For every 6 participants, iterations of a Bayesian model will be performed 
to associate the administered dose with the frequency of dose-limiting 
hypoglycemia or other adverse events. The model will adaptively reallocate the 
dose increments for the next 6 participants. To provide more precise and 
reproducible estimates of dose-limiting toxicity, the trial will modify the size of 
dose increments based on the developing model to allocate more administrations 
to areas of the model with greatest uncertainty, or to higher doses in the absence 
of dose-limiting hypoglycemia. This will provide more observations and the 
greatest information density at doses of greatest interest that are most relevant 
to the determination of maximally tolerated dose.  Safety and tolerability will be 
assessed for approximately 4 hours after each insulin administration. 
Objectives: 
Determine the maximum tolerated dose of intranasal insulin in healthy 
participants  
Endpoints: 
Primary:  The primary safety outcome will be severe hypoglycemia as defined by 
blood glucose <45mg/dL. 
 
Secondary:  Secondary endpoints will include the change in blood glucose, insulin, 
and C-peptide levels for up to 4 hours after drug administration to evaluate for 
any systemic absorption of intranasal insulin.   
Study Population: 
24 healthy subjects, male or female, 18 years of age or older 
Phase: 
I 
Study Site: 
Single site, Michigan Medicine, University of Michigan 

 
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Description of 
Study Intervention: 
HUMULIN R U-500 (insulin human injection, Eli Lily, NDC 0002-8501-01) to be 
given intranasally at differing doses depending on study day. Sterile diluent to 
prepare the zero insulin dose and lower concentrations of intranasal insulin will 
also be acquired through Eli Lily (NDC 0002-0800). 
A total of 11 possible doses will be tested, ranging from 0 to 1000 U insulin. Each 
volunteer will come for 5 visits.  
Study Duration: 
One year 
Participant 
Duration: 
Up to 60 days from the day of screening to last visit.  
 
 

 
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1.2 SCHEMA 
 
 
Figure 1.2.1. Study visit schedule                                                                                                                              
 
 
 
 
 
Figure 1.2.2. Intranasal insulin dose allocation if no study participants  experiences severe 
hypoglycemia       
 
 
 

 
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1.3 SCHEDULE OF ACTIVITIES (SOA) 
 
Schedule of Events table 
  
  
  
  
  
  
Study Visits 
Screening 
1 
2 
3 
4 
5 
Study Day(s) 
-28 
0 
7+/-3 
14+/-3 
21+/-3 
28+/-3 
Provide study overview and 
participant responsibilities 
X 
 
 
 
 
 
Informed consent 
X 
 
 
 
 
 
Medical history and 
inclusion/exclusion criteria review 
X 
 
 
 
 
 
Previous and concomitant 
medications 
X 
X 
X 
X 
X 
X 
Physical examination 
X 
 
 
 
 
 
Vital signs1 
X 
X 
X 
X 
X 
X 
Urine pregnancy test (female subjects 
of child bearing potential) 
X 
X 
X 
X 
X 
X 
Insertion of a short term intravenous 
catheter 
 
X 
X 
X 
X 
X 
Study drug administration2 
 
X 
X 
X 
X 
X 
Clinical laboratory screening labs3 
X 
 
 
 
 
 
POC serum glucose4 
 
X 
X 
X 
X 
X 
Serum C-Peptide5 
 
X 
X 
X 
X 
X 
Serum Insulin5 
 
X 
X 
X 
X 
X 
Adverse event reporting 
 
X 
X 
X 
X 
X 
 
1 Vital signs include blood pressure, pulse rate, respiration rate, and temperature at supine position. 
2 Study drug will be administered intranasally. 
3 Clinical laboratory screening labs include clinical comprehensive metabolic panel and CBC. 
4 Point-of-care (POC) serum glucose glucometry will be performed at 0, 15, 30, 60, 120, 180, and 240 minutes after 
each intranasal insulin administration.  Sample will be collected +/- 10 minutes of scheduled assessment. 
5 Blood and plasma samples will be collected, aliquoted, and frozen at 0, 15, 30, 60, 120, 180, and 240 minutes 
after each intranasal insulin administration for blood insulin and C-Peptide measurements by the University of 
Michigan Pharmacokinetics Core.  Sample will be collected +/- 10 minutes of scheduled assessment. 
 
 

 
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2 INTRODUCTION 
2.1 STUDY RATIONALE 
Sudden cardiac arrest is responsible for 1 in 5 U.S. deaths (1). Among patients who achieve return of 
spontaneous circulation after OHCA, less than 1 in 3 survive. Irreversible brain injury is the primary 
cause of death. Although many pharmacologic therapies are neuroprotective in animal models, none 
have been successfully translated to clinical cardiac arrest care. A major barrier is the inability to deliver 
drugs to the brain within the therapeutic window, which is often minutes after cardiac arrest onset. For 
cardiac arrest occurring outside the hospital, we anticipate this will require bystanders or first 
responders (police and firefighters) to deliver the therapy in addition to performing cardiopulmonary 
resuscitation (CPR) and defibrillation. However, the feasibility, safety, and effectiveness of this approach 
must first be explored. 
 
There is a substantial body of pre-clinical data demonstrating a potential neuroprotective effect of 
intranasal insulin in varying models of acute ischemic and chronic neurodegenerative diseases in which 
mechanisms of oxidative stress are implicated (2,3). Human clinical data evaluating neuroprotection by 
insulin is mostly limited to small studies of chronic administration in patients with Alzheimer's disease. A 
recent systematic review of these data reported on 7 exploratory clinical trials of intranasal insulin in 
293 participants (4). These were predominantly safety and feasibility trials, and they found intranasal 
insulin to be well tolerated and without safety concerns. Furthermore, they individually and collectively 
showed signals of efficacy on at least some cognitive outcome parameters. There are 8 recruiting trials 
on ClinicalTrials.gov of intranasal insulin in 500 planned patients with Alzheimer's disease, stroke, 
cognitive decline in multiple sclerosis, cognitive decline in HIV, and other cognitive disorders. Thus, a 
well-planned phase 1 study to explore the safety and tolerability of intranasal insulin in healthy 
participants is necessary prior to studying its neuroprotective effect in OHCA patients. 
 
This phase 1 study will evaluate the safety and tolerability of intranasal regular humulin insulin in 
healthy volunteers. The establishment of maximum tolerated dose for intranasal insulin in healthy 
participants will elucidate the doses to explore in future dose-finding OHCA efficacy studies. 
Understanding the maximum tolerated dose is also important because the proposed community use of 
the intervention by bystanders and first responders demands a larger margin of safety than typical 
drugs, as errors in administration and the potential for misdosing may increase in community use. 
Therefore, the maximum tolerated dose is needed to estimate the safety margin and therapeutic index 
of the intervention.  
2.2 BACKGROUND 
Given the high morbidity rate from sudden cardiac arrest in the U.S. (1), there is a critical need to pursue 
innovative interventions to improve OHCA survival. Despite modest improvements in the first half of the 
decade, outcomes have not changed in the past 5 years based on data from the Cardiac Registry to 
Enhance Survival (5). Similarly, recent reports from the Resuscitation Outcomes Consortium and the 
Heart Rescue Project show no improvement in survival with good neurologic outcome since 2012 (6, 7). 

 
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A novel and disruptive approach is needed to discover, translate, and implement new therapies to 
improve the outcome of cardiac arrest. 
  
When resuscitation is attempted outside the hospital in the U.S., only 8.5% of patients survive with good 
neurologic function (8). Sudden cardiac arrest is the most time sensitive human heart condition, and 
time to treatment is the most critical factor in successful translation of cardiac arrest interventions. In 
fact, the only therapies during cardiac arrest that are proven to increase survival with good neurologic 
function are early bystander CPR and early defibrillation, both of which require early, time-sensitive 
intervention. Moreover, pharmacologic therapies such as epinephrine and antiarrhythmic drugs that are 
given intravenously or intraosseously by paramedics much later during OHCA (>15-20 min after 911 call) 
have not improved survival with good neurologic function in double-blinded randomized clinical trials 
(9,10).  These observations coupled with robust pre-clinical mechanistic data support our premise that 
neuroprotective drug therapies are most effective if given within minutes of brain injury.  To do so, it is 
critical that we design the novel neuroprotective therapies that can be delivered by those who are first 
on the scene—bystanders and first responders (police, firefighters)—rather than paramedics, who often 
arrive outside the therapeutic window for maximum neuroprotection. 
2.3 RISK/BENEFIT ASSESSMENT 
2.3.1 KNOWN POTENTIAL RISKS 
  
Anticipated adverse events associated with the use of intranasal insulin may include an allergic reaction, 
hypoglycemia, nose soreness, nasal side effects (nasal congestion, rhinorrhea, minor bleeding, nasal or 
pharyngeal stinging, rhinitis, sneezing), headache, dizziness, weakness, aspiration, upper respiratory 
tract infections, rash, and gastrointestinal symptoms such as nausea or vomiting (4).   Other adverse 
reactions seen with intravenous administration of insulin, including hypokalemia, have not been 
previously identified with intranasal administration of insulin.  
  
Peripheral intravenous access and collection of blood samples can be associated with presyncope, 
syncope, bruising, anemia, thrombophlebitis, and in rare instances, infections. 
2.3.2 KNOWN POTENTIAL BENEFITS 
There are no direct benefits to study participants . The tangible benefit to society is that this study 
may help bring a neuroprotective agent one step closer to eventual clinical use by first responders to 
use in OHCA.  This can potentially help countless lives.  
2.3.3 ASSESSMENT OF POTENTIAL RISKS AND BENEFITS 
Although hypoglycemia has never been demonstrated with intranasal insulin at lower dose in humans, 
the potential for this safety concern exists in this study. Mechanisms of insulin transport into the brain 
through the olfactory epithelium is more efficient than vascular absorption of regular insulin into the 
systemic circulation, but Kullmann et al have demonstrated small but measurable increases in serum 
insulin with intranasal administration and have shown these to represent dose-dependent spillage (11). 

 
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There is insufficient data to show whether this dose response is linear, and the very small changes in 
insulin level detected did not manifest as a measurable change in glucose (11).  Of note, a minority of 
the 19 studies in which participants received 160 U of intranasal insulin also reported clinically 
insignificant but measurable decreases in serum glucose ranging from 0.2 to 0.4 mmol/L. Based on these 
data, we have developed an initially conservative dose escalation study that has the potential to adapt 
the magnitude of dose escalation with accumulating data if lower dose tiers demonstrate the same 
overwhelming safety signals seen in the prior data.  Point-of-care (POC) serum glucometry will be 
performed at 0, 15, 30, 60, 120, 180, and 240 minutes after each intranasal insulin administration.   
Hypoglycemia as defined by a blood glucose of <45mg/dL will be treated with 50 ml of intravenous 
D50W and food. The subject will also be monitored until achieving two consecutive measurements of 
blood glucose > 70mg/dL, measured at 30 minutes apart. If blood glucose falls to ≤70 mg/dL but not <45 
mg/dL, the blood glucose will be monitored every 30 minutes until it returns to >70 mg/dL.   
 
 

 
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3 OBJECTIVES AND ENDPOINTS 
OBJECTIVES 
ENDPOINTS 
JUSTIFICATION FOR 
ENDPOINTS 
Primary 
Determine the maximum tolerated 
dose of intranasal insulin in healthy 
participants  
Primary endpoint: The primary 
safety outcome will be severe 
hypoglycemia as defined by blood 
glucose <45mg/dL at any post 
administration glucose check during 
a study visit. 
 
The American Diabetes 
Association defined severe 
hypoglycemia as “severe 
cognitive impairment 
requiring external assistance 
for recovery” without 
assigning a blood glucose 
cutoff (12).  As such, we chose 
blood glucose < 45mg/dL as 
the cutoff for severe 
hypoglycemia based on prior 
clinical studies (13). 
Secondary 
Characterize the pharmacokinetics  
and pharmacodynamics (PK-PD) of 
intranasal insulin in healthy 
participants  
Secondary endpoints will include the 
change in blood glucose, insulin, and 
C-peptide levels for up to 4 hours 
after drug administration to evaluate 
for any systemic absorption of 
intranasal insulin.   
Prior studies have not 
characterized the PK-PD of 
conventional insulin 
formulation doses above 160 
U administered intranasally. 
 
 

 
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4 STUDY DESIGN 
4.1 OVERALL DESIGN 
This is a phase 1 study to determine the safety and tolerability of 100 – 1000 U regular short-acting 
intranasal insulin in 24 healthy adult volunteers. The primary endpoint will be severe 
hypoglycemia using a numerical cutpoint of blood glucose <45 mg/dL as a binary variable. The secondary 
endpoints will include the change in blood glucose, serum insulin, and serum C-peptide levels for up to 4 
hours after drug administration to evaluate for any systemic absorption of intranasal insulin.     
  
Experiments will be conducted at the Michigan Clinical Research Unit, which offers all the clinical 
research services necessary to study investigators at Michigan Medicine. Participants will be serially 
exposed to escalating doses of intranasal regular insulin.  
 
Participants will be asked to participate in 6 administrations over 5 sessions with a 1-week washout 
interval between each session. The first session will include an initial control administration of 0 U 
followed by 4 hours of observation, then followed by the first dose-escalation administration and 
another 4 hours of observation. Subsequent visits will involve a single dose-escalation administration 
per visit. The starting escalation schedule will use dose increments of 200 units for a schedule over 6 
administrations of 0 U, 200 U, 400 U, 600 U, 800 U, and 1000 U.  Any subject experiencing a 
hypoglycemic episode meeting the definition of serum glucose level <45 mg/dL or having any related or 
possibly related serious adverse event will not be given a subsequent dose escalation.   
  
Adaptive Design: For every 6 participants, iterations of a Bayesian model will be performed to associate 
the administered dose with the frequency of dose-limiting hypoglycemia or other adverse events. The 
model will adaptively reallocate the dose increments for the next 6 participants. To provide more 
precise and reproducible estimates of dose-limiting toxicity, the trial will modify the size of dose 
increments based on the developing model to allocate more administrations to areas of the model with 
greatest uncertainty, or to higher doses in the absence of dose-limiting hypoglycemia. This will provide 
more observations and the greatest information density at doses of greatest interest that are most 
relevant to the determination of maximally tolerated dose. Safety and tolerability will be assessed for 
approximately 4 hours after each insulin administration. 
  
Approach: The maximum tolerated dose between 100 – 1000 U of regular short-acting insulin will be 
determined with an adaptive dose-escalation phase I safety study performed in 24 healthy volunteers. 
The primary safety outcome will be severe hypoglycemia using a numerical cutpoint of serum glucose 
<45 mg/dL. Secondary endpoints will include the change in blood glucose, serum insulin, and serum C-
peptide levels for up to 4 hours after drug administration to evaluate for any systemic absorption of 
intranasal insulin.    
 

 
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4.2 SCIENTIFIC RATIONALE FOR STUDY DESIGN 
The most effective cardiac arrest therapies are those delivered within minutes of cardiac arrest onset by 
bystanders and first responders. By optimizing the time to initiating therapies already proven effective—
CPR and defibrillation—and adding safe, effective, and easily administered neuroprotective drug 
therapy, thousands of lives will be saved each year.  
 
Nose-to-Brain Insulin Delivery: Although the brain is highly vascular, systemic intravenous delivery of 
neuroprotective therapeutics is problematic, especially for large therapeutic peptides due to the blood-
brain barrier. Intravenous administration also creates pragmatic barriers to very early administration in 
OHCA because vascular access requires skills and equipment not accessible to bystander and most first 
responders that first attend to these patients, and because blood flow is limited. 
 
The unique physiological properties of direct drug delivery into the 
brain through novel intranasal pathways offer the potential to 
address both of these problems. These pathways have been known 
for years, but are only recently being fully evaluated in clinical trials 
of neurotherapeutics. These pathways are distinct from the better 
known use of intranasal administration as a route to drug delivery to 
the circulation of smaller and lipophilic molecules. Rather, the direct 
nose-to-brain pathways provide a noninvasive strategy to deliver 
drugs rapidly across the nasal mucosa directly into the central 
nervous system (14,15). These direct pathways were first 
demonstrated in the 1980’s and have been confirmed through increasingly elegant methods in recent 
years. Figure 1 illustrates the intracellular, intercellular, and perivascular absorption pathways found in 
the olfactory epithelium. Trigeminal nerve endings and olfactory receptor neurons are interspersed 
among supporting cells. These convey chemosensory information to the central nervous system but also 
serve as pathways for direct drug delivery through extracellular or intracellular mechanisms (dashed 
arrows in Figure 1). Drugs can also be transported through the nasal mucosa to the central nervous 
system by entering perivascular channels (dashed lines surrounding blood vessels in Figure 1). After 
crossing the olfactory epithelium and reaching the lamina propria, drugs enter channels created by 
olfactory ensheathing cells surrounding the olfactory nerves, where they can access the cerebrospinal 
fluid and rapidly distribute via bulk flow mechanisms (15). 

 
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Direct confirmation of direct-to-brain drug delivery 
in the absence of vascular absorption has been 
demonstrated in humans by delivery of intranasal 
insulin and tracing subsequent levels in both 
cerebrospinal fluid and the blood (16). As shown in 
Figure 2, increases in cerebrospinal fluid insulin are 
seen with nasal administration of insulin but not 
placebo, whereas concentrations of insulin in the 
blood are identical for nasal administration of insulin 
vs. placebo. This indicates that intranasal insulin 
enters the brain directly without entering the blood, allowing dosing of the target organ without 
systemic side effects like hypoglycemia. These pathways have also been confirmed by indirect but 
functional methods. In a study using functional magnetic resonance imaging, intranasal administration 
of four-fold doses of insulin resulted in a dose-dependent modulation of resting state regional brain 
activity diffusely in both cortex and midbrain, again without any evidence of clinically-relevant systemic 
effect (11). This confirms that insulin is present, physiologically relevant, and safe when delivered 
directly to the brain after nasal administration.    
4.3 JUSTIFICATION FOR DOSE 
There is substantial clinical safety data for intranasal insulin in human subjects, but there is no data 
suggesting the maximum tolerated dose. The majority of existing safety data involve doses of intranasal 
regular insulin in daily repeated administrations from 20 to 160 U. As reviewed recently by Schmid et al, 
there were no episodes of hypoglycemia in 1,053 subjects in 38 studies in this dose range (17). Although 
these data are reassuring, there are at least two important reasons to explore higher dose ranges. 
  
The first reason is to understand the upper limits of the dose that might be explored in future dose-
finding efficacy studies. Planned animal studies will refine the dose efficacy response in models of OHCA, 
but existing data demonstrate efficacy and safety in other animal models in doses that scale to human 
doses ranging from 300 U to 1000 U (depending on the method of scaling and parameters chosen).  It is 
important to determine if the maximum tolerated dose in humans is within or above these doses. 
  
Understanding the maximum tolerated dose is also important because the proposed community use of 
the intervention by bystanders and first responders demands a larger margin of safety than typical 
drugs, as errors in administration and the potential for inaccurate dosing may increase in community 
use. A maximum tolerated dose is needed to estimate the safety margin and therapeutic index of the 
intervention. The therapeutic dose will be estimated from ongoing animal experiments.  
4.4 END OF STUDY DEFINITION 
A participant is considered to have completed the study after he/she has completed all study visits as 
shown in the Schedule of Activities, Section 1.3. 
 

 
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5 STUDY POPULATION 
5.1 INCLUSION CRITERIA 
Participants will be healthy adult volunteers who have provided written informed consent. Sex balance 
will be sought through deliberate equitable selection patterns. There will be no preferential or 
discriminatory selection of participants on the basis of race. 
  
     Each subject must meet the following criteria to be enrolled in this study: 
1. Male or female volunteers at least 18 years of age and older, inclusive, in good health based on 
medical history, physical exam, and routine laboratory testing. 
2. Female participants must have negative urine pregnancy test or be surgically sterilized or 
postmenopausal. Criteria for menopause are surgical menopause (hysterectomy, 
oophorectomy) or age > 45 years with the absence of menses for greater than 12 months. Tubal 
ligation with menses within the past 12 months is not considered to be surgical sterilization. 
3. Body mass index (BMI) between 18 kg/m2 and 35 kg/m2. 
4. Willing and able to stay at the clinical research facility as required by the protocol. 
5. Willing and able to comply with the investigational nature of the study and able to communicate 
well with investigators. 
6. Ability to comprehend and willing to provide written informed consent in accordance with 
institutional and regulatory guidelines 
5.2 EXCLUSION CRITERIA 
Participants who meet any of the following criteria will be excluded from the study: 
1. Known allergy to insulin. 
2. Preexisting diabetes. 
3. Current or previous use of diabetes medication or insulin. 
4. Any nasal disease or congestion that may interfere with intranasal drug absorption. 
5. Baseline hypoglycemia (blood glucose ≤ 65 mg/dL) or hyperglycemia (blood glucose > 200 
mg/dL) as evident from the screening labs. 
6. Active serious disease, such as liver disease, kidney disease, uncontrolled hypertension, clinically 
significant hypokalemia, and significant or unstable medical illness 
7. Blood donation in excess of 500 mL within 60 days prior to the first dose of study medication. 
8. Treated with an investigational drug within 30 days. 
9. Individuals with inadequate venous access. 
5.3 LIFESTYLE CONSIDERATIONS 
Nothing to eat or drink for at least 2 hours prior to each study visit. 
Abstain from alcohol for at least 12 hours before the start of each dosing session. 

 
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5.4 SCREEN FAILURES 
Individuals who do not meet the criteria for participation in this trial (screen failure) will not be 
rescreened given that this is a healthy participants study.  
5.5 STRATEGIES FOR RECRUITMENT AND RETENTION 
The University of Michigan maintains a database of healthy male and female volunteers 
(https://www.umhealthresearch.org/ ). The database includes approximately 40,000 volunteers. The 
initial strategy will include a targeting request to volunteers that are likely to meet inclusion criteria 
based on information stored in this database. Given the study plan to recruit up to 24 volunteers, it is 
highly likely that this approach will be sufficient. However, other strategies such as the use of 
Institutional Review Board's (IRB) approved flyers may be posted throughout the University of Michigan, 
Ann Arbor campus to solicit potential healthy volunteers. The scheduling of procedures will be carefully 
orchestrated by the study team to improve subject retention. 
  
Participants will be compensated at the local competitive rate for healthy volunteers in phase I 
investigational drug studies. The full rate will be prorated over a maximum of 6 research visits (one 
screening visit and 5 study visits).  Phone calls, texts, or email reminders will be sent ahead of their 
appointment dates. 
5.6 CONSIDERATIONS RELATED TO COVID-19 
To reduce risks to participants and study staff related to COVID-19 infection, the study will comply with 
institutional and governmental precautions and restrictions in place at the time the study is being 
conducted.  In addition, the study will ask that all participants be vaccinated against COVID-19 prior to 
the first study visit.  The study may also perform point-of-care screening for COVID-19 infection using a 
saliva test at the time of the study visits depending on community prevalence and contemporaneous 
guidance.  
 
 

 
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6 STUDY INTERVENTION 
6.1 STUDY INTERVENTION(S) ADMINISTRATION 
 
Regular insulin (Humulin R U500, Eli Lilly, Indianapolis IN) at a concentration of 500 U/mL will be used to 
create  ≤2 mL doses containing between 0 – 1000 U. The ≤2 mL doses will be administered intranasally 
using a 5 mL syringe divided into ≤1 mL per nostril. Intranasal administration will be performed with the 
participants supine with the neck extended (a modified Mygind position)(19, 20, 21). to mimic the 
position of comatose OHCA patients. Participants will be instructed to sniff following intranasal 
administration and rest in supine position for 15 minutes.  
 
A total of 11 possible doses will be tested, ranging from 0 to 1000 U insulin. 
Each volunteer will come for 5 study visits. In the first visit, the volunteer is administered placebo. They 
will receive the first assigned intranasal insulin dose 4 hours later followed by another 4 hours of 
observation. During visits 2-5, they will receive additional escalating doses based on the dosing protocol. 
 
6.1.2 DOSING AND ADMINISTRATION 
 
Rules for assigning patients 
Within subject rules (subject progressing through tiers for cohort): 
1. 
If a volunteer experiences severe hypoglycemia (blood glucose <45 mg/dL), they will be assigned a 
dose 100 U less for the next round and for all subsequent rounds. 
2. 
If a volunteer experiences severe hypoglycemia a second time, they will be withdrawn from the 
study. 
 
Study wide rules (determine which dose tiers are allowable for all future volunteers): 
1. 
All doses from 0 to 1000 U by increments of 100 U will be eligible for testing in the study. 
2. 
If there is greater than 0.9 probability that a dose has a greater than 0.1 toxicity rate, that dose will 
be removed for all future cohorts (see below).   
 
 
Allocation Rules 
Initial Allocation and Strategy: 
Overall, the philosophy of allocation is to assign as many patients as possible to higher doses, while also 
limiting exposure to toxic doses. The trial will start the first cohort with 200, 400, 600, 800, and 1000 U. 
 
Allocation Adjustment Rules: 
After each hypoglycemic event, we will recalculate the toxicity probabilities for that dose to see if a dose 
removal threshold has been met. 
 

 
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If a dose meets the criteria for removal, that dose and all higher doses will be removed from future 
participants and cohorts. For the next cohort or patient within a cohort, the highest additional doses 
available will be used. For example, if 700 U was removed, then 200, 300, 400, 500, 600 U would be the 
new future allocation.  
 
If a cohort completes with no observed episodes of dose-limiting toxicity (DLT), the smallest (and any 
smaller) doses will drop, and the highest unused dose will replace it. For example, after zero episodes of 
DLT in Cohort 1 at any dose, Cohort 2 will use 400, 600, 800, 900, and 1000 U. Doses 100 and 200 U will 
be dropped.  
 
If a cohort has any DLT, but no dose is dropped (only 1 DLT in two different doses for example), the 
initial allocation rules will be used for the next cohort (200, 400, 600, 800, 1000 U). 
 
4 Cohort Example - No DLT (Demonstrated by Schema Figure 1.2.2) 
Cohort 1: Participants  #1 to 6 
The doses will be 0, 200, 400, 600, 800, and 1000 U.  
 
Cohort 2:  Participants #7 to 12 
If there were 0 events in Cohort 1, then the doses for Cohort 2 will be 0, 400, 600, 800, 900, and 1000 U.  
200 U will be dropped and 900 U will be entered. 
 
Cohort 3:  Participants #13 to 18 
If there were 0 events in Cohorts 1 and 2, then the doses for Cohort 3 will be 0, 600, 700, 800, 900, and 
1000 U. 400 U will be dropped and 700 U will be entered. 
  
Cohort 4:  Participants  #19 to 24 
Same as Cohort 3. 
Other situations (more than one DLT) 
Please also refer below to rules for discontinuing a dose. 
 
If a dose (or doses) is dropped during or after a cohort, the highest available unused doses will be used 
for the next patients within that cohort and the next cohort. For example, if 1000 U is dropped, the 
future doses will be 200, 400, 600, 800 and 900 U. Or, if 800 U and all higher doses are dropped, then 
200, 400, 500, 600, and 700 U will be the next set.  
As one example, if there is 1 DLT in a different dose in each of the 4 cohorts, the trial will simply allocate 
using the initial rules for all cohorts, with the exception of the within subject adjustment rule noted 
above.  
For example, if there are no DLTs in Cohort 1, then Cohort 2 will use 400, 600, 800, 900, and 1000 U. If 
Cohort 2 also has no DLTs, then 300 and 400 U will drop and Cohort 3 will use 600, 700, 800, 900, and 

 
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1000 U. Even if there are no DLTs in Cohort 3 given that allocation, we will continue that allocation as it 
utilizes the 5 highest available doses.  
If higher doses are dropped, they cannot be reconsidered in the study. 
If lower doses are dropped, they can re-enter the study. This would occur if higher doses needed to be 
dropped by having DLTs over the threshold. The lower doses may need to re-enter in order for each 
subject to have 5 potential doses.  
 
6.2 PREPARATION/HANDLING/STORAGE/ACCOUNTABILITY 
6.2.1 ACQUISITION AND ACCOUNTABILITY 
HUMULIN R U-500 (insulin human injection, NDC 0002-8501-01) will be purchased as commercial supply 
through Eli Lilly and Company. Storage and accountability will be maintained by Research Pharmacy, 
Michigan Medicine. Diluent to prepare the 0 U/mL dose and lower concentrations of intranasal insulin 
will be acquired through Eli Lilly and Company (Sterile Diluent, NDC 0002-0800) or similar manufacturer 
(lilly_distribution@lilly.com). 
6.2.2 FORMULATION, APPEARANCE, PACKAGING, AND LABELING 
HUMULIN R U-500 is available as single 20 mL multi-use vials. The concentration is 500units/mL 
6.2.3 PRODUCT STORAGE AND STABILITY 
Unopened vials of HUMULIN R U-500 will be stored in a refrigerator (36° to 46°F [2° to 8°C]) protected 
from freezing and light, with a do not shake notice. Opened vials of HUMULIN R U-500 will also be 
stored in a refrigerator (36° to 46°F [2° to 8°C]) protected from freezing and light and used within 40 
days then subsequently discarded. Syringes containing ≤1 mL volume at doses of 0 (diluent), 50, 100, 
150, 200, 250, 300, 350, 400, 450, and 500 U (undiluted) will be prepared and stored in a refrigerator for 
no more than four hours total at (36° to 46°F [2° to 8°C]) and/or room temperature, protected from 
freezing and light. A prior stability study demonstrated at least 28-day stability at this temperature. (18) 
 
6.2.4 Preparation 
No special preparation is necessary for HUMULIN R U-500 and sterile diluent measured to the 
aforementioned doses under a laminar flow hood by Research Pharmacy. 
6.3 MEASURES TO MINIMIZE BIAS: RANDOMIZATION AND BLINDING 
No randomization or masking procedures will be used in this study.  
6.4 STUDY INTERVENTION COMPLIANCE 
The study related activities, including drug administration and observation, will be performed with 
support from the Michigan Clinical Research Unit with an extensive track record of data integrity and 
compliance with regulatory procedures. Given that participants will be seen weekly, study compliance 

 
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will be reviewed at each visit. A record of any study queries, discrepancies, or deviations from the 
protocol will be maintained and provided to the study site and PI to resolve and document as a part of 
the study record.  There are no subject diaries or study related activities that occur outside of subject 
visits. 
6.5 CONCOMITANT THERAPY 
6.5.1 RESCUE MEDICINE 
If a subject’s blood glucose level falls <45 mg/dL, he/she will be treated with 50 ml of intravenous D50W 
and provided food. He/she will be monitored until achieving two consecutive measurements of blood 
glucose > 70mg/dL, measured at 30 minutes apart. If blood glucose falls to ≤70 mg/dL but not <45 
mg/dL, the blood glucose will be monitored every 30 minutes until it returns to >70 mg/dL.   
 
 

 
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7 STUDY INTERVENTION DISCONTINUATION AND PARTICIPANT DISCONTINUATION/WITHDRAWAL 
7.1 DISCONTINUATION OF STUDY INTERVENTION 
The rules for stopping or changing an individual dosing regimen are given above in the Dosing and 
Administration section. 
 
Rules for discontinuing a dose 
We are interested in proceeding with a dose if we remain confident that the Dose Limiting Toxicity 
(blood glucose < 45 mg/dL) is < 0.1 (1 in 10). 
 
Using the Bayesian Toxicity Monitoring software from MD Anderson (https://ibl.mdanderson.org/BTM/)  
 
We define Toxicity parameters as: 
Probability of DLT is represented by θ 
Maximum probability of DLT allowed (θmax): 0.1 
 
When calculating the probability of DLT at a dose, we will use a Beta(0.2,0.8) prior. This is a vague prior 
with a median of 0.2 and a 95% credible interval from 0 to 0.94. There is greater density over 
probabilities of DLT near zero in this non-symmetric distribution. Each DLT is added to the A parameter 
of the beta distribution, and each patient who is DLT free is added to the B parameter of the B 
distribution. Therefore, with the prior included, if 24 participants have DLT on a dose, the probability of 
DLT can be described with a Beta(0.2,24.8) distribution. In this setting, the posterior estimate for DLT 
probability is 0.008 (or 0.8%). In addition, the probability that that dose has a DLT proportion over 0.1 is 
only 0.006 (or 0.6%).  
 
Prob(θ >θmax) ≥ 0.9 (Or that there is 90% or greater probability that the DLT is greater than 0.1) 
 
We define the probability that Prob(θ >θmax) as the final output of the trial for each dose. For each dose, 
we will also calculate the probability that toxicity is greater than 0.1 at that dose. 
 
The below table (SB1A) illustrates the probability that DLT is greater than 0.1 based on the number of 
observed events.  

 
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 In practice, this means if 2 DLTs are observed for a dose in the first cohort, the dose is dropped. To drop 
a dose in subsequent cohorts, see the shaded area of the above table. To delete a dose, one must have 
3 total observed DLTs in Cohorts 1 and 2, or 4 total DLTs in Cohorts 1-3, or 5 total DLTs in Cohorts 1-4. 
 
 
Rules for discontinuing or modifying the overall study 
There are no binding rules to discontinue the overall study. However, if the 500 U dose tier was 
dropped, there would only be 100, 200, 300, and 400 U available. In this case, the PI will consult with the 
 other investigators to present an amended plan for future study conduct or overall discontinuation. 
 
7.2 PARTICIPANT DISCONTINUATION/WITHDRAWAL FROM THE STUDY 
Participants may be withdrawn from the study at any time for voluntary reasons or discontinued from 
study treatment at the request of the study investigators. Reasons for withdrawal or termination may 
include but are not limited to: 1) Subject requests discontinuation of study drug or withdrawal; 2) 
Subject becomes pregnant; 3) Subject requires treatment that is not permitted per protocol due to a risk 
for a drug interaction. 
7.3 LOST TO FOLLOW-UP 
Participants who do not return to their clinic appointment for a required study visit will be contacted by 
telephone immediately to reschedule their appointment. They will be counseled on the importance of 
maintaining the assigned visit schedule and ascertain whether they wish to continue in the study.  
  
Before a subject is deemed lost to follow-up, the investigator or designee will make every effort to 
regain contact with the subject (where possible, 3 telephone calls and, if necessary, a certified letter to 
the participant's last known mailing address or local equivalent methods). These contact attempts will 
be documented in the participant's medical record or study file. 
  

 
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Should the subject continue to be unreachable, he or she will be considered to have withdrawn from the 
study with a primary reason of lost to follow-up.  Data collected on study participants to the point of 
withdrawal will remain a part of the study record. 
 
 

 
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8 ASSESSMENTS 
8.1 EFFICACY ASSESSMENTS 
Initial screening will be completed by telephone after a potential subject seeks interest in study 
participation. This initial screening will include review of the study protocol inclusion/exclusion criteria.  
If the subject meets eligibility criteria, they will be asked to come into the research clinic to start the 
informed consent process and undergo screening laboratory testing.   
 
8.1.1 Screening Visit 
All participants will be informed of the nature and purpose of the study, and their written informed 
consent will be obtained prior to any study related activities.   The investigator or designated qualified 
individual will perform the physical examinations. A physical examination will include measurement of 
body weight and height, general assessment of the skin, nose/throat, heart, lungs, and abdomen. The 
subject’s total body weight and supine vital signs (heart rate, blood pressure, temperature, and 
respiratory rate) will be recorded. 
 
Clinical Laboratory Testing and Urine hCG 
● Serum comprehensive metabolic panel will be done including blood levels of albumin, blood 
urea nitrogen, calcium, carbon dioxide, chloride, creatinine, glucose, potassium, sodium, 
total bilirubin and protein, and liver enzymes (alanine aminotransferase, alkaline 
phosphatase, and aspartate aminotransferase). 
● Serum hemoglobin will be measured. 
● Urine human chorionic gonadotropin (hCG) will be measured for females who are not 
diagnosed as postmenopausal. 
 
8.1.2  Study Visits 
Participants (in cohorts of 6) will present to the Michigan Clinical Research Unit on their scheduled 
dosing day, and will be asked to have not consumed any food for at least 2 hours and any alcohol for at 
least 12 hours prior to this visit. A urine pregnancy test will be performed to rule out pregnancy prior to 
administration of the intranasal insulin dose to applicable female participants. The subject’s total body 
weight and supine vital signs (heart rate, blood pressure, temperature, and respiratory rate) will be 
recorded. Eligible Participants will proceed with the study procedures after review of inclusion and 
exclusion criteria.  
Participants will be asked to participate in 6 administrations over 5 sessions with a 1-week washout 
interval between each session. The first session will include an initial control administration of 0 U with 4 
hours of observation, then followed by the first dose-escalation intranasal insulin administration and 
another 4 hours of observation. Subsequent visits will involve a single dose-escalation administration 
per visit as described in 6.1.2 Dosing and Administration. Any subject experiencing severe hypoglycemic 
episode will be given 100 U less than the last administered dose for the remainder of the study.  Any 
participant having a second event, will be withdrawn from the study. 
 

 
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Participants will have peripheral intravenous access established with the placement of a short-term IV 
catheter for serial blood sampling and administration of supplemental glucose if needed. 5 mL of venous 
blood samples (3 mL as waste and 2 mL for blood glucose, insulin, and C-Peptide analyses) will be 
collected at 0, 15, 30, 60, 120, 180, and 240 minutes after each intranasal drug administration. Blood 
glucose levels will be measured with a point of care glucometer. The remaining blood samples will be 
centrifuged, aliquoted, and frozen for serum insulin and C-Peptide analyses by the University’s 
Pharmacokinetics Core.   
 
If a subject’s blood glucose level falls <45 mg/dL, he/she will be treated with 50 ml of intravenous D50W 
and provided food. He/she will be monitored until achieving two consecutive measurements of blood 
glucose > 70mg/dL, measured at 30 minutes apart. If blood glucose falls to ≤70 mg/dL but not <45 
mg/dL, the blood glucose will be monitored every 30 minutes until it returns to >70 mg/dL.  Participants 
will not be released to home until any symptoms of hypoglycemia have resolved.  
Participant-reported adverse events will also be tracked as supplemental outcomes.  
 
8.2 SAFETY AND OTHER ASSESSMENTS 
● 
Vital Signs (blood pressure, pulse, respiratory rate, temperature) 
● 
Clinical laboratory tests including blood chemistry and hematology parameters 
● 
Urinalysis pregnancy screening test(s) in women   
● 
Adverse events 
● 
Concomitant medication use 
● 
Physical exam 
 
8.3 ADVERSE EVENTS AND SERIOUS ADVERSE EVENTS 
8.3.1 DEFINITION OF ADVERSE EVENTS (AE) 
The internationally accepted definition of an adverse event (AE) is any untoward medical occurrence in a 
patient or clinical investigation subject administered a pharmaceutical product and that does not 
necessarily have a causal relationship with this treatment. An AE can therefore be any unfavorable and 
unintended sign (including an abnormal laboratory finding), symptom, or disease temporally associated 
with the use of a medicinal product. 
8.3.2 DEFINITION OF SERIOUS ADVERSE EVENTS (SAE) 
A serious adverse event (SAE) is any AE occurring at any dose that results in any of the following 
outcomes: a life-threatening situation, death, inpatient hospitalization or prolongation of existing 
hospitalization, persistent or significant disability or incapacity, congenital anomaly or birth defect, or 
important medical events such as those that may not result in death, be life-threatening, or require 
hospitalization may be considered an SAE when based upon appropriate medical judgment. It 
jeopardizes the subject or requires medical or surgical intervention to prevent one of the outcomes 
listed above. 

 
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8.3.3 CLASSIFICATION OF AN ADVERSE EVENT 
8.3.3.1 SEVERITY OF EVENT 
The severity of adverse events will be graded using the Common Terminology Criteria for Adverse 
Events (CTCAE) – NIH/NCI  
          Grade 1:    
 
Mild AE 
Grade 2:    
 
Moderate AE 
     
Grade 3:    
 
Severe AE 
     
Grade 4:    
 
Life-Threatening or Disabling AE 
     
Grade 5:    
 
Death related to AE 
8.3.3.2 RELATIONSHIP TO STUDY INTERVENTION 
The study physician will use clinical judgment to define a necessary medical course. AEs of interest are 
those that occur after the first dose of study medication. The relationship of AEs to the investigational 
product should be determined by the Investigator using the definitions below: 
● 
Definitely related:  clearly associated with study drug/treatment 
● 
Probably related:  likely associated with study drug/treatment 
● 
Possibly related:  may be associated with the study drug or other treatment 
● 
Unlikely to be related 
● 
Unrelated to the study drug 
8.3.3.3 EXPECTEDNESS 
The investigators will be responsible for determining whether an AE is expected or unexpected. 
An AE will be considered unexpected if the nature, severity, or frequency of the event is not 
consistent with the risk information previously described for the study agent in the protocol, 
informed consent, or labeling for the Humulin R U-500 
8.3.4 TIME PERIOD AND FREQUENCY FOR EVENT ASSESSMENT AND FOLLOW-UP 
The intranasal insulin dose administration will be managed by the study personnel. The participant will 
be directly monitored by study staff for the initial 4 hours after dose administration. AE assessments will 
occur during each study visit.  
8.3.5 ADVERSE EVENT REPORTING 
Out of range clinical laboratory findings (e.g., clinical chemistry, hematology, urinalysis) or findings on 
other assessments (vital signs) per se are not reported as AEs. However, those abnormal findings that 
are deemed clinically significant by the investigator or are associated with signs and/or symptoms must 
be recorded as AEs (and recorded as an SAE if they meet the criteria of being serious; see Section 8.3.2). 
The investigator should exercise medical and scientific judgment in deciding whether an out of range 
clinical laboratory finding or finding on other assessment is clinically significant. 

 
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All AEs occurring within 24 hours of study drug dosing and all SAEs occurring up until the last day of 
study participation will be recorded on the online AE case report form (CRF) through the study database. 
The investigators or Study Coordinator or designee is responsible for entering any and all AEs and SAEs 
into the database as soon as he/she becomes aware of the event and updating the information (e.g., 
date of resolution, action taken) in a timely manner. All non-serious AEs must be recorded on the 
electronic AE CRF within 5 days from the time it was discovered by the site study personnel.  
  
Reporting of AEs and SAEs will follow the IRB standard AE reporting timeline.  
8.3.6 SERIOUS ADVERSE EVENT REPORTING 
SAEs associated with intranasal insulin will be reported to the IRB in accordance with the University of 
Michigan IRBMED reporting guidelines and to the FDA according to the regulations found in 21 CFR 
312.32.   
8.3.7 REPORTING EVENTS TO PARTICIPANTS 
Participants who experience an AE will be told at the time the event is identified.   
8.3.8 EVENTS OF SPECIAL INTEREST 
Not applicable. 
8.3.9 REPORTING OF PREGNANCY 
Any participant with a positive pregnancy test will be notified of the test result and withdrawn or 
excluded from the study. 
 
 

 
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8.3.10  DEFINITIONS OF SELECTED OR ANTICIPATED ADVERSE EVENTS 
The adverse events identified below are anticipated and will be tracked. 
 
Allergic reaction 
Aspiration 
Nasal irritation  
 
Sneezing 
 
Warm feeling in nostrils 
 
Congestion/Rhinitis 
 
 
 
Lightheadedness 
 
Nausea and vomiting 
Nosebleeds 
 
Hypoglycemia  
Headache 
Weakness 
Upper respiratory infection 
Rash 
 
8.4 UNANTICIPATED PROBLEMS 
8.4.1 DEFINITION OF UNANTICIPATED PROBLEMS (UP) 
Any incident, experience or outcome that is unexpected in nature, severity or frequency that is related 
or possibly related to study participation will be deemed an unanticipated problem. The impetus for 
actions related to the unanticipated problem will depend on the information that suggests that the 
research study procedure places participants at greater risk of harm than previously recognized. 
8.4.2 UNANTICIPATED PROBLEM REPORTING 
Any unexpected AE and unanticipated problem will be reported to the University of Michigan IRBMED 
according to their reporting guidelines.   Upon becoming aware of any incident, experience, or outcome 
(not related to an adverse event) that may represent an unanticipated problem, the investigator should 
assess whether the incident, experience, or outcome represents an unanticipated problem. The 
incident, experience, or outcomes is considered unanticipated if it meets all of the following criteria: 
 

 
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Unexpected (in terms of nature, severity, or frequency); 
Related or possibly related to participation in the research; and 
Suggests that the research places subjects or others at a greater risk of harm than was previously known 
or recognized. 
Unanticipated problem Reporting: Per 21 CFR 312.66, 312.53 (c)(1)(vii), and 56.108(b)(1), should an 
Unanticipated problem occur during the investigation, the investigator will promptly report all 
unanticipated problems involving risks to human subjects or others to the IRB /FDA. 
8.4.3 REPORTING UNANTICIPATED PROBLEMS TO PARTICIPANTS 
Reporting unanticipated problems to participants will occur as directed by the UM IRBMED. 
 
 

 
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9 STATISTICAL CONSIDERATIONS 
9.1 STATISTICAL HYPOTHESES 
● 
Primary Efficacy Endpoint(s): Severe hypoglycemia (DLT) as defined by blood glucose <45mg/dL. 
The maximum dose that is under the threshold of a 0.9 probability of having a DLT proportion of greater 
than 0.1 will be considered the maximum tolerated dose for this study.   
● 
Secondary Efficacy Endpoint(s): Change in blood glucose, insulin, and C-peptide levels for up to 4 
hours after drug administration to evaluate for any systemic absorption of intranasal insulin.    
Our main secondary hypothesis is the secondary efficacy endpoints are not associated with increasing 
doses of insulin. For example, change in blood glucose from baseline to 4 hours will be similar in the 
placebo and highest doses. 
9.2 SAMPLE SIZE DETERMINATION 
 
Overall, for any dose we estimated whether the study would end, with different assumptions of the 
probability of toxicity for a given dose. These ranged from 0.01 to 0.25.  It is possible, however, that a 
dose tier may not be desirable despite having not been dropped. For example, there will be about an 
82% probability that the toxicity rate is > 0.1 if 4 out of 24 participants experience a DLT, then the point 
estimate using the Bayesian model will be that arm has a 0.17 probability of DLT (slightly above the raw 
observed rate of 0.125 due to the Bayesian prior).  
The design has approximately 80% “power” at the margin (scenario 5). That is, if the true rate of DLT is 
0.1 at a dose, the DLT will NOT be declared toxic using the pre-specified definition (Prob(θ >0.1) ≥ 0.9  
just under 80% of the time. 
It is important to note that the table below is for a single dose with 24 observations. As the trial 
progresses, we will generate independent estimates of the probabilities for each dose.  The average 
number of patients indicates that the dose will stop early some proportion of the time. The average 
number of toxicities provides the average number of DLTs observed within a simulated trial when the 
true probability of toxicity is given (for example 0.1 in scenario 5). 
 
 
 
 

 
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Table: Operating Characteristics for a Dose Assuming N of 24 
Scenario 
Prob.Of.Tox Prob 
Early.Stop 
ProbDeclare.Tox Avg.N.Patients Avg.N.Tox Obs.Tox.Rate 
1 
0.01 
0.0016 
0.0016 
23.9724 
0.2397 
0.0104 
2 
0.025 
0.0105 
0.0105 
23.8235 
0.5956 
0.0272 
3 
0.05 
0.0453 
0.0467 
23.2792 
1.164 
0.0583 
4 
0.075 
0.1062 
0.1125 
22.3836 
1.6788 
0.0921 
5 
0.1 
0.1898 
0.2062 
21.1965 
2.1196 
0.127 
6 
0.125 
0.2892 
0.3196 
19.8036 
2.4754 
0.1617 
7 
0.15 
0.3962 
0.4415 
18.2978 
2.7447 
0.1952 
8 
0.2 
0.6031 
0.669 
15.277 
3.0554 
0.2563 
9 
0.25 
0.7684 
0.8336 
12.6203 
3.1551 
0.3089 
 
9.3 POPULATIONS FOR ANALYSES 
Male or female volunteers at least 18 years of age and older, who meet study inclusion and exclusion 
criteria.  
9.4 STATISTICAL ANALYSES 
9.4.1 GENERAL APPROACH  
For each dose DLT probability θ, we will calculate the (Prob(θ >0.1). The maximum dose that is under the 
threshold of a 0.9 probability of having a DLT proportion of greater than 0.1 will be considered the 
maximum tolerated dose for this study.  
 
We will also report the observed toxicity rate for each dose. Depending on the number of DLT observed, 
we may fit a dose response model that uses data from all doses to fit a dose-toxicity curve. We also will 
plan to use dose response models for the analyses of the secondary endpoints. 

 
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9.4.2 ANALYSIS OF THE PRIMARY EFFICACY ENDPOINT(S) 
Primary analyses of these clinical trial data will be descriptive, including critical event rates reported 
with 95% confidence intervals. 
9.4.3 ANALYSIS OF THE SECONDARY ENDPOINT(S) 
First, we will graphically plot the relationship between the secondary endpoint and dose. We will 
explore these plots to determine a reasonable functional form (i.e. linear, Emax, log transformed, log-
logistic) for dose-response. We will consider dose as a linear predictor in addition to the most 
reasonable functional form. However, we will not fit other models if the linear predictor is reasonable. 
 
We will fit generalized linear models to predict change in glucose from baseline given dose. We will use 
a random effects model (with a random intercept for each subject). The main predictor variable will be 
dose. The main covariate will be the baseline value for the covariate for variables that are measured 
longitudinally (i.e. model predicts 4 hour glucose, and baseline glucose is a covariate in the model).  
9.4.4 SAFETY ANALYSES 
Secondary analyses include modeling of dose-safety curves based on primary and secondary safety 
outcomes, and modeling of pharmacokinetics/pharmacodynamics based on dose and serial serum 
insulin measurements if there is sufficient signal. Analyses of the effects of intranasal insulin on 
indicators of glycemic variability will also be explored. 
9.4.5 BASELINE DESCRIPTIVE STATISTICS 
The baseline values of labs, vital signs, and our descriptive characteristics will be summarized in a table 
with means and 95% confidence intervals or proportion with 95% confidence intervals. 
9.4.6 PLANNED INTERIM ANALYSES 
The dosing plan for the next cohort will be determined after each cohort completes. Participants within 
any cohort will have their dosing plan adjusted in the event one of the planned doses for that cohort is 
dropped due to excess DLTs in the other participants within that cohort. 
9.4.7 SUB-GROUP ANALYSES 
We will conduct stratified analyses by gender for the primary and secondary analyses, unless the gender 
ratio is greater than 3:1 (i.e. over 75% most common sampled gender). 
9.4.8 TABULATION OF INDIVIDUAL PARTICIPANT DATA 
 The full dataset will be de-identified for future use.  
9.4.9 EXPLORATORY ANALYSES 
 None planned.  
 

 
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10 SUPPORTING DOCUMENTATION AND OPERATIONAL CONSIDERATIONS 
10.1 REGULATORY, ETHICAL, AND STUDY OVERSIGHT CONSIDERATIONS 
10.1.1 INFORMED CONSENT PROCESS 
All participants will be informed of the volunteer nature and purpose of the study. Their written 
informed consent will be obtained in a private setting, during the pre-study screening procedures 
conducted prior to the first dosing. The informed consent form will contain all elements required by the 
International Conference on Harmonization Good Clinical Practices Guidelines in addition to any other 
elements required by state, local or institutional policy. 
10.1.1.1 CONSENT/ASSENT AND OTHER INFORMATIONAL DOCUMENTS PROVIDED TO PARTICIPANTS 
All participants will provide an IRB approved informed consent prior to any research procedures. A 
signed informed consent document will be obtained during the initial screening visit. 
10.1.1.2 CONSENT PROCEDURES AND DOCUMENTATION 
Informed consent procedures will be performed in a private setting. As part of the consent process, 
participants will be asked to summarize what is being asked of them to ensure they understand the 
study, have had sufficient time to contemplate the study, have not been coerced, and understand the 
voluntary nature of their participation. The original copy of the informed consent document will be 
maintained in the individual subject study binder. 
10.1.2 STUDY DISCONTINUATION AND CLOSURE 
Participants may withdraw from the study at any time for voluntary reasons or discontinued from study 
treatment at the request of the study investigators. Reasons for withdrawal or termination may include 
but are not limited to 1) Subject requests discontinuation of study drug or withdrawal; 2) Subject 
becomes pregnant; 3) Subject requires treatment that is not permitted by the study protocol. 
10.1.3 CONFIDENTIALITY AND PRIVACY 
The investigators will abide by detailed guidance provided by the IRB on data management and security 
considerations. 
10.1.4 FUTURE USE OF STORED SPECIMENS AND DATA 
There will be no stored specimens for future use.   
 
Data collected for this study will be analyzed and stored in a secure location in the Emergency Medicine 
Research Office. After the study completion, study records will be retained for a duration in accordance 
with relevant regulations and institutional policies. This period of time complies with regulations within 
45 CFR 46.115(b), and permits sufficient time for an audit of records if deemed necessary by regulatory 
authorities. A de-identified dataset will be prepared after study completion and archived in the event 
further analyses are necessary to develop the treatment in. 

 
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10.1.5 KEY ROLES AND STUDY GOVERNANCE 
Define the roles generically without names. 
The roles and responsibilities of the investigators and study coordinators include, but are not limited to 
assuring that the trial is conducted according to the study protocol, identifying, recruiting, and enrolling 
participants, obtaining individual informed consent, collecting and entering study data into the study 
database, following participants through study completion, maintaining essential research documents, 
and assuring regular IRB approvals and renewals,.  Additionally, the principal investigator has the overall 
responsibility for the study and is responsible for ensuring all participating staff members are adequately 
trained and competent to perform his/her job.   
10.1.6 SAFETY OVERSIGHT 
Medical personnel will be present throughout the drug administration and study visits. Safety 
monitoring and reporting of AEs and SAEs will follow IRB procedures. All observed and volunteered 
adverse reactions will be documented in the study records. All adverse events will be graded using the 
Common Terminology Criteria for Adverse Events (CTCAE) – NIH/NCI. 
10.1.7 CLINICAL MONITORING 
The study procedures will be performed with support from the Michigan Clinical Research Unit with an 
extensive track record of data integrity and compliance with regulatory procedures. To assure adequate 
protection of the rights of human subjects, per 21 CFR §312.50, 312.53, this study will be monitored by 
the University of Michigan Institute for Clinical and Health Research (MICHR).  Routine monitoring will 
be scheduled at appropriate intervals, with more frequent visits occurring at the beginning of the study. 
An initiation visit will take place, followed by routine monitoring visits. Additional visits can be scheduled 
at the request of the Project Manager or the Sponsor-Investigator. The established monitoring plan will 
ensure the quality and integrity of the data through pre- investigation visits and periodic site visits to 
verify adherence to the protocol, completeness and accuracy of study data and samples collected, 
proper storage, dispensing and inventory of study medication, and compliance with regulations.  
 
10.1.8 QUALITY ASSURANCE AND QUALITY CONTROL 
Quality control procedures will be implemented beginning with the data entry system. Data quality 
control checks will be run on the generated database. Any missing data or data anomalies will be 
communicated to the investigator or study coordinator for clarification/resolution. 
10.1.9.1 DATA COLLECTION AND MANAGEMENT RESPONSIBILITIES 
Data collection and management will be performed consistent with good clinical practice. The 
investigator-sponsor of the Investigational New Drug will ensure that all investigators, study 
coordinators, and nursing staff are appropriately trained. Electronic case report forms will be created 
and programmed with real time logic and range data entry checks in a RedCap database (Vanderbilt, 
Nashville, TN). Electronic direct data entry into the electronic case report forms at the bedside will be 
performed for all clinical data. Send-out laboratory data will be entered later from source documents.     

 
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10.1.9.2 STUDY RECORDS RETENTION 
Study records will be maintained in a study binder in a locked cabinet with access limited to the study 
staff under the direction of the principal investigator.  After the study completion, the archived data will 
be stored for at least 2 years after approval of an NDA or determination that an NDA will not be pursued 
for this use of intranasal insulin. This period of time complies with regulations at 21 CFR 312.62(c), and 
permits sufficient time for an audit of records if deemed necessary by regulatory authorities. Copies of 
these data will be destroyed after this period of time. 
10.1.10 PROTOCOL DEVIATIONS 
The study will be conducted according to the study protocol. If a protocol deviation occurs, such as a 
visit being missed, the investigator must decide whether to proceed, for example, whether or not to 
complete the visit outside of the protocol-defined window. The nature and reasons for the protocol 
deviation will be recorded in the subject’s CRF and submitted to the IRB according to institutional 
guidelines. 
10.1.11 PUBLICATION AND DATA SHARING POLICY 
All data, results, and intellectual property rights in the data and results derived from the study will be 
the property of the University of Michigan, who may utilize the data in various ways, such as for 
submission to government regulatory authorities or disclosure to other investigators. If the results of 
this study are reported in medical journals or at meetings, the identities of all participants will remain 
confidential. 
10.1.12 CONFLICT OF INTEREST POLICY 
The University of Michigan defines a conflict of interest (COI) as a situation where a faculty or staff 
member is in a position to influence the business, research, or other decisions of the University in 
relationship to an outside organization that could lead directly or indirectly to financial gain for that 
individual or the family of that individual, or give improper advantage to others to the detriment of the 
University. Study personnel will abide by the COI policies outlined in the Standard Practice Guide that 
detail procedures to guide disclosure of outside interests and managing COI that is identified. 
10.2 ADDITIONAL CONSIDERATIONS 
Not applicable. 
 
 

 
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10.3 ABBREVIATIONS 
AE 
Adverse Event 
CFR 
Code of Federal Regulations 
COI 
Conflict of Interest 
CRF 
Case Report Form 
DLT 
Dose Limiting Toxicity 
FDA 
Food and Drug Administration 
GCP 
Good Clinical Practice 
IND 
Investigational New Drug 
IRB 
Institutional Review Board 
OHRP 
Office for Human Research Protections 
PI 
Principal Investigator 
SOA 
Schedule of Activities 
US 
United States 
 
 

 
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10.4 PROTOCOL AMENDMENT HISTORY 
Version 
Date 
Description of Change  
Brief Rationale 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
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