A Phase II, Single Center, Randomized, Double-Blind, Placebo-Controlled Study of the Safety and the Therapeutic Effectiveness of Intranasal Glulisine in Amnestic Mild Cognitive Impairment and Probable Mild Alzheimer's Disease
Phase: PHASE2
Conditions: Mild Cognitive Impairment, Alzheimer's Disease (AD)
Sponsor: HealthPartners Institute

A Phase II, Single Center, Randomized, Double-Blind, Placebo-Controlled Study of the 
Safety and the Therapeutic Effectiveness of Intranasal Glulisine in Amnestic Mild 
Cognitive Impairment and
Probable Mild Alzheimer’s disease.
Clinical Study Protocol
US IND Number: 124247
Amendment 4 Date: November 11, 2016
Amendment 3 Date: October 12, 2015
Amendment 2 Date:       July 2 2015
Amendment 1 Date:       20 April 2015
Original Protocol Date:      25 September 2014 
Sponsor
HealthPartners Center for Memory and Aging
640 Jackson Street
St. Paul, MN 55101
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 HealthPartners Center for Memory and Aging is 
permitted without prior written authorization from
HealthPartners Center for Memory and Aging.

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 hospital 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 and 812, general standards of good clinical 
practice and hospital EC/IRB requirements.
_______________________________________
Principal Investigator
____________________
Date 

                   
                                          Table of Contents 
Contents 
1. 
Introduction ........................................................................................................................ 10 
1.1 Background and Rationale ................................................................................................10 
2. 
Summary of Device Description ........................................................................................ 12 
3.1. Primary Objective..........................................................................................................13 
3.2. Secondary Objectives ....................................................................................................13 
3.3 
Exploratory Objectives..................................................................................................13 
4. 
Endpoint(s)......................................................................................................................... 13 
4.1. Primary Endpoints.........................................................................................................13 
4.2. Safety.............................................................................................................................14 
5. 
Study Design ...................................................................................................................... 14 
5.1. Outcome Measures ......................................................................................................14 
5.2. Study Duration.............................................................................................................14 
6. 
Study Population ................................................................................................................ 15 
6.1. Eligibility Criteria..........................................................................................................15 
6.1.1. 
Inclusion Criteria......................................................................................................................... 15 
6.1.2. 
Exclusion Criteria........................................................................................................................ 15 
1. 
Study assessments and procedures..................................................................................... 16 
7.1. Demographic and Baseline Assessments.......................................................................16 
7.1.1. 
Visit 1; Screening Visit Fasting................................................................................................... 16 
7.1.2. 
Visit 2; Baseline Visit ................................................................................................................. 17 
7.1.3. 
Visit 3; Initial Treatment Visit; (Week 0).................................................................................... 17 
7.1.4. 
Visit 4; Follow-Up Safety Visit; (Week 1).................................................................................. 17 
7.1.5. 
Visit 5; Treatment and Adherence; (Week 4).............................................................................. 18 
7.1.6. 
Visit 6; Treatment and Adherence; (Week 8).............................................................................. 18 
7.1.7. 
Visit 7; Treatment and Adherence; (Week 12)............................................................................ 18 
7.1.8. 
Visit 8; Treatment and Adherence (Week 16)............................................................................. 19 
7.1.9. 
Visit 9; 5-month check-in (Week 20).......................................................................................... 19 
7.1.10. 
Visit 10; 6 month follow-up (Week 24) ...................................................................................... 19 
7.1.11. 
Visit 11; Final Safety and Discharge; Fasting (Week 28) ........................................................... 19 
7.2. Early Withdrawal...........................................................................................................20 
7.3. Safety.............................................................................................................................20 
7.3.1. Physical Examination ....................................................................................................20 
7.3.2. Neurological Examination.............................................................................................20 
7.3.3. Vital Signs .....................................................................................................................20 
7.3.4. Weight ...........................................................................................................................20 
7.3.5. ECG ...............................................................................................................................21 
7.3.6. Laboratory Samples.......................................................................................................21 
7.3.6.1 
HOMA-IR – Measure for Insulin Resistance .............................................................................. 21 
7.4. Neuropsychological Assessment ...................................................................................21 
7.4.1. 
ADAS-Cog 13 Summary............................................................................................................. 22 
7.4.2. 
Digit Span subtest from the Wechsler Adult Intelligence Scale -4th Edition (WAIS-IV)............ 22 
7.4.3. 
Controlled Oral Word Association Test (COWAT).................................................................... 23 
7.4.4. 
Trail Making Test, Parts A & B .................................................................................................. 23 
7.4.5. 
Montreal Cognitive Assessment (MOCA) .................................................................................. 23 
7.4.6. 
Clinical Dementia Rating (CDR) Scale....................................................................................... 23 
7.4.7. 
Columbia-Suicide Severity Rating Scale (C-SSRS).................................................................... 24 
7.4.8. 
15-Item Geriatric Depression Scale (GDS) ................................................................................. 24 
7.4.9. 
The Functional Activities Questionnaires (FAQ)........................................................................ 24 
7.4.10. 
Animal Naming........................................................................................................................... 24 
7.4.11. 
Wechsler Memory Scale Logical Memory.................................................................................. 24 
7.4.12 
Alberta Smell Test....................................................................................................................... 24 
7.4.13 
Sleep Questions........................................................................................................................... 25 
7.4.14 
Epworth Sleepiness Scale (ESS) ................................................................................................. 25 
7.5. Pharmacogenetics..........................................................................................................25 

8. 
Investigational product(s)................................................................................................... 25 
8.1. Description of Investigational Product..........................................................................25 
8.2. Handling and Storage ....................................................................................................25 
8.3. Treatment Assignment...................................................................................................26 
8.4. Packaging and Labeling.................................................................................................26 
8.5. Occupational Safety.......................................................................................................26 
9. 
Concomitant medications and non-drug therapies ............................................................. 26 
9.1. Permitted Medications...................................................................................................26 
9.2. Prohibited Medications..................................................................................................27 
10. 
Subject completion and withdrawal ................................................................................... 27 
10.1. Subject Completion .......................................................................................................27 
10.2. Subject Withdrawal .......................................................................................................27 
11. 
Adverse events (AE) and Serious adverse events (SAE)................................................... 28 
11.1. Definition of AE............................................................................................................28 
11.2. Definition of SAE..........................................................................................................28 
11.2.1. 
Clinical Laboratory Abnormalities & Other Abnormal Assessments as AEs & SAEs................ 28 
11.2.2. 
Time Period and Frequency of Detecting AEs and SAEs ........................................................... 28 
12. 
Data analysis and statistical considerations........................................................................ 28 
12.1. Analysis Overview ........................................................................................................28 
12.2. Statistical Analysis of Efficacy Outcomes ....................................................................29 
12.3. Statistical Analysis of Safety Outcomes........................................................................30 
12.4. Study Power...................................................................................................................30 
13. 
STUDY CONDUCT CONSIDERATIONS....................................................................... 30 
13.1. Regulatory and Ethical Considerations, Including the Informed Consent Process .......30 
13.2. Data Safety Monitoring Board.......................................................................................31 
13.3. Quality Assurance..........................................................................................................31 
13.4. Study Closure ................................................................................................................31 
13.5. Records Retention..........................................................................................................31 
13.6. Provision of Study Results and Information to Investigators........................................31 
13.7. Data Management..........................................................................................................31 
13.8. Device Accountability...................................................................................................31 
References ...............................................................................................................................32 

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. 
ADAS-Cog 13 - an outcome measure for global cognition in clinical trials for Alzheimer’s 
Disease.
Amnestic Mild Cognitive Impairment (aMCI) - A condition characterized by subject memory 
complaints and objective neuropsychological testing demonstrating abnormalities >1.0 SD 
affecting memory.  There is preservation of instrumental activities of daily living without 
functional limitations.  This population has a 40% change of developing Alzheimer’s Disease 
over a 2 year period.  
Alberta Smell Test (AST) - A test to identify the olfactory abilities of an individual.
Alzheimer’s disease (AD) – A chronic progressive neurodegenerative condition resulting in 
memory impairment, loss of function, and progressive deterioration in other cognitive domains 
including language, perceptual skills, attention, construction, orientation, and problem solving.
Animal Naming - a word fluency test measuring executive functioning and language ability.
Mild Alzheimer’s disease - Patients meeting NINCDS-ADRDA criteria for probable AD with a 
CDR=1
Columbia-Suicide Severity Rating Scale (C-SSRS) - A scale designed to quantify the severity 
of suicidal ideation and behavior.
Controlled Oral Word Association Test (COWAT) - a short, paper/pencil measure of 
phonemic fluency and executive functioning from the Multilingual Aphasia Examination.
Clinical Dementia Rating (CDR) - a numeric scale used to quantify the severity of symptoms
of dementia (i.e. stage).
Data Safety Monitoring Board (DSMB) - an independent group assigned to review safety data 
to monitor for incidence of trends that would warrant termination of the trial.
Diagnostics and Statistical Manual-IV(DSM-IV TR) – a diagnostic manual published by the 
American Psychiatric Association that provides a common language and standard criteria for the 
classification of mental disorders including Alzheimer’s Disease.
Digit Span subtest from the Wechsler Adult Intelligence Scale -4th Edition (WAIS-IV) - is a 
brief measure of auditory attention, concentration, and working memory.  
Epworth Sleepiness Scale (ESS) – A measure to identify excessive daytime sleepiness.

Geriatric Depression Scale (GDS) - a screening test for depression in older adults.
Glulisine - a rapidly absorbed insulin analogue lacking the zinc ingredient commonly found in 
insulin formulations and characterized by a unique amino acid sequence that distinguishes it 
from other commercially available insulin compounds.  
The Functional Activities Questionnaires (FAQ) – a measure of instrumental activities of 
daily living (IADLs).
Hachinski Ischemia Score - a screening tool used to differentiate vascular causes of dementia 
from neurodegenerative causeV$VFRUHRILVVXJJHVWLYHof a vascular etiology.
Intranasal (IN) - a method of drug delivery that is particularly applicable to conveying centrally 
acting medications into the central nervous system.
Mini Mental State Exam (MMSE) - a 30 point cognitive screening tool that assesses 
orientation, working memory, short term memory, visuospatial construction, and language. 
Montreal Cognitive Assessment (MOCA) - a 30 point screening instrument for cognitive
impairment that has a 90% sensitivity for MCI and 100% sensitivity for AD.
National Institute of Neurological and Communicative Disorders and Stroke and the 
Alzheimer’s disease and Related Disorders Association (NINCDS-ADRDA) criteria -
criteria used to diagnose definite, probable, possible and unlikely Alzheimer’s disease for 
research and clinical purposes.   These criteria specify eight cognitive domains that may be 
impaired in AD: memory, language, perceptual skills, attention, constructive abilities, 
orientation, problem solving and functional abilities.  These require that symptoms are confirmed 
by neuropsychological testing or screening in probable AD. 
Trail Making Test, Parts A & B - a brief paper and pencil measure of attention, processing 
speed, and mental flexibility (or set-shifting).
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.   
WMB Logical Memory - The Wechsler Memory Scale (WMS) is a neuropsychological test 
designed to measure different memory functions.

PROTOCOL   SUMMARY
PROTOCOL TITLE
A Phase II, Single Center, Randomized, Double-Blind, Placebo-
Controlled Study of the Safety and the Therapeutic Effectiveness of 
Intranasal Glulisine in Amnestic Mild Cognitive Impairment and
Probable Mild Alzheimer’s Disease.
SHORT TITLE 
MCI-AD IN Insulin Study 
STUDY PHASE
Phase II
STUDY OBJECTIVES AND PURPOSE 
Primary Objective: To measure the chronic effects of IN insulin glulisine on cognition and 
function in subjects with aMCI and probable mild AD over a 6 month period.
Secondary Objectives: 
x
To measure the effect of IN insulin glulisine on mood in subjects with aMCI and mild AD 
over a 6 month period.
x
To measure the safety and efficacy of IN glulisine in aMCI and mild AD subjects with
non-insulin dependent diabetes over a 6 month period.   
Exploratory Objectives:
x
To measure the effect of IN delivery of insulin glulisine on parieto-temporal and posterior 
cingulate/precuneus glucose metabolism in subjects with aMCI and mild AD over a 6 
month period.
x
To measure the chronic effect of IN delivery of insulin glulisine on AD-specific 
cerebrospinal (CSF) biomarkers (Abeta42, tau, and phosphotau) in subjects with aMCI 
and mild AD over a 6 month period.
x
To measure the chronic effect of IN delivery of insulin glulisine on sleep in subjects with 
aMCI and mild AD over a 6 month period.
x
To measure the chronic effect of IN delivery of insulin glulisine on olfaction in subjects 
with aMCI and mild AD over a 6 month period.
STUDY DESIGN 
Study Type
Safety
Control Type
Placebo
Study Indication Type
Treatment
Blinding Schema
Double-Blind
Device
Pressurized Olfactory Delivery (POD) device
Study Design
Randomized, double-blinded control
Planned Duration of 
Subject Participation 
8 months
ENDPOINTS
Primary
x
ADAS-Cog 13
x
CDR-SOB
x
FAQ  
Secondary
x
Digit Span Forward

x
Trailmaking Test Parts A&B
x
Controlled Oral Word Association Test 
(COWAT)
x
Animal Naming
x
Weschler Memory Scale (WMS)
x
Geriatric Depression Scale (GDS) 
Exploratory
x
CSF Abeta, tau, phosphotau
x
FDG-PET metabolism posterior 
parietiotemporal, posterior cingulate, and 
precuneus regions 
x
Sleep questionnaire/Epworth Sleepiness 
Scale (ESS) 
x
Alberta Smell Test 
INVESTIGATIONAL PRODUCTS, DOSE AND MODE OF ADMINISTRATION
Investigational 
Product
Glulisine  20 IU/IN (.1ml/10 units IN in each nostril) BID
Placebo
Sterile Normal Saline  20 IU/IN  placebo (.1ml IN in each nostril) BID
SUBJECT SELECTION
Targeted Accrual
Approximately 90 randomized subjects.  We estimate will need to 
consent 130 participants in order to reach this goal.
Inclusion Criteria
1. Male or female subject with a clinical and research diagnosis of amnestic-mild cognitive 
impairment (1, 2) OR probable mild AD in accordance with NINCDS-ADRDA criteria (3)
2. Subject has a MOCA score of 18-27.
3. Subject has a Hachinski Ischemia Score < 4.
4. Subject is > 50 and <90 years of age.
5. Female subjects must be at least 2 years post-menopausal or surgically sterile.
6. The subject must be proficient in speaking, reading and understanding English in order to 
comply with procedural testing of cognitive function, memory and physiology.
7. Subject has a dedicated family member /caregiver, who will be able to attend all visits and 
report on subject’s status.
8. Subject and family member/caregiver have both provided fully informed written consent prior 
to participation. In the event that subject is legally unable to provide informed written 
consent due to deterioration in cognitive abilities, fully informed written consent must be 
provided by a legally authorized representative.
9. If AD, subject must have a brain CT or MRI in the initial diagnostic work up or subsequent 
care that is compatible with the diagnosis of probable Alzheimer’s Disease.
Exclusion Criteria
1. Subject has medical history and/or clinically determined evidence of other CNS disorders 
including, but not limited to brain tumor, active subdural hematoma, seizure disorder, 
multiple sclerosis, dementia with Lewy bodies, vascular dementia, corticobasal syndrome, 
progressive supranuclear palsy, Parkinson’s disease, multiple system atrophy, frontotemporal 
dementia, normal pressure hydrocephalus, Huntington’s disease, or Jakob-Creutzfeldt disease 

presenting as dementia.
2. Subject has medical history and/or clinically determined disorders: current B12 deficiency, 
chronic sinusitis or any untreated thyroid disease, significant head trauma, or history of 
difficulty with smell and/or taste prior to AD diagnosis.
3.   Subject has history of any of the following: moderate to severe pulmonary disease, poorly 
controlled congestive heart failure, significant cardiovascular and/or cerebrovascular events 
within previous 6 months, condition known to affect absorption, distribution, metabolism, or 
excretion of drugs such as any hepatic, renal or gastrointestinal disease or any other clinically 
relevant abnormality that inclusion would pose a safety risk to the subject as determined by 
Investigator.
4. Subject has had previous nasal and/or oto-pharyngeal surgery and severe deviated septum
and/or other anomalies.
5. Subject has history of any psychiatric illness, with the exception of major depressive and 
anxiety disorder (according to DSM-IV TR) currently in remission or stable with treatment 
for > 2 years, or any other psychiatric condition that inclusion would pose a safety risk to the 
subject as determined by investigator.  
6. Subject is currently taking any medications, herbals and food supplements that are 
medically/clinically contraindicated as determined by investigator in order to comply with 
procedural testing of cognitive function as well as ensure study safety. See list of prohibited 
medications and compounds.
7.   Subject has undergone a recent change (<1month) in their prescribed acetylcholinesterase 
inhibitor (e.g. donepezil, rivastigmine, galantamine) or mematine.
8. Subject has undergone a recent change (<1 month) in their SSRI or anti-depressant 
medication.
9. Subject has current or recent drug or alcohol abuse or dependence as defined by DSM-IV TR.
10. Screening laboratory results that are medically relevant, in which inclusion would pose a 
safety risk to the subject as determined by investigator.
11. The subject has participated in any other research study at least 3 months prior to this study.
12. The subject has an insulin allergy.

1.
INTRODUCTION
1.1 Background and Rationale
Alzheimer’s disease (AD) is the most common cause of dementia, representing 60-80% of all 
cases and afflicting 5.4 million individuals within the United States. While characteristic amyloid 
plaques (AP) and neurofibrillary tangles (NFT) represent the pathognomic signature of AD, the 
AD brain is characterized by a severe impairment of insulin-signaling, including deficits of 
insulin, insulin-like growth factor, insulin receptors as well as central resistance to insulin action 
(4-8). Disruption of central nervous system (CNS) insulin signaling has been increasingly 
associated with AD pathogenesis, and consequently this disease has been referred to as a type III 
diabetes of the brain (9) Furthermore, longitudinal cohort studies have shown that diabetes 
doubles the risk of AD,(10) and have linked high blood sugar with increased dementia risk (11).
Flurodeoxyglucose positive emission tomography (FDG-PET) studies of subjects with peripheral 
insulin resistance demonstrate a cerebral pattern of frontal, parietotemporal, and cingulate 
cortical hypometablism that mimics AD (12)
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 (13).  Central insulin and insulin 
receptors (IRs) have been established as differing from that of the systemically occurring 
counterparts that specifically regulate glucose utilization. Although central insulin does induce 
glucose uptake in the brain, it also functions in the modulation of various neurotransmitters and 
receptors involved in executive function and the long-term potentiation of memories. For 
example, systems with impaired insulin signaling pathways have demonstrated inhibition of 
acetylcholine biosynthesis and subsequently have incurred debilitating effects on neuronal 
plasticity (7, 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 (13, 15).
Brain deposition of beta-amyloid ($ȕ) plaques and neurofibrillary tangles (NFTs) are both 
critical pathological signatures of AD, but are also modulated by CNS insulin activity. Increased 
insulin concentration upregulates the production of insulin-degrading enzyme (IDE); a
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hyperphosphorylation as well as decreases the number of synaptic insulin receptors (IRs) (5).
The downregulation of dendritic surface IRs IURPDFFXPXODWLQJ$ȕFRPSURPLVHVV\Qaptic 
function in brain regions associated with memory and cognitive function, leading to the 
disruption in the insulin-signaling pathway that is characteristic of AD (16). Insulin further
inhibits neurofibrillary tangle production by maintaining a phosphorylation equilibrium between 
kinase and phosphatase activity (8, 17-19).
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 BBB that may 
degrade or limit the amount arriving at the target (20-27). Increased insulin in healthy adults has 
been detected in CSF as soon as 10 minutes after IN delivery while not measurably increasing 

systemic blood-glucose levels (22, 28). Similar effects have been achieved using IN insulin with 
human subjects of altered brain insulin concentrations (29, 30).
Clinical trials of intranasal insulin in AD have demonstrated therapeutic effects involving
memory, attention and cognition without significantly altering serum insulin or glucose levels. A
clinical trial consisting of 26 memory impaired subjects (13 with AD and 13 with mild cognitive 
impairment) and 35 normal controls showed that IN insulin 20 IU or 40 IU resulted in 
improvements on two declarative memory tasks compared to placebo within 15 minutes of drug 
administration (29). IN insulin administered at 20 IU resulted in greater story recall whereas 
doses at 40 IU more favorably improved word list recall. There was no impact on IN insulin on 
serum glucose or insulin levels. Another study of 24 early AD/mild cognitive impairment 
subjects showed that 20 IU BID of intranasal insulin resulted in sustained benefit in over a 21 
day period (30). Furthermore, IN insulin induced favorable changes in the serum amyloid-beta 
40/42 ratio while having no impact on systemic glucose or insulin levels. Most recently, 
Craft and colleagues have shown improved memory and function as represented by the ADAS-
Cog and ADCS-ADL, respectively in AD patients treated during a four month clinical trial (31).
These changes in memory and function were associated with favorable changes in CSF tau and 
tau-Ab42 ratio. Additionally, IN insulin minimized progression in loss of cerebral 
hypometabolism as measured by FDG-PET.
Therapeutic response to IN insulin may be modified by both insulin type as well as ApoE4 
carrier status. A study of 38 normal subjects showed that rapid-acting insulin aspart (Insulin 
NovoLog HM, Novo Nordisk) resulted in greater declarative memory improvements compared 
to regular insulin over 8 an week period (32). Additional clinical trials have suggested memory 
benefits associated with IN insulin are diminished among ApoE4 carriers diagnosed with AD 
(29).
Glulisine is a rapidly absorbed insulin analogue lacking the zinc ingredient commonly found in 
insulin formulations. This analogue differs from human insulin in that the amino acid asparagine
at position B3 is replaced by lysine and the lysine in position B29 is replaced by glutamic acid.
For this study, we have chosen glulisine due to prior speculation that zinc-containing compounds 
may be toxic to olfactory neurons (33, 34) as well as prior studies demonstrating improvements 
in declarative memory with RA compared to regular insulin.
To test the hypothesis regarding the acute safety and efficacy of glulisine, the HealthPartners 
Center for Memory and Aging performed a double blinded, randomized, placebo-controlled, 
cross-over pilot study in 9 subjects with mild-moderate AD. We further sought to address the 
responsiveness of ApoE4 carriers to this treatment, and so all subjects were carriers of this 
polymorphism. The study drug was intranasally administered using the LMA Mucosal 
Atomization Device (MAD) and was not associated with any serious adverse events. Treated 
subjects made fewer Trails B errors relative to controls. Otherwise, there were no significant 
difference between IN glulisine and placebo for cognitive tests of learning/memory, 
attention/executive function, language, or visuospatial function. Fingerstick glucose was not 
impacted by IN glulisine, but the drug resulted in a 19% decrease in insulin levels compared to 
placebo. The conclusion was that ApoE4 subjects were unresponsive to acute IN glulisine, but 
the drug was safe and well-tolerated. 

In the current study, we are investigating the chronic effects of IN glulisine 20 IU administered 
two times daily in adults with amnestic-mild cognitive impairment (a-MCI) and mild 
Alzheimer’s disease (AD). The investigation will enroll n=90 subjects and follow them over a 6 
month period. This study is characterized by three major changes: 1.) the IN glulisine will be 
administered using the Pressurized Olfactory Delivery (POD) Device; 2.) the study will include 
non-insulin dependent diabetics; 3.) the study will incorporate biomarkers for amyloidosis 
(cerebrospinal fluid Abeta42) and neurodegeneration (cerebrospinal tau/phosphotau and FDG-
PET).
In the acute pilot study, we had employed the LMA MAD device in the acute setting, but for this 
chronic trial, there is concern about variable, user-dependent drug delivery due to dependency 
upon subject positioning and inconsistent volume ejection. The POD device is more practical for 
a 6 month clinical trial. The POD device is powered with a standard hydrofluoroalkane (HFA) 
meter valve to administer drug into the nasal cavity rather than the mechanical ability of the user, 
which results in greater inter-user consistency.
This study will also include a subsample of subjects with non-insulin dependent diabetes. Studies 
have shown that diabetics have twice the risk of Alzheimer’s disease (35) and FDG-PET scans 
demonstrate a pattern of hypometabolism resembling Alzheimer’s disease (12). We hypothesize 
that the subjects with peripheral deficits in insulin signaling will also be the ones to demonstrate 
deficits in central nervous system signaling. While diabetics comprise a significant portion of 
patients with AD, previous studies have excluded this population due to concerns regarding 
hypoglycemia in the setting of concomitant oral hypoglycemics or intranasal insulin. However, 
studies in non-diabetic subjects have failed to reveal any significant glucose lowering effects of 
IN insulin. There are minimal investigations addressing the effect of intranasal insulin on 
peripheral glucose in non-insulin dependent diabetes. One of the secondary objectives of this 
clinical trial is to demonstrate the safety and efficacy of IN glulisine in this population. Based on 
the multiple trials failing to reveal effects on peripheral glucose, we suspect that IN insulin will 
be well-tolerated in this population. 
Finally, investigations have shown that Alzheimer’s disease is characterized by the co-existence 
of cerebral amyloidosis with neurodegeneration, and that biomarkers of these abnormal changes 
can be used to support an AD diagnosis as well as longitudinally track disease progression (36).
Most recent AD clinical trials have included biomarkers from CSF and FDG-PET to provide 
evidence for biological drug actions related to amyloidosis and neurodegeneration (37, 38).
2.
Summary of Device Description
2.1. Pressurized Olfactory Delivery (POD) Device
The Pressurized Olfactory Delivery (POD) device is intended to be used with a variety of known 
and yet-to-be-known drugs. The POD device is designed specifically to deliver centrally acting 
drugs via the olfactory and trigeminal neural pathways in the roof of the nasal cavity and 
olfactory nasal epithelium.  
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.  Other problems associated with 

traditional nasal sprays include variable aerosolized product, dependence on user position, and 
high frequency of device non-compliance/misuse.  The POD device has specifically been 
developed to effectively and consistently deliver CNS therapeutics to the brain via the 
nasal/olfactory pathways.  The device is not currently FDA approved, but numerous studies 
support its role in intranasal brain delivery of radiolabeled and therapeutic compounds.
3.0.
OBJECTIVES
3.1.
Primary Objective
x
To measure the chronic effects of IN insulin glulisine on cognition and function in subjects 
with aMCI and probable mild AD over a 6 month period.
3.2.
Secondary Objectives
x
To measure the effect of IN insulin glulisine on mood in subjects with aMCI and mild AD 
over a 6 month period.
x
To measure the safety and efficacy of IN glulisine in aMCI and mild AD subjects with non-
insulin dependent diabetes over a 6 month period.  
3.3
Exploratory Objectives
x
To measure the effect of IN delivery of insulin glulisine on posterior parietotemporal and 
cingulate/precuneus glucose metabolism in subjects with aMCI and mild AD over a 6 month 
period.
x
To measure the chronic effect of IN delivery of insulin glulisine on AD-specific 
cerebrospinal (CSF) biomarkers (Abeta42, tau, and phosphotau) in subjects with aMCI and 
mild AD over a 6 month period.
x
To measure the chronic effect of IN delivery of insulin glulisine on sleep (Sleep 
questionnaire and ESS) in subjects with aMCI and mild AD over a 6 month period 
x
To measure the chronic effect of IN delivery of insulin glulisine on olfaction in subjects with 
aMCI and mild AD over a 6 month period.
4.
ENDPOINT(S)
4.1.
Primary Endpoints
x
Change in global measure of cognition as measured by the Alzheimer’s disease Assessment 
Scale-Cognitive (ADAS-Cog-13) in subjects with aMCI and mild AD between baseline and
visit 7 and between baseline and visit 10.
x
Change in Clinical Dementia Rating-Sum of Boxes (CDR-SOB) between screen/baseline and 
final assessment visit.

x
Change in daily functioning as measured by the Functional Assessment Questionnaire (FAQ) 
in subjects with aMCI and mild AD between baseline and final assessment visit.
4.2.
Safety
x
Incidence and severity of serious adverse events (SAEs) and adverse events (AEs).
x
Frequency of change in clinically-significant vital signs, physical exam or neurological 
exam. 
x
Change from baseline in 12-lead ECG.
x
Plasma glucose level <70mg/dl. 
5.
STUDY DESIGN
This study is a single center, phase II randomized, double-blind, placebo-controlled clinical trial 
designed to assess the efficacy of intranasally (IN) administered glulisine versus placebo in 
patients with aMCI and probable mild AD. 
After written informed consent has been obtained from the subject and family member/caregiver, 
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 (Visit 1) will be scheduled for a 
baseline visit (visit 2). During the baseline visit, subjects will undergo neuropsychological testing 
with the ADAS-Cog 13 battery and supplemental tests including the Trailmaking Test Parts A & 
B, Digit Span, Controlled Oral Word Association Test, Animal Naming, and WMS logical 
memory to establish a baseline.  Repeat cognitive assessments will be performed at 12 and 24 
weeks.  Patients will also be assessed on two additional measures for olfaction (Alberta smell 
test) and sleep measures (Sleep Questionnaire and ESS) at baseline, 12 weeks, and 24 weeks.
Patients enrolled in the AD biomarker substudy will undergo a baseline FDG-PET scan and CSF 
biomarkers (Abeta42, tau, phosphotau) at baseline and 24 weeks.
A total of n=90 subjects will be randomized to receiving either IN glulisine (n=45) or placebo 
(n=45) two times daily (morning and evening, separate by at least 8 hours).  In addition, subjects 
will be block randomized based on ApoE4 status, presence of non-insulin dependent diabetes,
and MOCA score (18-22 vs. 23-27). Subjects will receive either 0.2 ml IN insulin (20 IU) 
(0.1ml/10 units IN in each nostril) or placebo administered two times daily over a 6 month 
period.
5.1.
Outcome Measures
Neuropsychological, functional, and depression batteries will be performed at screen/baseline,
Visit 7 and at Visit 10 (refer to section 7.4 for full description) to assess primary endpoint. A
subset of subjects will undergo testing for CSF biomarkers (Abeta42, tau, phosphotau) and FDG-
PET imaging at baseline and Visit 10 to address exploratory endpoints.
5.2.
Study Duration

Study participation will last approximately 8 months, consisting of a minimum of 7 days but no 
more than 4 week screening phase and baseline visit, and a total of 9 treatment/safety visits over 
6 months. If study visit window exceeds 28 days, study drug will be resupplied which may 
require additional study drug pickup visits. Patients who discontinue treatment will continue 
follow-up measures as possible and will be analyzed as randomized per the intent to treat 
principal.
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 of the following criteria are 
met:
1. Male or female subject with a clinical and research diagnosis of amnestic-mild cognitive 
impairment (1, 2)OR probable mild AD in accordance with NINCDS-ADRDA criteria (3).
2. Subject has a MOCA score of 18-27.
3. Subject has a Hachinski Ischemia Score < 4.
4. Subject is > 50 and <90 years of age.
5. Female subjects must be at least 2 years post-menopausal or surgically sterile.
6. The subject must be proficient in speaking, reading and understanding English in order to 
comply with procedural testing of cognitive function, memory and physiology.
7. Subject has a dedicated family member/study partner, who will be able to attend all visits and 
report on subject’s status.
8. Subject and family member/study partner have both provided fully informed written consent 
prior to participation. In the event that subject is legally unable to provide informed written 
consent due to deterioration in cognitive abilities, fully informed written consent must be 
provided by a legally authorized representative.
9. If AD, subject must have a brain CT or MRI in initial diagnostic workup or subsequent care 
that is compatible with the diagnosis of probable Alzheimer’s disease.
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 evidence of other CNS disorders 
including, but not limited to brain tumor, active subdural hematoma, seizure disorder, 
multiple sclerosis, dementia with Lewy bodies, vascular dementia, corticobasal syndrome, 
progressive supranuclear palsy, Parkinson’s disease, multiple system atrophy, frontotemporal 
dementia, normal pressure hydrocephalus, Huntington’s disease, or Jakob-Creutzfeldt disease 
presenting as dementia.
2. Subject has medical history and/or clinically determined disorders: current B12 deficiency, 
chronic sinusitis or any untreated thyroid disease, significant head trauma, or history of 
difficulty with smell and/or taste prior to AD diagnosis.
3.   Subject has history of any of the following: moderate to severe pulmonary disease, poorly 

controlled congestive heart failure, significant cardiovascular and/or cerebrovascular events 
within previous 6 months, condition known to affect absorption, distribution, metabolism, or 
excretion of drugs such as any hepatic, renal or gastrointestinal disease or any other clinically 
relevant abnormality that inclusion, would pose a safety risk to the subject as determined by 
investigator.
4. Subject has had previous nasal and/or oto-pharyngeal surgery and severe deviated septum
and/or other anomalies.
5. Subject has history of any psychiatric illness, with the exception of major depressive and 
anxiety disorder (according to DSM-IV TR) currently in remission or stable with treatment 
for > 2 years, or any other psychiatric condition that inclusion would pose a safety risk to the 
subject as determined by investigator.  
6. Subject is currently taking any medications, herbals and food supplements that are 
medically/clinically contraindicated as determined by investigator in order to comply with 
procedural testing of cognitive function as well as ensure study safety. See list of prohibited 
medications and compounds.
7.  Subject has undergone a recent change (<1month) in their prescribed acetylcholinesterase 
inhibibitor (e.g. donepezil, rivastigmine, galantamine) or mematine.
8.  Subject has undergone a recent change (<1 month) in their SSRI or anti-depressant 
medication.
9. Subject has current or recent drug or alcohol abuse or dependence as defined by DSM-IV TR.
10. Screening laboratory results that are medically relevant, in which inclusion would pose a 
safety risk to the subject as determined by investigator.
11. The subject has participated in any other research study at least 3 months prior to 
12. The subject has an insulin allergy. 
1. Study assessments and procedures
A summary of study events and procedures is outlined in the Study Visit Table.  Demographic,
Screening, and Baseline Assessments are described in Sections 7.1.1-7.1.2, the Treatment Phase 
is outlined in Sections 7.1.3-7.1.10, and the Safety monitoring are detailed in Section 7.1.11,;
details of neuropsychological assessments are outlined in Section 7.4. Note that all visits are 
non-fasting unless otherwise noted.
7.1.
Demographic and Baseline Assessments
7.1.1. Visit 1; Screening Visit Fasting
The following procedures will be performed at this visit:
x
Obtain written informed consent from study partner and subject (or subject’s legally 
authorized representative) prior to any study related procedures.
x
Collect laboratory samples for screening assessment, for specific tests refer to Table 1.
x
Administer MOCA.
x
Obtain Hachinski Score.
x
Administer Geriatric Depression Scale (GDS).
x
Review Inclusion/Exclusion Criteria.

x
Review medical history, as it pertains to inclusion/exclusion criteria, such as research 
diagnosis, disease severity, and course of AD.
x
Obtain subject’s demographic information (date of birth, gender, race, education, etc.).
x
Obtain details of medications taken over the course of the last 30 days.
x
Complete physical exam, including neurological exam.  Collect vital signs, height and weight 
prior to ECG and blood draw.
x
Perform a standard 12-lead ECG.
7.1.2. Visit 2; Baseline Visit 
The following procedures will be performed at this visit:
x
Review Inclusion/Exclusion Criteria.
x
Collect vital signs and weight.
x
Collect concomitant medication information and record AEs/SAEs.
x
Neuropsychological testing with ADAS-Cog 13 and supplemental cognitive assessments.
x
Administer CDR
x
Administer Columbia-Suicide Severity Rating Scale (C-SSRS).
x
Administer Functional Assessment Questionnaire (FAQ).
x
Administer Alberta Smell Test.
x
Administer sleep measures (Sleep questionnaire and ESS)
x
Study Drug administration training.
x
Hypoglycemia rescue training.
x
Sub-study PET scan. For participating subjects, scan will be obtained within 1 week (± 3 
days).
x
Sub-study LP/ CSF. For participating subjects, procedure will be performed within 1 week (± 
3 days).
7.1.3. Visit 3; Initial Treatment Visit; (Week 0)
The following procedures will be performed at this visit:
x
Review Inclusion/Exclusion Criteria.
x
Collect vital signs and weight.
x
Collect concomitant medication information and record AEs/SAEs.
x
Study Drug administration training.
x
Study drug dispensing /diary.
x
Administer study dose in clinic with subject/study partner.
x
Obtain fingerstick blood glucose pre study dose and post study dose at 15 min, 30 min and 1 
hour.
x
Visit 4 scheduled within 1 week (± 3 days).
7.1.4. Visit 4; Follow-Up Safety Visit; (Week 1)
The following procedures will be performed at this visit:

x
Collect concomitant medication information and record AEs/SAEs.
x
Collect vital signs and weight
x
Obtain laboratory studies.
x
Administer study dose in clinic by subject/ study partner.
x
Obtain fingerstick blood glucose pre study dose and post study dose at 15min, 30 min and 1 
hour. 
x
Study drug administration training. 
x
Study drug dispensing/diary.
x
Visit 5 scheduled within 3 weeks (± 3 days). 
7.1.5. Visit 5; Treatment and Adherence; (Week 4)
The following procedures will be performed at this visit:
x
Collect vital signs, and weight.
x
Collect concomitant medication information and record AEs/SAEs.
x
Review training of study drug administration.
x
Study drug dispensing/diary.
x
Visit 6 will be scheduled within 1 month (± 3 days).
7.1.6. Visit 6; Treatment and Adherence; (Week 8)
Following procedures will be performed at this visit.
x
Collect vital signs and weight 
x
Collect concomitant medication information and record AEs/SAEs.
x
Review training of study drug administration.
x
Study drug dispensing /diary.
x
Visit 7 scheduled within 1 month (± 7 days).
7.1.7. Visit 7; Treatment and Adherence; (Week 12)
Following procedures will be performed at this visit.
x
Collect vital signs and weight.
x
Collect concomitant medication information and record AEs/SAEs.
x
Neuropsychological testing with ADAS-Cog 13 and supplemental cognitive assessments.
x
Administer CDR.
x
Administer FAQ.
x
Administer GDS.
x
Administer C-SSRS.
x
Administer sleep measures (Sleep questionnaire and ESS)
x
Administer Alberta smell test
x
Review training of study drug administration.
x
Study drug dispensing/diary.

x
Visit 8 will be scheduled within 1 month (± 3 days).
7.1.8. Visit 8; Treatment and Adherence (Week 16)
Following procedures will be performed at this visit.
x
Collect vital signs and weight.
x
Collect concomitant medication information and record AEs/SAEs.
x
Review training of study drug administration.
x
Study drug dispensing/diary.
x
Visit 9 will be scheduled within 1 month (± 3 days).
7.1.9. Visit 9; 5-month check-in (Week 20)
Following procedures will be performed at this visit.
x
Collect vital signs and weight.
x
Collect concomitant medication information and record AEs/SAEs.
x
Review training of study drug administration. 
x
Study drug dispensing/diary.
x
Visit 10 will be scheduled within 1 month (± 7 days).
7.1.10. Visit 10; 6 month follow-up (Week 24)
Following procedures will be performed at this visit.
x
Collect vital signs and weight.
x
Collect concomitant medication information and record AEs/SAEs.
x
Neuropsychological testing with ADAS-Cog 13 and supplemental cognitive assessments.
x
Administer CDR.
x
Administer FAQ.
x
Administer GDS.
x
Administer Alberta smell test
x
Administer sleep measures (Sleep questionnaire and ESS)
x
Obtain repeat FDG-PET study in subgroup. Scan will be obtained within 1 week (± 3 days).
x
Obtain repeat spinal fluid analysis in subgroup. Procedure will be performed within 1 week 
(± 3 days).
x
Visit 11 scheduled within 1 month (± 7 days).
7.1.11. Visit 11; Final Safety and Discharge; Fasting (Week 28)
Following procedures will be performed at this visit.
x
Collect vital signs and weight.
x
Complete physical and neurological exam. 

x
Collect concomitant medication information and record AEs/SAEs.
x
Collect laboratory samples, for specific tests refer to in Table 1.
x
Perform a standard 12-lead ECG. 
x
Administer C-SSRS.
7.2.
Early Withdrawal
If subject withdraws from the study after the screening visit, but before visit 3, no further 
evaluations are necessary.  If subject withdraws from the study after visit 3, all safety 
assessments will be performed (see section 7.3). 
7.3.
Safety
For all safety assessments described below, any clinically significant change will be recorded as 
an AE or SAE.
7.3.1. Physical Examination
Complete physical examination will be performed at visits, 1 and 11, 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 and 11, 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 visits.  For within subject consistency, brachial artery pressure 
will be obtained in the routine fashion, the same arm should be used for all study measurements. 
Blood pressure and heart rate to be measured after subject has been sitting quietly for a minimum 
of 5 minutes.  Diastolic blood pressure will be measured at the disappearance of Korotkoff 
sounds. Vitals sign will be monitored by clinical staff during each visit of the study. 
In addition, vital signs will be measured pre and post study dose on Visits 3 and 4. The 
investigator will be notified for any baseline changes in blood pressure >20 mmHg systolic and 
>10mmHg diastolic 
A pre-dose fingerstick blood glucose will be measured followed by measurements post- study 
drug at 15 minute, 30 minute and 1 hour during visit 3 and 4. The investigator will be notified for 
any baseline changes. $EORRGJOXFRVHޒPJG/ will be considered clinically significant.
Vital signs will be taken prior to ECG and blood draw.
7.3.4. Weight
Body weight will be measured at all visits. 

7.3.5. ECG
A standard 12-lead ECG will be performed on subjects at visit 1 and 11 
7.3.6. Laboratory Samples
APOE genotyping procedure is discussed in Section 7.5.
All subjects will be required to fast for a minimum of 12 hours prior to collection of blood
sampling on designated fasting visits.
7.3.6.1 HOMA-IR – Measure for Insulin Resistance
Calculation of HOMA-IR with Blood glucose and insulin level during Visit 1 using the 
following formula(45, 46);
HOMA-IR = [glucose (nmol/L) * insulin (μU/mL)/22.5]
See Table 1 for specific list of laboratory assessments.
Table 1 
Laboratory Assessments
Procedure
Visit 1; *Visit 3;
*Visit 4; 
Visit 11;
Early 
Withdrawal
Basic Metabolic Panel 
(Na, K, CO2, Cl, BUN, 
Creatinine, Glucose and 
Ca)
X
X
X
X
Insulin Level
X
X
X
CBC w/ Diff
X
X
X
Fingerstick Blood Glucose
Baseline, post study dose, 
15 min, 30 min and 1hour
X
X
HbA1c
X
APOE status 
X
Thyroid Stimulating 
Hormone (TSH)
X
Vitamin B12
X
*Blood samples to be collected after study dose is administered.
7.4.
Neuropsychological Assessment
See Table 2 for specific list of neuropsychological Assessment.

Table 2                Neuropsychological Assessment 
Procedure
Screen
Baseline 
Phase
Treatment Phase
Follow-up
Early 
Withdrawal
Visit 1
Visit 2 
Visit  7
Visit 10
Visit 11
FAQ
X
X
X
GDS
X
X
X
X
ADAS-Cog 13
X
X
X
CDR
X
X
X
MOCA
X
C-SSRS
X
X
X
X
Digit span 
forward/backward
X
X
X
Trailmaking Parts 
A&B
X
X
X
COWAT
X
X
X
Animal naming
X
X
X
WMS Logical 
Memory 
X
X
X
AST
X
X
X
Sleep 
Questionnaire
X
X
X
ESS
X
X
X
7.4.1. ADAS-Cog 13 Summary
The ADAS-Cog was developed as an outcome measure for global cognition in clinical trials for 
Alzheimer’s disease. The ADAS-Cog assesses multiple cognitive domains including memory, 
language, praxis, and orientation (47). Overall, the ADAS-Cog has proven successful for its 
intended purpose with higher scores indicating greater cognitive impairment.  The modified 
ADAS-Cog 13-item scale (48) includes all original ADAS-Cog items with the addition of a 
number cancellation task and a delayed free recall task, for a total of 85 points. The purpose of 
these additional items increases the number of cognitive domains and range of symptom severity
to best assess subjects affected by the earlier stages of Alzheimer’s disease (e.g. amnestic MCI 
and mild AD).
7.4.2. Digit Span subtest from the Wechsler Adult Intelligence Scale -4th Edition (WAIS-
IV)
The WAIS-IV Digit Span test is a brief measure of auditory attention, concentration, and 
working memory.  Subjects are read a random list of numbers at a rate of one per second and 
immediately asked to repeat them in a forward sequence, a backward sequence, or to rearrange 
them in numeric order starting with the lowest number.  Digit strings gradually expand and 
become lengthier until the subject makes consistent errors, at which point the test is 
discontinued.  The test yields numerous scores for interpretation.

7.4.3. Controlled Oral Word Association Test (COWAT) 
The COWAT is a short, paper/pencil measure of phonemic fluency and executive functioning 
from the Multilingual Aphasia Examination. 
7.4.4. Trail Making Test, Parts A & B
The Trail Making Test is a brief paper and pencil measure of attention, processing speed, and 
mental flexibility (or set-shifting).  The test has been well validated for use with a wide range of 
subject populations, is easy to administer, and has been shown to be sensitive in detecting subtle 
changes in central nervous system compromise and specifically changes in frontal lobe or 
“executive” functions.  Part A requires subjects to connect a series of numbered dots in order as 
quickly as possible.  Part B requires subjects to connect numbered and lettered dots as quickly as 
possible by alternating back and forth between the two without making mistakes.  Scores include 
both time to completion and various error types.
7.4.5. Montreal Cognitive Assessment (MOCA)
The Montreal Cognitive Assessment (MoCA) was designed as a rapid screening instrument for 
mild cognitive dysfunction. It assesses different cognitive domains: attention and concentration, 
executive functions, memory, language, visuoconstructional skills, conceptual thinking, 
calculations, and orientation. The time to administer the MoCA is approximately 10 minutes. 
The total possible score is 30 points, and scores of 26 or above are considered normal.
7.4.6. Clinical Dementia Rating (CDR) Scale
The CDR is a 5-point scale used to characterize six domains of cognitive and functional 
performance applicable to Alzheimer disease and related dementias: Memory, Orientation, 
Judgment & Problem Solving, Community Affairs, Home & Hobbies, and Personal Care
http://alzheimer.wustl.edu/cdr/aboutcdr.htm. The necessary information to make each rating is 
obtained through a semi-structured interview of the patient and a reliable informant or collateral 
source (e.g., family member). 
The CDR table (http://knightadrc.wustl.edu/cdr/PDFs/CDR_Table.pdf) provides descriptive 
anchors that guide the clinician in making appropriate ratings based on interview data and 
clinical judgment. In addition to ratings for each domain, an overall CDR score may be 
calculated through the use of an algorithm. This score is useful for characterizing and tracking a 
patient's level of impairment/dementia: 
CDR-0.5 = very mild dementia
CDR-1 = mild
CDR-2 = moderate
CDR-3 = severe
In addition, the total CDR ratings for each of the six cognitive/functional domains can be added 
to create a CDR sum of boxes (SOB). 

7.4.7. Columbia-Suicide Severity Rating Scale (C-SSRS)
The Columbia–Suicide Severity Rating Scale (C-SSRS) was designed to quantify the severity of 
suicidal ideation and behavior (49).  In this study, suicidal ideation and behavior will be 
prospectively assessed using the C-SSRS. The C-SSRS will be administered by trained raters at 
specified time points, as indicated in the Visit Flow Chart, as well as at unscheduled visits as 
clinically indicated. Any subjects demonstrating evidence of suicidality will prompt immediate 
consultation with the site’s on-call psychiatrist for assistance with decision-making and potential 
referral to behavioral health services. 
7.4.8. 15-Item Geriatric Depression Scale (GDS) 
The 15-item GDS, a paper, interview-based depression screening tool will be administered to the 
subject and score by a qualified, trained rater. 
7.4.9. The Functional Activities Questionnaires (FAQ) 
The Functional Activities Questionnaire (FAQ) measures instrumental activities of daily living 
(IADLs), such as preparing balanced meals and managing personal finances. Since functional 
changes are noted earlier in the dementia process with IADLs that require a higher cognitive 
ability compared to basic activities of daily living (ADLs) this tool is useful to monitor these 
functional changes over time.
7.4.10. Animal Naming
Animal Naming is a word fluency test measuring executive functioning and language ability. 
The subject is asked to list all of the animals he/she can think of in the next 60 seconds. 
7.4.11. Wechsler Memory Scale Logical Memory
The Wechsler Memory Scale (WMS) is a neuropsychological test designed to measure different 
memory functions in a person. Logical memory represents one of 7 memory subtests that are 
included within the WMS-IV.  Of all the subtests, logical memory is the largest discriminator 
between the normal aging population and mild dementia (50). Subjects are read two thematically 
independent stories and later asked to recall each story immediately and later thirty minutes after 
the reading.  Patients struggling with recall of story contents are provided cues to assist with 
recognition.
7.4.12 Alberta Smell Test
The Alberta Smell Test (AST) is a quick, easy to use method to test the ability to identify smells 
presented to one nostril at a time (51). Over the course of the test, the subject is presented with 8
different odors of the Mr. Sketch scented markers namely, licorice, cinnamon, mint, orange, 
lemon, grape, melon, and raspberry. In 20 trials, the different scented markers are held up to the 
participants’ right or left nostril with their eyes closed. The subjects then select the scent of the 
marker from a list of the 8 scents. Each smell correctly identified will be recorded as a correct 
response with a maximum score of 20. A score of 2/10 or less in either nostril suggests 
impairment.

7.4.13 Sleep Questions
A set of 4 questions will be asked to the caregiver/spouse that looks at areas of sleep quality 
including sleep duration, sleep latency and sleep efficiency for the past month. The questions 
address the patient's usual bedtime and wake-up time, as well as how long it takes to fall asleep 
and how many hours of sleep are obtained per night. The caregiver will answer these questions 
for the subject.
7.4.14 Epworth Sleepiness Scale (ESS)
Epworth sleepiness scale (ESS) is a simple self-administered questionnaire that is widely used to 
measure the daytime sleepiness of an individual (52). It has 8 standardized questions that assess 
the likelihood of a subject dozing off or falling asleep (on a scale of 0-3) in daily living 
situations. A total of 0-24 score can be obtained. Excessive daytime sleepiness is assumed if a 
subject reaches a score of 10 and above. The caregiver will answer these questions for the 
subject.
7.5.
Pharmacogenetics
APOE genotyping will be performed at Visit 1, following informed consent and will be used as 
part of the randomization.  Sample will be tested at an external genetic processing facility, and 
results will be returned to testing site.  The identity of study participants will be withheld from 
outsourced company completing testing.  Samples will be identified by participant initials and a 
number identifier specific to this study.  
8.
INVESTIGATIONAL PRODUCT(S)
8.1.
Description of Investigational Product
The Center for Memory and Aging will utilize the following investigational products:
x
Intranasal delivery
x
Insulin glulisine
x
Placebo saline
x
POD Device
8.2.
Handling and Storage
The study drug (glulisine/saline) will be given to participant/care partner after training for 
administration.
The study drug on site will be kept per label recommendations and institutional Standard 
Operational Policy, specifically, but not limited to temperature controlled secure area.
Participants will be instructed to keep study drug at room temperature.

At investigational site, the study drug will be stored according to manufacturer 
recommendations. To ensure that a stable temperature and/or conditions are maintained, 
refrigerated study drug is electronically monitored for temperature control. .  A log will be 
securely stored at the HealthPartners Center for Memory and Aging. Study staff will be 
responsible for safeguarding and maintaining the master log. In the event of a medical 
emergency requiring a blind break, unblinded study staff will be contacted by a member of the 
investigative team and the appropriate information will be relayed only to those responsible for 
patient’s immediate medical care (e.g. ED physician).
8.3.
Treatment Assignment
Randomization will be stratified by MOCA score (high = 23-27; low = 18-22), presence of non-
insulin dependent diabetes, and APOE4 status a priori by the permuted block method.  The trial 
is a double blinded study so that the subject, the investigator, and the trial coordinator will be 
unaware to which treatment group the subject has been randomized. To facilitate subject 
blinding, all subjects will undergo the same follow-up procedures. One investigative team 
member at the site will be unblinded to randomization and prepare the study dose for each
subject. This person will not have responsibility for obtaining any study data.   An electronic 
database will be used for automated randomization of participants using a table generated by the 
statistician.
8.4.
Packaging and Labeling
Actual pharmacy-product indicator will be decided by pharmacy in order to maintain investigator 
objectivity.
All study drug and placebo will be labeled according to the following specifications:
x
Protocol identifier/IRB approval/account/study number.
x
Participant ID #.
x
“Store at room temperature.”
x
“Last day to use” date.”
x
“Caution: New Drug--Limited by Federal (or United States) law to investigational use”.
x
Study contact # 651-254-0769.
8.5.
Occupational Safety
No known significant safety risks exist to site personnel in direct or indirect contact with the 
study drug.
9.
CONCOMITANT MEDICATIONS AND NON-DRUG THERAPIES
9.1.
Permitted Medications
Any medication not listed in list of Prohibited/Conditional Medications 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.

Table 2: Prohibited Medications
Anticholinergics
Antihistamines (centrally-acting)
Barbiturates
Benzodiazepines
Insulin
Muscle relaxants
Neuroleptics
Opiates
9.2.
Prohibited Medications
Subjects taking prohibited medications at time of screening will not be allowed to participate in 
study, unless such treatment is discontinued within 30 days. These medications include 
anticholinergics, and centrally acting antihistamines (e.g. Benadryl), opiates, benzodiazepines, 
barbiturates, neuroleptics, muscle relaxants, and insulin. For randomized subjects who take a 
prohibited med (episodic or PRN) will be considered for study continuation on an individual 
basis. Randomized subjects who take a prohibited med (long-term or for >7 days per 
concomitant illness/episode) will be withdrawn from study.  Randomized subjects needing to 
initiate a conditional medication or alter dose of conditional medication during study will be 
considered on an individual basis. 
10.
SUBJECT COMPLETION AND WITHDRAWAL
10.1.
Subject Completion
Subjects completing all 11 study visits 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 document the reason(s) for withdrawal.  In the event a subject chooses 
to withdraw from study before Visit 11, the safety procedures described in Section 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 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. All subjects randomized to the study will be included in the analysis as 
part of their assigned study group regardless of treatment completion, as is consistent with the 
intent-to-treat principle. .
Subject’s participation may be terminated at the discretion of the investigator.  Individuals may 
be withdrawn for the following reasons:
x
Clinically significant adverse events.
x
Lost to follow-up.
x
Protocol violations.
x
Inability to tolerate study medication.

x
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:
x
Results in study withdrawal.
x
Is associated with clinical signs or symptoms.
x
Leads to treatment or to further diagnostic tests.
x
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:
x
Death.
x
Life-threatening situation.
x
Hospitalization or prolongation of hospitalization.
x
Disability or incapacitation.
x
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.
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.
12.
DATA ANALYSIS AND STATISTICAL CONSIDERATIONS
12.1.
Analysis Overview
Measures of central tendency and spread will be computed for the study population and 
by treatment group for each outcome variable. These summaries will describe patient 
characteristics, confirm that randomization produced a balanced design, and confirm that 
the planned analyses are appropriate for the distribution of the outcome variables. 

Missing data are expected to be minimal. Therefore, the primary analyses will follow an 
intent-to-treat principal and include all randomized subjects regardless of intervention 
compliance and missing data. We will use linear mixed models, an analytic approach that 
can accommodate unbalanced data and use all available observations in the analysis by 
assuming data are missing at random. Sensitivity analyses will include evaluation of 
missing assumptions using pattern mixture models and multiple imputation and a per-
protocol approach to accommodate subjects who do not adhere to the intervention 
protocol. 
All statistical tests will be conducted in SAS Version 9.4 and will be evaluated using a 
two-sided 0.05 level of significance. 
12.2.
Statistical Analysis of Efficacy Outcomes
Previous research has shown that the primary outcomes (ADAS-Cog 13, CDR-SOB, and 
FAQ) to be highly correlated. Therefore, to increase power and account for this 
correlation, the primary analysis will be a multivariate, linear mixed-effects regression. 
Specifically, the change in cognition and function measures between baseline and week 
12 and between baseline and week 24 will serve as the primary outcome variables. We 
will include intervention arm, intervention week, intervention arm*week, age, and 
baseline test scores as fixed effects. The efficacy of intranasal (IN) insulin on cognition 
and function will be tested on the intervention arm*week interaction. Age and baseline 
test scores are included because of the possibility of unequal baseline values. Random 
intercept effects will be included to account for the nested structure of the data, multiple 
outcomes and observation points. 
First, the assumptions of such a model will be verified. That is, the dependent variables 
will be tested for: (1) multivariate normality, (2) homoscedasticity, (3) equality of 
covariance matrices, and (4) independent observations. These outcome variables have 
been analyzed after log-transformation in previous studies because they have a 
distribution skewed to the right. We will confirm that this transformation is suitable for 
our study. If outcome variables are log-transformed, efficacy measures will correspond to 
percent change of cognitive outcomes. 
To protect against type I error, efficacy of IN insulin will be considered if an overall 
cognitive decline is observed relative to placebo. Efficacy measures for composite 
cognitive and functional decline with corresponding 95% confidence intervals and p-
values will be reported.
For the analysis of secondary cognition and function measures (Digit Span, Trailmaking, 
COWAT, Animal Naming, and WMS), secondary mood measure (GDS), and exploratory 
smell measure (AST) we will follow the same approach as for the primary outcomes. We 
have not included these tests as primary endpoints because they measure more specific 
effects of AD language, memory, mood, or olfaction impairment and, therefore, may be 
less sensitive to overall cognitive change in MCI/AD patients. Each outcome measure 
will be tested in its own linear mixed-effects regression model with measures from 
baseline to week 12 and baseline to week 24 as the outcome variables. The efficacy of IN 

insulin on each measure will be tested on the intervention arm*week interaction. Efficacy 
measures with corresponding 95% confidence intervals and p-values will be reported. 
Exploratory measures of sleep (Sleep Questionnaire and ESS) and biomarkers (CSF 
Abeta, tau, and phosphotau) will follow the same approach as for the primary outcomes. 
Two multivariate linear mixed-effects regression models will be fit with change in either 
sleep or biomarker measures from baseline to visit 7 and baseline to visit 10 as the 
outcome variables. The efficacy of IN insulin on sleep will be tested on the intervention 
arm*week interaction. Efficacy measures for composite sleep decline with corresponding 
95% confidence intervals and p-values will be reported. 
Exploratory PET metabolism will be measured in four cortical ROIs primarily affected 
by AD: precuneus, posterior cingulate, and bilateral parietotemporal. A multivariate 
regression model will again be applied to the imaging outcome measures. Specifically, 
standardized change in posterior cerebral metabolism in the four ROIs from baseline to 6 
months will serve as the outcomes. Intervention arm, age, and baseline test scores will be 
used as fixed effects. Tests of assumptions and interpretation will be similar to that 
described for primary outcomes. Overall cerebral metabolism and metabolism at each of 
the four cortical ROIs efficacy measures with corresponding 95% confidence intervals 
and p-values will be reported.
12.3.
Statistical Analysis of Safety Outcomes
Adverse event rates will be modeled using Poisson regression with a natural logarithm 
link function. If over dispersion is identified, a negative binomial model will be used 
instead. Incidence rate ratios and 95% confidence intervals will be reported. Differences 
in baseline to 3-month and 6-month change in vital signs and 12-lead ECG will be 
assessed using Student’s two-sample t-test.
12.4.
Study Power
A total of n=90 subjects will be randomized to receiving either IN glulisine (n=45) or 
placebo (n=45) two times daily. The study is powered on the three primary outcomes, 
ADAS-Cog 13, CDR-SOB, and FAQ.
Control
Group Mean
Standard 
Deviation
Minimum 
Detectable 
Difference
Power
ADAS-Cog 13
19.2
4.6
3
88.426
CDR-SOB
7.9
1.3
1.5
99.972
FAQ
11.56
3.6
2
74.100
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). 

13.2.
Data Safety Monitoring Board 
The DSMB will be an independent group who are not participating in the trial and have no direct 
affiliation with HealthPartners Center for Memory and Aging. They will serve as an advisory
panel to HealthPartners Center for Memory and Aging. The DSMB will be comprised of an 
independent behavioral neurologist, endocrinologist, and intranasal insulin researcher. The 
DSMB Charter will be established prior to initiation of the study. The DSMB responsibilities 
include but are not limited to the following:
x
Monitoring the study for compliance to the protocol.
x
Stopping the study if the rate of SAE’s raises safety concerns.  The details will be specified 
in the DSMB charter. 
During the course of the trial, the DSMB will review accumulating safety data to monitor for 
incidence of treads that would warrant termination of the trial. The frequency of the DSMB 
meetings, responsibilities, membership, and procedures will be documented in the DSMB 
charter.
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. 
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 (interim analysis) is 
complete.  Study staff will not be unblinded in regards to individual subject’s randomization 
status until after database lock.
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 Accountability 

A Device Tracking Log will be maintained at the investigational site. POD Device will be 
recorded on the log upon delivery to the investigational site and will be stored in a secured area. 
The Device Tracking Log will be updated as each device is delivered, dispensed, returned and 
the reason for the return.  Serial numbers, expiration date and model number of devices delivered 
to the site will also be recorded.  
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