Measurement of Insulin Levels in Cerebral Spinal Fluid (CSF) of Healthy Adults After a Single Intranasal Dose
Phase: PHASE1
Conditions: Healthy
Sponsor: HealthPartners Institute

 
 
Measurement of Insulin Levels in CSF of 
Healthy Adults after a Single Intranasal 
Dose 
Short title: INI-CSF 
 
 
Clinical Study Protocol 
NCT05866367 
US IND Number: 158094 
Serial Number: 0010 
Version 1.3, September 12th, 2024 
Sponsor 
Leah R Hanson, PhD 
Julia Johnson, MD (Study Physician) 
Neuroscience Research, HealthPartners Institute 
295 Phalen Blvd, 
Saint Paul, MN 55130 
 
This study will be conducted in compliance with the protocol, IND regulations and other applicable 
regulatory requirements. 
 
 
 
 
 
 
Confidential Information 
No use or disclosure of this document outside Neuroscience Research at HealthPartners Institute is 
permitted without prior written authorization from Neuroscience Research. 
 
 

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PROTOCOL SIGNATURE PAGE 
 
 
 
 
 
 
 
I have read this protocol and agree to adhere to the requirements. I will provide copies of this 
protocol and pertinent information to the study personnel under my supervision and my local ethics 
committee/institutional review board (EC/IRB). I will discuss this material with them and ensure they 
are fully informed regarding the study medication and the conduct of the study according to this 
protocol, applicable law, applicable regulatory requirements including 21 CFR parts, 50, 54, 56, 312 
and 812, general standards of good clinical practice and local EC/IRB requirements. 
 
 
 
 
 
 
 
 
 
_______________________________________                              
Principal Investigator 
 
 
 
____________________ 
Date 
 
 
 

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Table of Contents 
List of Tables 3 
List of Figures 4 
Definitions 
4 
Protocol Summary ........................................................................................................................................ 5 
1 
Introduction .......................................................................................................................... 7 
1.1 
Background and Rationale ............................................................................................................ 7 
1.1.1 
Intranasal Insulin ................................................................................................................... 7 
1.1.2 
Intranasal Insulin and Devices ............................................................................................... 7 
1.1.3 
Safety of Intranasal Insulin .................................................................................................... 8 
2 
Summary of Device Description ............................................................................................ 8 
2.1 
Intranasal SipNose Device Overview ............................................................................................ 8 
2.1.1 
Intranasal SipNose Device ..................................................................................................... 8 
2.1.2 
SipNose Utility, Reliability, and Safety ................................... Error! Bookmark not defined. 
3 
Study Objectives.................................................................................................................... 9 
3.1 
Primary Objective.......................................................................................................................... 9 
3.2 
Secondary Objective ..................................................................................................................... 9 
4 
Study Endpoints .................................................................................................................... 9 
4.1 
Primary Endpoints ......................................................................................................................... 9 
4.2 
Secondary Endpoints .................................................................................................................... 9 
4.3 
Safety ............................................................................................................................................ 9 
5 
Study Design .......................................................................................................................... 9 
5.1 
Justification for Study Design, Dose and Endpoints ...................................................................... 9 
5.1.1 
Justification for Study Design and Dose ................................................................................ 9 
5.1.2 
Justification for Endpoints .................................................................................................. 10 
5.2 
Study Duration ............................................................................................................................ 10 
5.3 
Study Drug Administration .......................................................................................................... 10 
5.3.1 
Drug Administration with SipNose ...................................................................................... 10 
5.3.2 
Device Dose Loading and Administration ........................................................................... 10 
6 
Study Population ................................................................................................................. 10 
6.1 
Eligibility Criteria ......................................................................................................................... 10 
6.1.1 
Inclusion Criteria ................................................................................................................. 10 
6.1.2 
Exclusion Criteria ................................................................................................................. 10 

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Study Assessments and Procedures .................................................................................... 11 
7.1 
Demographic and Baseline Assessments .................................................................................... 11 
7.1.1 
Screening Visit 1 .................................................................................................................. 11 
7.1.2 
Lumbar Puncture Visit 2: In-Person Visit (2 week ± 7 days after Visit 1) ............................ 12 
7.1.3 
Phone Visit 1: (following a day after lumbar puncture) ...................................................... 13 
7.1.4 
Follow-up Visit 3 (Within a week of Visit 2 ± 5 days). ......................................................... 13 
7.1.5 
Ad hoc Visit if Symptomatic: ............................................................................................... 13 
7.2 
Early Withdrawal ......................................................................................................................... 13 
7.3 
Safety .......................................................................................................................................... 13 
7.3.1 
Physical Examination ........................................................................................................... 13 
7.3.2 
Neurological Examination ................................................................................................... 13 
7.3.3 
Vital Signs: ........................................................................................................................... 13 
7.3.4 
Weight: ................................................................................................................................ 14 
7.3.5 
Laboratory Samples: ........................................................................................................... 14 
7.3.6 
Point-of-Care Glucose Testing: ........................................................................................... 14 
7.3.7 
Pregnancy Test .................................................................................................................... 14 
7.3.8 
Contraceptive Counseling ................................................................................................... 14 
7.3.9 
Suicidality: Columbia Suicide Severity Rating Scale (C-SSRS) .............................................. 14 
7.3.10 
Nasal Irritation Scale ........................................................................................................... 15 
7.3.11 
Pharmacokinetic Sampling .................................................................................................. 15 
8 
Investigational Product(s) ................................................................................................... 16 
8.1 
Description of Investigational Product ....................................................................................... 16 
8.2 
Handling and Storage .................................................................................................................. 16 
8.3 
Packaging and Labeling ............................................................................................................... 16 
8.4 
Occupational Safety .................................................................................................................... 17 
9 
Concomitant Medications and Non-Drug Therapies .......................................................... 17 
9.1 
Permitted Medications ............................................................................................................... 17 
9.2 
Prohibited Medications ............................................................................................................... 17 
10 
Subject Completion and Withdrawal .................................................................................. 17 
10.1 
Subject Completion ..................................................................................................................... 17 
10.2 
Subject Withdrawal ..................................................................................................................... 17 
11 
Adverse Events (AE) and Serious Adverse Events (SAE) ..................................................... 18 
11.1 
Definition of AE ........................................................................................................................... 18 

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11.2 
Definition of SAE ......................................................................................................................... 18 
11.2.1 
Clinical Laboratory Abnormalities & Other Abnormal Assessments as AEs & SAEs ........... 18 
11.2.2 
Time Period and Frequency of Detecting AEs and SAEs ..................................................... 19 
11.2.3 
Device Failures and Malfunctions ....................................................................................... 19 
12 
Data Analysis and Statistical Considerations ...................................................................... 19 
12.1 
Statistical Analysis Overview ....................................................................................................... 19 
12.2 
Statistical Analysis of Primary and Secondary Endpoints ........................................................... 19 
13 
Study Conduct Considerations ............................................................................................ 19 
13.1 
Regulatory and Ethical Considerations, Including the Informed Consent Process ..................... 19 
13.2 
Data Safety Monitoring Plan ....................................................................................................... 19 
13.2.1 
Stopping Criteria ................................................................................................................. 20 
13.3 
Quality Assurance ....................................................................................................................... 20 
13.4 
Study Closure .............................................................................................................................. 20 
13.5 
Records Retention ....................................................................................................................... 21 
13.6 
Provision of Study Results and Information to Investigators ..................................................... 21 
13.7 
Data Management ...................................................................................................................... 21 
13.8 
Device and Drug Accountability .................................................................................................. 21 
14 
References .......................................................................................................................... 22 
Summary of Changes .................................................................................................................................. 25 
Appendix A. 
Intranasal Insulin Studies Table .......................................................................................... 26 
Appendix B. 
Schmid et al. 2018, The safety of intranasal human insulin: a review ................................ 27 
Appendix C. 
Novolin R Label Prescribing Information ............................................................................ 28 
Appendix D. 
Regular Insulin, ClinicalKey Drug Monograph ..................................................................... 29 
Appendix E. 
Born et.al 2002 – Sniffing Neuropeptides: A transnasal approach to the human brain .... 30 
Appendix F. 
HealthPartners Investigator-Initiated Intranasal Insulin Studies ........................................ 31 
Appendix G. 
Insulin Stability Experiment Report & Raw Data ................................................................ 32 
 
List of Tables 
Table 1: REDACTED ............................................................................................ Error! Bookmark not defined. 
Table 2: Study Design ....................................................................................................................................... 9 
Table 3: Study Visit Schedule ......................................................................................................................... 15 
Table 4: Prohibited Medications .................................................................................................................... 17 
 

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List of Figures 
REDACTED 
Definitions 
Adverse event (AE) 
Any undesirable patient experience that may include but is 
not limited to an abnormal sign, symptom, illness, abnormal 
laboratory value, or other medical event. 
Cerebrospinal Fluid (CSF) 
Clear liquid that flows around the brain and spinal cord. 
Columbia-Suicide Severity Rating 
Scale (C-SSRS) 
A scale designed to quantify the severity of suicidal ideation 
and behavior. 
Data Safety Officer (DSO) 
An independent individual with relevant experience 
assigned to review safety data to monitor for incidence of 
trends that would warrant termination of the trial. 
Intranasal (IN) 
A method of drug delivery that is particularly applicable to 
delivering centrally acting medications into the central 
nervous system. 
Intranasal Insulin (INI) 
Human insulin that is administered to the nose in effort to 
directly target delivery to the central nervous system  
Lumbar Puncture (LP) 
A medical procedure which involves inserting a needle 
between two vertebrae of the lower back to collect fluid 
surrounding the spinal cord and brain. 
Serious adverse events (SAE) 
Any symptom, sign, illness or experience that develops 
during the study and results in a life-threatening situation, 
hospitalization, significant disability, and other events 
determined by the investigator to be significant. 
 
 
 
 
 
 
 

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Protocol Summary 
PROTOCOL TITLE 
Measurement of Insulin Levels in CSF of Healthy Adults after a Single 
Intranasal Dose 
SHORT TITLE  
INI-CSF 
STUDY PHASE 
Phase I 
STUDY OBJECTIVES AND PURPOSE  
Primary Objectives:  
• 
To measure the concentration of insulin that reaches central nervous system in humans after 
intranasal administration 
 
Secondary Objectives:  
• 
To describe the effect of single intranasal insulin dose on serum insulin in healthy adults 
 
STUDY DESIGN  
Study Type 
Pharmacokinetics 
Control Type 
None 
Study Indication Type 
Pharmacokinetics 
Blinding Schema 
Unblinded 
Study Drug 
Regular, Human Insulin [rDNA origin] USP solution for injection 100 units/mL 
Device 
SipNose  
Route 
Intranasal 
Study Design 
Open-label, randomized  
Planned Duration of 
Subject Participation 
4-5 weeks 
(Treatment duration: 1 dose) 
PRIMARY ENDPOINTS 
• 
Cerebrospinal Fluid (CSF) Insulin Levels at pre-defined times (0-40 Min)  
SECONDARY ENDPOINTS 
• 
Serum Insulin levels at pre-defined times (0-40 Min)  
 
 

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INVESTIGATIONAL PRODUCTS, DOSE AND MODE OF ADMINISTRATION 
Investigational Product 
Regular insulin (Novolin-R) in 2 different doses (80, and 40 IU) (see Table 
2) 
 
INI Dose 
CSF Collection Times 
40 IU (n=6) 
2 x 200 uL 
0, 10, 20, 30, 40 min 
80 IU (n=6) 
2 x 200 uL 
Wait 2 min 
2 x 200 uL 
0, 10, 20, 30, 40 min 
 
SUBJECT SELECTION 
Targeted Accrual 
Up to 12 subjects.  We estimate a need to consent 25 participants to 
reach this goal. 
INCLUSION CRITERIA 
1. Subject is between ≥18 and ≤ 35 years of age  
2. Subject’s body mass index is between >=18.5 and <=24.9, or can safety undergo a lumbar 
puncture at the discretion of the radiologist 
3. Subject must be proficient in speaking English to comply with instructions and measures for 
the study 
4. Subject can provide written informed consent 
5. Female subjects must have either: (1) a negative pregnancy test at the screening and 
treatment visits OR (2) be at least 2 years post-menopausal / surgically sterile. 
EXCLUSION CRITERIA 
1. Subject has medical history and/or clinically determined disorders: chronic sinusitis, previous 
nasal and/or oto-pharyngeal surgery and severe deviated septum and/or other anomalies. 
2. Subject has history of any of the following: active and significant central nervous system, 
psychiatric illness, pulmonary, or cardiovascular disorders or any other clinically relevant 
abnormality that inclusion would pose a safety risk to the subject as determined by 
investigator 
3. Subject has participated in a clinical trial investigation within 3 months of this study. 
4. Subject has an insulin allergy 
5. Subject has Insulin-dependent diabetes 
6. Subject is pregnant or breast feeding 
7. Contraindication to spinal tap or other safety factors that preclude lumbar puncture in the 
investigator’s opinion 

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1 Introduction 
1.1 Background and Rationale 
1.1.1 Intranasal Insulin  
Intranasal (IN) delivery offers a non-invasive route to deliver large molecules such as insulin directly to the 
brain while minimizing systemic exposure. Peptides, proteins, vaccines, drug treatments and ions of 
various sizes are able to pass along the olfactory and trigeminal nerves and are deposited directly into the 
CNS without having to pass through the blood-brain barrier (BBB), which may degrade or limit the amount 
arriving at the target 1-8. CSF insulin levels in healthy adults have been detected as early as 10 minutes 
following IN delivery (Appendix A) while not measurably increasing systemic blood-glucose levels 3,9. 
Similar observations of the safety of intranasal insulin (INI) I have been made in memory impaired adults 
10,11. 
Originally thought to exist solely in the periphery, insulin has since been determined to be instrumental in 
the overall health and function of the CNS 12.  Central insulin and insulin receptors (IRs) have been 
established as differing from that of the systemically occurring counter parts that specifically regulate 
glucose utilization. In rodents, insulin receptors and insulin-sensitive glucose transporters are selectively 
co-localized in brain areas responsible for memory, thus providing a platform for insulin signaling whereby 
selective increases in cerebral glucose utilization could modulate memory 13,14. Consistent with evidence of 
insulin functioning as a neuromodulator for memory-related function is the high-density of IRs in the 
hippocampus and cerebral cortex, brain regions integral to the formation, retention and recall of 
information 12,15. Systems with impaired insulin signaling pathways have demonstrated inhibition of 
acetylcholine biosynthesis and subsequently have incurred debilitating effects on neuronal plasticity 16,17.  
Increased insulin resistance and glucose intolerance has been observed in a multitude of 
neurodegenerative processes including Alzheimer’s disease 14, Parkinson’s disease 18, and Huntington’s 
disease 19.   
Clinical studies with INI in the last 20 years have studied it as a therapeutic option for multiple diseases 
including Alzheimer’s dementia (AD), Parkinson’s disease, and Post-traumatic stress disorders. In addition, 
studies in healthy humans and patients have shown effects on metabolism, including reductions in food 
intake, body weight, improvements of glucose uptake, and possible influence on mood, addiction, and 
sleep 20. Recent studies have shown that INI also reduces white matter damage in the brains of patients 
with Alzheimer’s Disease21. Appendix A provides an overview of the multiple studies conducted with INI. In 
Appendix B, Tables 1 (acute INI, 38 studies) and 2 (chronic INI, 18 studies) from the Schmid review provide 
key effects of INI on functional outcomes such as cognition, or memory.  
The delivery of drugs to the CNS remains a development challenge mainly due to the blood-brain barrier 
limiting access. Commercially available aerosol nasal devices such as the LMA MAD device are not 
specifically engineered to facilitate nose-brain delivery, and consequently deposit most of the drug within 
the lower nasal cavity, resulting in suboptimal CNS penetration.  Furthermore, there is concern about 
variable, user-dependent drug delivery due to dependency upon subject positioning and inconsistent 
volume ejection. 
1.1.2 Intranasal Insulin and Devices 
REDACTED   

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1.1.3 Safety of Intranasal Insulin 
Novolin R has been approved for subcutaneous or intravenous use since 1991. The most common side 
effect noted with these two routes is hypoglycemia (Regular Insulin, Drug Monograph; Appendix D). A 
recent review of safety of IN human insulin trials revealed no safety concerns, with no serious adverse 
events or symptomatic hypoglycemia in a total of 1092 individuals studied28. In Appendix B, Tables 1 
(acute) and 2 (chronic) of this review provide a summary of the human studies with INI. The majority of 
the studies used regular Insulin (Novolin R or Actrapid, Humulin R, Insuman Rapid, H-insulin 100) with 
similar excipients and none are believed to be in a range that causes harm 29. 
Study of pharmacokinetics of INI in mice showed only 3% of INI entered the circulation and no peripheral 
metabolic effects were detected up to a day after IN administration30.  Treatment with 40 IU INI in humans 
found no significant changes in circulating insulin levels, suggesting that INI does not pass into the 
circulation 3,9. A recent study in cardiac surgery patients, intranasal administration of insulin at doses of 40 
IU and 80 IU did not cause hypoglycemia intraoperatively31. Though some studies with higher doses 
(160IU) have shown small detectable increase in insulin levels, no significant change in blood glucose 
levels were noted32. Notably, intranasally administered insulin improves memory in healthy adults and 
Alzheimer’s patients without altering blood levels of insulin or glucose 11,33-39. 
The most common side effect with INI administration noted is transient local or nasal irritation. In 
Appendix B, please refer to Table 3 of the Schmid et al., 2018 review for a summary of the local or nasal 
effects of INI in humans. In 2012, Craft et al reported, that number of adverse events was higher for the 20 
IU and 40 IU when compared to the placebo group; however, there were no SAE’s and mostly minor AEs 
such as rhinitis were reported (Table 4, 35). Similar minor AEs were reported in the 2017 by Craft et al using 
INI (Supplementary table 2, 39). Similar findings were reported in recent meta-analyses40 and the most 
recent trial on INI26, which showed no clinically relevant adverse events during the chronic administration 
of 40 IU of insulin with two different administration devices. Overall, INI at the proposed dose in this 
protocol, is expected to be safe with limited side effect profile. We do plan to monitor the subjects for 
hypoglycemia.  
 
2 Summary of Device Description 
2.1 Intranasal SipNose Device Overview 
REDACTED 
2.1.1 Intranasal SipNose Device 
REDACTED 
2.1.1.1 
Reliability 
REDACTED 
2.1.1.2 
Safety 
REDACTED 
2.1.1.3 
Device-Drug Contact and Safety 
REDACTED 

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3 Study Objectives 
3.1 Primary Objective 
• 
To measure the concentration of insulin that reaches cerebrospinal fluid in humans after 
intranasal administration 
3.2 Secondary Objective 
• 
To describe the effect of single intranasal insulin dose on serum insulin in healthy adults 
4 Study Endpoints 
4.1 Primary Endpoints 
• 
Cerebrospinal Fluid Insulin Levels at pre-defined times (0-40Min)  
4.2 Secondary Endpoints 
• 
Serum Insulin levels at pre-defined times (0-40 Min)  
4.3 Safety 
• 
Incidence and severity of serious adverse events (SAEs) and adverse events (AEs) 
• 
Frequency of change in clinically-significant vital signs 
• 
fingerstick glucose level <70mg/dl  
5 Study Design 
This Phase 1 is a single center, open-label, randomized pharmacokinetic study designed to describe the 
central nervous system delivery of intranasal insulin to CSF using anIN delivery device.  
Table 1: Study Design 
INI Insulin Dose 
Collection Times 
40 IU (n=6) 
2 x 200 uL (0.2ml) 
0, 10, 20, 30, 40 min 
80 IU (n=6) 
2 x 200 uL (0.2ml) 
Wait 2 min 
2 x 200 uL (0.2ml) 
0, 10, 20, 30, 40 min 
 
After written informed consent has been obtained, subjects will be screened to assess study eligibility 
based on study inclusion/exclusion criteria. Subjects who are eligible at the end of the screening visit will 
be randomized and assessed for outcome measures, including lumbar puncture and CSF/blood draw 
following a single dose of INI administered by the trained research staff. Randomization will be stratified 
by gender to ensure a balanced distribution of male and female participants within each group. A Block 
randomization scheme will be employed to ensure that the final group sizes are equivalent. 
5.1 Justification for Study Design, Dose and Endpoints 
5.1.1 Justification for Study Design and Dose 
In this study, we will be measuring the concentrations of insulin in the CSF of 12 healthy adults after IN 
administration of one of two doses of insulin: 40 IU and 80 IU (Table 2). Multiple clinical trials of INI have 
ranged from 10 IU -160 IU for a variety of disorders, have been found to be safe in healthy adults45 and are 
the basis for dose selection in this study. The higher dose (80 IU) may be the maximum feasible dose for 

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clinical studies, while the lower dose is the most encountered dose in clinical studies (both acute/chronic). 
The INI doses proposed are both safe, and are known to improve function or outcomes in the respective 
disorders. In addition, our research group has completed (NCT01436045, NCT02432716, NCT02503501, 
NCT04028960) or has ongoing (NCT04115384, NCT04251585) INI based clinical trials with similar doses 
and designs that are summarized in Appendix F.  
Figure 7. REDACTED 
5.1.2 Justification for Endpoints 
The key objective of this study is to provide essential information on the ability of device to target INI to 
the brain and spinal cord as measured in CSF collected by lumbar puncture, which is the primary endpoint 
for the study. Born et al (2002)3 (Appendix E) demonstrated detection of insulin after intranasal 
administration of 40 IU using a metered dose spray pump. Our study will measure the concentrations of 
insulin that reach the CSF following administration using an intranasal delivery system. The secondary 
endpoint will provide additional information regarding the concentration of insulin in serum.   
5.2 Study Duration 
Study participation will last approximately 4-5 weeks, consisting of a screening/baseline visit, 
treatment/lumbar puncture visit, a follow-up/final assessment visit.  
5.3 Study Drug Administration 
5.3.1 Drug Administration with SipNose 
REDACTED  
5.3.2 Device Dose Loading and Administration 
REDACTED 
 
6 Study Population 
6.1 Eligibility Criteria 
6.1.1 Inclusion Criteria 
A subject will be included for consideration in this study only if all the following criteria are met: 
1. Subject is between ≥18 and ≤ 35 years of age 
2. Subject’s BMI is between >=18.5 and <=24.9, or can safely undergo a lumbar puncture at the 
discretion of the radiologist 
3. Subject must be proficient in speaking English to comply with instructions and measures for the 
study 
4. Subject can provide written informed consent 
5. Female subjects must have either: (1) a negative pregnancy test at the screening visit and 
treatment visit OR (2) be at least 2 years post-menopausal / surgically sterile. 
6.1.2 Exclusion Criteria 
A subject will not be included for consideration in this study if any of the following criteria are met: 
 
1. Subject has medical history and/or clinically determined disorders: chronic sinusitis, previous nasal 
and/or oto-pharyngeal surgery and severe deviated septum and/or other anomalies. 

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2. Subject has history of any of the following: active and significant central nervous system, 
psychiatric illness, pulmonary, or cardiovascular disorders or any other clinically relevant 
abnormality that inclusion would pose a safety risk to the subject as determined by investigator 
3. Subject has participated in a clinical trial investigation within 3 months of this study. 
4. Subject has an insulin allergy 
5. Subject has Insulin-dependent diabetes 
6. Subject is pregnant or breast feeding 
7. Contraindication to spinal tap or other safety factors that preclude lumbar puncture in the 
investigator’s opinion 
7 Study Assessments and Procedures 
A summary of study events and procedures is outlined in the Study Visit Table (Table 3). 
7.1 Demographic and Baseline Assessments 
7.1.1 Screening Visit 1 
The following procedures will be performed at this visit: This may also be divided into two parts, with 
virtual and in-person visits. Virtual activities (e.g., consent, review of medical history/medications) can be 
completed on a separate day(s) from other study procedures, provided that consent occurs within 3 
months of the lumbar puncture and all other screening activities are completed within the specified 
window (21-7 days prior to lumbar puncture visit/Visit 2). All activities will take approximately 2 hours to 
complete. No study procedures, virtual or otherwise, will be completed until a fully executed consent form 
has been obtained. Participants will be given the option to have a dose of sterile saline administered by 
research staff using the device if they want to experience the sensation of the device before the lumbar 
puncture visit. 
• 
Obtain written informed consent Review Inclusion/Exclusion Criteria 
• 
Review medical history, as it pertains to inclusion/exclusion criteria 
• 
Pregnancy test for women of childbearing potential 
• 
Review guidance on the use of effective birth control from the time of study enrollment until 30 
days after the final visit. 
• 
Obtain subject’s demographic information (date of birth, gender, race, and ethnicity). 
• 
Collect vital signs, height and weight 
• 
Obtain details of medications taken over the course of the last 30 days 
• 
Physical/neuro examination including examination of the nares and nasal passages 
• 
Blood draw for laboratory investigations 
• 
C-SSRS 
• 
12-Lead Standard ECG 
• 
Saline test dose (optional) 
• 
Pre-procedure instructions 
• 
Provide Information sheet provided relating to pre-lumbar puncture hydration 
• 
Visit #2 scheduled after 2 weeks ±7 days 

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7.1.2 Lumbar Puncture Visit 2: In-Person Visit (2 week ± 7 days after Visit 1) 
The subject will be check in at the Regions Hospital outpatient care unit (OPCU). The visit will be at the 
General Radiology Department (where the lumbar puncture will be performed and study data collected) 
and will return to the OPCU for post-operative monitoring.  
The following procedures will be performed at this visit: 
• 
Review Inclusion/Exclusion Criteria 
• 
Collect concomitant medication information and record AEs/SAEs 
• 
Collect vital signs and weight 
• 
Pregnancy test for women of childbearing potential 
• 
Review guidance on the use of effective birth control until 30 days study after the final visit. 
• 
Glucose finger stick test (once before study drug administration and twice after) 
• 
Clinician will discuss procedure with the subject  
• 
Lumbar puncture and collection of CSF for insulin  
o CSF samples will be obtained at predefined times (0-40 min). CSF samples will be collected 
FIRST at all time points. 
• 
Blood draw and collection of serum for insulin  
Serum samples will be obtained at predefined times (0-40 Minutes). CSF Samples will be 
collected SECOND at all time points.  
• 
Study Drug administration 
• 
Nasal Irritation Scale  
• 
Patient will lay on back for a total of 2 hours post-lumbar puncture  
• 
Visit #3 scheduled within a week ± 5 days 
7.1.2.1 
Fluoroscopy-Guided Lumbar Puncture Procedure  
CSF samples will be collected according to standard operating procedures for fluoroscopy-guided lumbar 
puncture as observed by the General Radiology department at Regions Hospital. The procedure will be 
performed by a trained clinician (i.e., an interventional radiologist or  an experienced member of their 
team), who will obtain verbal consent (in addition to the research informed consent 
obtained/documented at Visit 1). CSF will be collected serially using gravity drainage. Sterile extension 
tubing connected to the LP needle will dispense CSF into sterile collection tubes for pharmacokinetic 
analysis. Samples will be collected at 5 times: once prior to the administration of a single dose of 
intranasal insulin and at once each at 10, 20, 30, and 40 minutes post-drug administration. Approximately 
1-2ccs of CSF will be collect at all 5 time points for a total of 10ccs of CSF. The tubing connected to the 
needle will be flushed out between sample collections to avoid contamination between specimen 
timepoints. The subject will lie in prone position throughout the procedure with appropriate measures 
taken to minimize discomfort (e.g., provision of warm blankets, use of positioning sponges, etc.). 
 Subjects will lie on their back 2 hours after the procedure for standard-of-care monitoring with nursing 
staff, and periodic collection of vital signs. Participants will receive instructions from the clinical team on 
techniques for preventing and treating post-LP headache (e.g., drinking water and caffeinated beverages, 
using over-the-counter pain medications). They will also receive guidance on when to seek additional help 
for their headache and/or any other emergent symptoms requiring immediate medical attention such as 
fever, persistent headache, and weakness/numbness of the lower extremities. Subjects will receive a 
safety assessment phone call from study staff 1 day after the procedure.  

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7.1.3 Phone Visit 1: (following a day after lumbar puncture) 
• 
Study staff phone call to address AEs/SAEs Day 2 (+ 1 days) after visit 2/LP procedure 
• 
There will be screening questions related to headache, bleeding at the site, and infectious 
symptoms. 
7.1.4 Follow-up Visit 3 (Within a week of Visit 2 ± 5 days). 
The following procedures will be performed at this visit: This can be divided into two parts, with virtual 
and in-person visits 
• 
Collect concomitant medication information and record AEs/SAEs 
• 
Physical/neuro examination including examination of nares and nasal passages 
• 
Collect vital signs, weight 
• 
Pregnancy test for women of childbearing potential 
• 
Review guidance on the use of effective birth control from the time of study enrollment until 30 
days study after the final visit. 
• 
12-Lead Standard ECG 
• 
C-SSRS 
• 
Blood draw for laboratory investigations 
7.1.5 Ad hoc Visit if Symptomatic: 
• 
Collect vital signs, Weight 
• 
Physical Examination 
• 
Study staff to address AEs/SAEs 
7.2 Early Withdrawal 
If subject withdraws from the study after the screening visit, but before the lumbar puncture visit, no 
further evaluations are necessary.   
7.3 Safety 
For all safety assessments described below, any clinically significant change will be recorded as an adverse 
event (AE) or serious adverse event (SAE). 
7.3.1 Physical Examination 
Complete physical examination including examination of the nares/nasal passages will be performed at 
visits 1 and 3, or if the subject withdraws or is withdrawn from the study early.  Any abnormalities noted 
at Visit 1, will be documented as part of the subject’s medical history. 
7.3.2 Neurological Examination 
Neurological examination will be performed at visits 1, 3 or if the subject withdraws early.  Any 
abnormalities noted at Visit 1, will be documented as part of the subject’s medical history.   
7.3.3 Vital Signs: 
Vital signs will be recorded at all three study visits.  Diastolic blood pressure will be measured at the 
disappearance of Korotkoff sounds. Vitals sign will be monitored by clinical staff during screening and 
lumbar puncture visits of the study. 

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Serum Glucose will be examined at visit 1 (screening) and visit 3 (safety follow-up). Fingerstick glucose 
tests will be performed at visit 2 (LP procedure). A blood glucose ˂ 70 mg/dL will be considered clinically 
significant.  The likelihood of peripheral hypoglycemia is low based on numerous clinical trials performed 
showing no effects of INI on peripheral glucose. 
7.3.4 Weight: 
Body weight will be measured at all three study visits. 
7.3.5 Laboratory Samples: 
All the laboratory samples will be drawn independent of fasting or non-fasting status.  
When a patient withdraws from the study, samples collected prior to the date of withdrawal may still be 
analyzed, unless the patient specifically requests that the samples be destroyed or local laws require 
destruction of the samples. However, if samples have been tested prior to withdrawal, results from those 
tests will remain as part of the overall research data. Samples will be collected and processed according to 
the standard procedures. Key planned labs include: 
• 
Basic Metabolic Panel 
• 
Complete Blood Count 
• 
Blood Coagulation Tests 
7.3.6 Point-of-Care Glucose Testing: 
At Visit 2, the participant will receive a single dose of intranasal insulin administered prior to their lumbar 
puncture. Point-of-care fingerstick glucose testing will be performed once before the administration of the 
study drug (baseline glucose reading) and twice after the administration of the drug according to the 
following schedule: 
 
(1) 15 minutes after study drug dosing (between the 2nd and 3rd CSF/serum samples are collected) 
(2) 45 minutes study drug dosing (after the final CSF/serum samples are collected) 
7.3.7 Pregnancy Test 
All women of childbearing potential will have blood collected to test for pregnancy. This will be performed 
at the screening visit (Visit 1), treatment visit (Visit 2), and final follow-up visit (Visit 3). A negative 
pregnancy test (urine) must be confirmed on the day of Visit 2 prior to the administration of the study 
drug.  
7.3.8 Contraceptive Counseling  
All study participants (male and female) will be counseled on the use of effective forms of birth during 
study participation and in the 30 days following the final study visit. If a pregnancy occurs during this time 
period (in either the subject or the subject’s partner), the participant will be asked to inform the study 
team immediately.  
7.3.9 Suicidality: Columbia Suicide Severity Rating Scale (C-SSRS)  
The C-SSRS is a suicidal ideation rating scale to evaluate suicidality. It rates an individual's degree of 
suicidal ideation on a scale, ranging from "wish to be dead" to "active suicidal ideation with specific plan 
and intent”47. The C-SSRS will be administered by trained raters at specified time points, as indicated in 
Table 3 as well as when clinically indicated. Any subjects demonstrating evidence of suicidality will prompt 

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immediate consultation with the site’s on-call psychiatrist for assistance with decision-making and 
potential referral to behavioral health services. 
7.3.10 Nasal Irritation Scale 
Study team will ask subjects if they are experiencing any nasal irritation (schedule see Table 3) on a Likert 
scale of 0-10, with 0 being no irritation and 10 being severe irritation. The scale will be administered 5 
times at the following predetermined intervals: Once immediately before study drug administration, and 
again at 20 and 40 minutes post-administration. Research staff will also observe whether the subject has 
experienced any bleeding (Yes/No).  If Yes, they will be asked to categorized the bleeding as:  a) Bleeding 
which stops within 1 minute; b) Bleeding, taking 1-5 minutes to stop; c) Bleeding for 4-60 minutes, does 
not require medical intervention; d) Bleeding which requires medical intervention. 
7.3.11 Pharmacokinetic Sampling 
The study team will attempt to collect the CSF and serum samples as close in time as possible for each PK 
time point. Given differences in how serum/CSF are extracted for this study, there may be a slight 
difference between collection start and stop times. To ensure consistency across participants, the study 
team will initiate collection of the CSF samples first, followed by the serum samples, at each of the 5 PK 
time points.   
7.3.11.1 CSF Sampling 
It is our intention to collect samples at pre-defined times following a single dose administration. CSF will 
be collected at 0, 10, 20, 30 and 40 minutes for all study participants. CSF samples will be assessed for 
insulin using a one-step sandwich immunoassay with a limit of detection of 0.1 µIU/ml or 0.7 pmol/L 
7.3.11.2 Serum Samples 
It is our intention to collect samples at pre-defined times following a single dose administration. Serum 
will be collected at 0, 10, 20, 30 and 40 minutes for all study participants. Serum samples will be assessed 
for insulin using the same one-step sandwich immunoassay with a limit of detection of 0.1 µIU/ml or 0.7 
pmol/L.   
Table 2: Study Visit Schedule 
Assessment/ Procedure  
Visit 1: 
Screen 
 
Visit 2: 
Lumbar 
Puncture (LP) 
Phone Visit 1: 
Safety Check  
Follow-up 
Visit 3 
Ad Hoc Visit if 
Symptomatic 
Schedule – Week and Windows 
-2 
1 
Following day 
after LP 
Within 1 
week of 
Visit 2 
 
 
      ± 7 days 
      + 1 day 
  ± 5  days 
 
Informed consent  
X 
 
 
 
 
Review of inclusion/exclusion criteria  
X 
X 
 
 
 
Medical History, demographics 
X 
 
 
 
 
Physical/Neuro exam with 
examination of nares and nasal 
passages 
X 
 
 
X 
X 
Vital Signs (Blood pressure, heart rate, 
weight) 
X 
X 
 
X 
X 
Height 
X 
 
 
 
 

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Basic Metabolic Panel (Na, K, CO2, CL, 
BUN, Creatinine, Glucose and Ca) 
X 
 
 
X 
 
Labs INR/PT/PTT 
X 
 
 
X 
 
Complete Blood Count 
X 
 
 
X 
 
Pregnancy Test (in females)  
X 
X 
 
X 
 
Counseling on effective birth control 
X 
X 
 
X 
 
Randomization 
 
X 
 
 
 
Lumbar Puncture 
 
X 
 
 
 
Glucose Finger Stick  
(0 Min, 15 Min, 45 Min) 
 
X 
 
 
 
Nasal Irritation Scale (0, 20, 40 min) 
 
X 
 
 
 
CSF Samples and Insulin Levels 
(0-40 Min) 
 
X 
 
 
 
Serum insulin Levels 
(0-40 Min)  
 
X 
 
 
 
C-SSRS, 12-Lead Standard ECG 
X 
 
 
X 
 
Pre-Procedure instructions 
X 
 
 
 
 
Study Drug Administration (single 
dose) 
 
X 
 
 
 
Concomitant Medication 
X 
X 
X 
X 
X 
Adverse Events 
X 
X 
X 
X 
X 
8 Investigational Product(s) 
8.1 Description of Investigational Product 
The research staff will utilize the following investigational products: 
• 
Intranasal delivery route 
• 
Regular insulin (Novolin-R –U-100; Insulin Full Prescribing Information; Appendix C) 
• 
SipNose Device  
 
8.2 Handling and Storage 
The study drug will be kept at study site per label recommendations and institutional Standard 
Operational Policy, specifically, but not limited to temperature controlled secure area. 
To ensure that a stable temperature and/or conditions are maintained, site staff will verify and document 
temperature at a minimum of three times per week or monitored continuously if automated systems such 
as TempTrak are used.  An electronic log will be securely stored at the study site. Study staff will be 
responsible for safeguarding and maintaining the master log.  
8.3 Packaging and Labeling 
Only designated study staff will be responsible for handling all investigational drug and devices; study 
participants will not be preparing/administering the drug themselves. The study drug will not be 
individually labeled; the study team may use the same vial of insulin for multiple participants in order to 
minimize waste. 
All study drug will be labeled according to the following specifications: 
• 
Protocol identifier/IRB approval/account/study number 

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• 
Quantity statement 
• 
“Caution: New Drug—Limited by Federal (or United States) law to investigational use” 
All study devices will be individually packaged and labeled by the manufacturer as follows: 
REDACTED 
8.4 
Occupational Safety 
No known significant safety risks exist to site personnel in direct or indirect contact with the study drug. As 
the lumbar puncture will employ fluoroscopy, clinical (non-research) staff will be exposed to radiation. The 
fluoroscopy technicians and radiologist will follow standard clinical protocols for radiation safety (e.g. 
wearing lead Personal Protective Garments). The amount/duration of radiation exposure for this research 
LP is not greater than what would be experienced during a routine LP. As fluoroscopy is only needed to 
assist with needle placement, study staff will not need to be present for this part of the procedure and can 
step out of the procedure room while the fluoroscopy machine is in use. Research staff can then reinter 
the room to assist with pre-dose sample collection/fingerstick glucose testing after the machine has been 
deactivated.  
9 Concomitant Medications and Non-Drug Therapies 
9.1 
Permitted Medications 
Any medication not listed in list of Prohibited Medications (Table 4) will be permitted during this study.  A 
record will be kept by site staff detailing doses and indication of any concomitant medications used by 
subjects. 
9.2 
Prohibited Medications 
Any subject with insulin-dependent diabetes taking insulin will be excluded from the study. Additional 
medications that are exclusionary and/or should be avoided during the study period are outline below in 
Table 4. 
Table 3: Prohibited Medications 
Prohibited Medications 
Barbiturates 
Benzodiazepines 
Insulin 
Muscle Relaxants 
Opioids 
Beta Blockers 
10 Subject Completion and Withdrawal 
10.1 Subject Completion 
Subjects completing all study visits (4) will be considered to have completed study. 
10.2 Subject Withdrawal 
Subject may withdraw from study at any time for any reason without penalty or be terminated from the 
study by the clinical investigator (see provisions for termination by study team.) Investigational team will 

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document the reason(s) for withdrawal.  In the event a subject chooses to withdraw from study before 
Visit 3, the safety procedures described in 7.3 will be performed (ideally within 3 days following subject’s 
decision to withdraw).  For all subjects who withdraw, all final safety assessments will be collected as 
possible regardless of time elapsed since previous visit.  In addition to the termination visit, subjects who 
withdraw early will be contacted within 7 days by study staff via telephone to assess development of new 
and/or ongoing AEs and concomitant medications.  Efforts will be made to recruit subjects to replace any 
withdrawals before lumbar puncture (or subjects for whom we are unable to get sufficient for analysis) so 
as to maintain n=6 per group. 
Subject’s participation may be terminated at the discretion of the investigator.  Individuals may be 
withdrawn for the following reasons: 
• 
Clinically significant adverse events 
• 
Lost to follow-up 
• 
Protocol violations 
• 
Inability to tolerate study medication 
• 
Other 
11 Adverse Events (AE) and Serious Adverse Events (SAE) 
11.1 Definition of AE 
An adverse event is any symptom, sign, illness or experience which develops or worsens in severity during 
the course of the study. Intercurrent illnesses or injuries should be regarded as adverse events. Abnormal 
results of diagnostic procedures are considered to be adverse events if the abnormality: 
• 
Results in study withdrawal 
• 
Is associated with clinical signs or symptoms 
• 
Leads to treatment or to further diagnostic tests 
• 
Is considered by the investigator to be of clinical significance 
 
11.2 Definition of SAE 
Adverse events are classified as either serious or non-serious. A serious adverse event is any event that 
results in: 
• 
Death, Life-threatening situation 
• 
Hospitalization or prolongation of hospitalization 
• 
Disability or incapacitation 
• 
Other events determined by investigator to be medically significant in which subject’s well-being is 
jeopardized (e.g. events that have high likelihood of escalating to the point of meeting criteria 
outlined above) 
 
11.2.1 Clinical Laboratory Abnormalities & Other Abnormal Assessments as AEs & SAEs 
Any new abnormal, vital, examination, or laboratory finding judged clinically significant by the investigator 
will be documented as an AE or SAE, if meeting the definitions for such.  Abnormal lab findings or other 
abnormal assessments associated with the disease under study will not be considered AEs or SAEs unless 
more severe than expected, as judged by the investigator. 

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11.2.2 Time Period and Frequency of Detecting AEs and SAEs 
Upon consenting, a subject is considered to be a participant in the study, and until that person either 
withdraws or completes study, AEs and SAEs will be recorded.  The investigational team will promptly 
report any AE/SAE as required per federal guidelines. 
11.2.3 Device Failures and Malfunctions 
Device Quality Control. All failures and malfunctions of the device will be documented.  
12 Data Analysis and Statistical Considerations 
12.1 Statistical Analysis Overview 
The population demographics (age, gender, etc.) and clinical values (vital signs, labs etc.) will be reported 
using the appropriate summary measures (e.g. mean, standard deviation, and proportions). 
12.2 Statistical Analysis of Primary and Secondary Endpoints 
Primary Endpoints: We will utilize immunoassay to analyze the CSF samples to determine the levels of 
Insulin concentration at the specified times. We will graph these values across time for each subject using 
line graphs. We will also graph the sample average Insulin concentration across time, and at different 
doses with error bars and report concentrations as mean ± SE. Finally, the concentrations will be analyzed 
using common pharmacokinetic measures including the peak concentration (Cmax), time to peak 
concentration (Tmax), and area under the curve (AUC). This type of analysis is very similar to that done in 
Dhuria’s 2009 publication in the Journal of Pharmaceutical Sciences, on which Leah Hanson and William 
Frey II were co-authors. Similar analysis will be conducted for secondary endpoints. 
AEs and SAEs will be categorized and summarized by study group. We will descriptively note if any group 
was more likely to have experienced a particular AE/SAE or generally experienced more AEs/SAEs 
throughout the course of the study. 
13 Study Conduct Considerations 
13.1 Regulatory and Ethical Considerations, Including the Informed Consent 
Process 
The study will be conducted in accordance with GCP.  Subject privacy requirements will also be observed 
as well as the fundamental concepts of the Declaration of Helsinki (e.g. IRB approval of the study, 
obtaining informed consent from all subjects, and meeting reporting requirements). The clinical trial will 
be registered on the clinicaltrials.gov website. 
13.2 Data Safety Monitoring Plan 
A data safety officer (DSO) will be an independent individual who is not participating in the trial and has no 
direct affiliation with the research team. The Data safety monitoring plan will be established prior to 
initiation of the study. The DSO responsibilities include but are not limited to the following: 
• 
Monitoring the study for compliance to the protocol.  
• 
Stopping the study if the rate of SAE’s raises safety concerns.  The details will be specified in the 
data safety monitoring plan.  

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During the course of the trial, the DSO will review accumulating safety data to monitor for incidence of 
trends that would warrant termination of the trial. The frequency of the DSO meetings, responsibilities, 
membership, and procedures will be documented in the data safety monitoring plan. 
13.2.1 Stopping Criteria 
Previous research suggests that the incidence of peripheral hypoglycemia in patients given INI at the 
dosages examined in this trial (40IU and 80IU) is low. However, the study team will obtain point-of-care 
blood glucose readings before and after administration of the single dose of intranasal insulin and use this 
information to determine the safety of continued study participation. Although the study team will 
document the blood glucose readings <70mg/dL as a safety event, levels this low will not necessitate 
withdrawal/study stoppage. A reading of <54mg/dL will be considered clinically significant regardless of 
symptoms.  
For individual subjects: Study activities will be stopped for participants with Hypoglycemia readings 
<54mg/dL as measured by point-of-care fingerstick glucose tests. If this value is reached, hypoglycemia 
rescue measures will be performed, and no additional CSF or blood samples will be collected. If a 
participant exhibits the following symptoms of dangerous hypoglycemia in the absence of clinically 
significant value, their participation will be similarly stopped: blurred vision, confusion, slurred speech, 
cold clammy skin, etc or that requires hypoglycemia rescue measures. 
The study team will follow our institution’s standard clinical protocol for the treatment of hypoglycemic 
events. Measures taken to restore glucose levels to >70mg/dL may include oral treatments (e.g., fruit juice 
or oral glucose gel) as appropriate. If a patient is unresponsive or otherwise not able to take oral 
treatments, they may be given IV dextrose or intramuscular glucagon. 
Participants with glucose levels >54 but less than <70 will not need to be withdrawn based on these values 
alone. A reading in this range will trigger an ad-hoc glucose test reading for safety monitoring in 
participants that are otherwise asymptomatic/mildly symptomatic but not requiring rescue measures. 
For dosing cohorts: Participants will be randomized to one of two arms for this study, receiving either a 
40IU or 80IU dose of insulin. If two participants in either arm (40IU or 80IU) experience a clinically 
significant glucose level (<54mg/dL) and/or experience severe/possibly life-threatening symptoms of 
hypoglycemia as defined in the protocol, the study will be paused and the Data Safety Officer will review 
data related to these events and recommend a course of action for the study. These options may include, 
but are not limited to: (1) Adjusting the arm to a lower dose for future enrollees (e.g., reducing the 
amount from 80IUs to 40IU, or 40IU to 20IUs, etc.), (2) Arm termination, and (3) Study termination. 
 
13.3 Quality Assurance 
In the event of a regulatory agency audit or inspection, site will allow the auditor/inspector access to all 
records documented and facilities utilized in conducting the study.  Site will also make accommodations 
(e.g. time, schedule) to discuss findings, concerns, and questions with auditor/inspector. 
13.4 Study Closure 
Upon completion of all subject visits, data entry and analysis, investigator will inform local IRB of study 
closure. 

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13.5 Records Retention 
All site records will be maintained and stored in a safe and secure location for a minimum of 15 years post 
study completion. 
13.6 Provision of Study Results and Information to Investigators 
Study results will be made available by the study statistician once analysis is complete.   
13.7 Data Management 
Data collection/reporting tools will be developed internally (i.e. CRFs and source documents).  Data 
collected and stored electronically will remain confidential and secure (e.g. secured server, encrypted 
data, password protected file) 
13.8 Device and Drug Accountability 
A Device Tracking and Drug Log will be maintained at the investigational site. Intranasal devices will be 
recorded on the log upon delivery to the investigational site and will be stored in a secured area. The 
Device and Drug Tracking Log will be updated as each device or drug is delivered, dispensed, returned and 
the reason for the return.  Serial numbers, expiration date and model number of devices or batch/lot 
numbers for drugs delivered to the site will also be recorded. 
 
 
 

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Appendix A. 
Intranasal Insulin Studies Table 
 
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Appendix B. Schmid et al. 2018, The safety of intranasal human 
insulin: a review   
 
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Appendix C. Novolin R Label Prescribing Information 
 
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Appendix D. Regular Insulin, ClinicalKey Drug Monograph 
 
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Appendix E. Born et.al 2002 – Sniffing Neuropeptides: A transnasal 
approach to the human brain    
 
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Appendix F. HealthPartners Investigator-Initiated Intranasal Insulin Studies  
 
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Appendix G. Insulin Stability Experiment Report & Raw Data  
 
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Appendix H. REDACTED