Study of Nasal Insulin to Fight Forgetfulness - Combination Intranasal Insulin and Empagliflozin Trial
Phase: PHASE2
Conditions: Mild Cognitive Impairment, Cognitive Impairment, Alzheimer Disease
Sponsor: Wake Forest University Health Sciences

Official Title:  Study of Nasal Insulin to Fight Forgetfulness - Combination Intranasal Insulin and Empagliflozin Trial  
NCT05081219
IRB Approval Date:  07/14/23

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Study of Nasal Insulin to Fight Forgetfulness (SNIFF)   
SNIFF Combination Intranasal Insulin and Empagliflozin Trial 
PROJECT DIRECTOR / IND SPONSOR 
Suzanne Craft, Ph.D. 
Wake Forest University Health Sciences/ Wake Forest School of Medicine 
Medical Center Boulevard 
Winston-Salem, NC  27157-1207 
suzcraft@wakehealth.edu 
Tel: 336-713-8832 
PROTOCOL VERSION 
Final Protocol v5.0 
July 14, 2023 

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TABLE OF CONTENTS 
1.0       INTRODUCTION ............................................................................................................ 9 
 
Primary Aim .............................................................................................................. 10 
 
Secondary Aim 1 ....................................................................................................... 10 
 
BACKGROUND AND SIGNIFICANCE............................................................................ 10 
 
Rationale for Insulin................................................................................................... 10 
 
Insulin as a Therapeutic Agent .................................................................................. 11 
 
Intranasal Pathways to the CNS ................................................................................ 11 
 
Intranasal Delivery System Devices .......................................................................... 12 
 
PRELIMINARY STUDIES ................................................................................................ 12 
 
Participants ............................................................................................................... 13 
 
Procedures ................................................................................................................ 14 
 
Safety and Compliance ............................................................................................. 14 
 
Statistical Analyses ................................................................................................... 14 
 
Results: Cognitive and Functional Outcome Measures ............................................. 15 
 
Results: AD Biomarkers ............................................................................................ 16 
 
Results: FDG-PET CMRglc ....................................................................................... 16 
 
Safety and Compliance ............................................................................................. 17 
 
Implications ............................................................................................................... 17 
 
PRELIMINARY STUDY 2 ................................................................................................ 17 
 
PRELIMINARY STUDY 3 ................................................................................................ 19 
 
Rationale for Dosage Selection ................................................................................. 20 
 
Rationale for Primary and Secondary Outcome Measures ........................................ 20 
 
Rationale for Design of Trial ...................................................................................... 20 
 
Rationale for Biofluids ............................................................................................... 20 
 
STUDY OVERVIEW ........................................................................................................ 20 
 
Study Population ....................................................................................................... 20 
 
Diagnosis Criteria ...................................................................................................... 20 
 
Inclusion Criteria ....................................................................................................... 21 
 
Exclusion Criteria ...................................................................................................... 21 
 
Recruitment and Retention Strategies ....................................................................... 22 
 
STUDY TIMELINE ........................................................................................................... 22 
 
DESCRIPTION OF STUDY VISITS ................................................................................. 22 
 
Baseline Visit 1 (Visit 2) ............................................................................................. 22 
 
Baseline Visit 2 (Visit 3; one week after Baseline Visit 1) .......................................... 23 
 
Phone Safety/Compliance Assessment 2 .................................................................. 23 
 
Post-treatment Visit 1 (Visit 5) ................................................................................... 23 
 
Post-treatment Visit 2 (Visit 6; one week after Post-treatment Visit 1) ....................... 24 
 
STUDY-SPECIFIC PROCEDURES ................................................................................. 24 
 
Cognitive Evaluation Instruments Administered to the Participant ............................. 24 
 
Clinical and Functional Evaluations ........................................................................... 24 
 STUDY METHODS .......................................................................................................... 25 

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Safety Assessments .................................................................................................. 25 
 
Physical and Neurological Examination ..................................................................... 25 
 
Electrocardiogram (ECG) .......................................................................................... 25 
 
Clinical Laboratory Evaluations ................................................................................. 25 
 
Actigraphy ................................................................................................................. 25 
 
Continuous Glucose Monitoring ................................................................................ 25 
 BIOMARKER STUDIES .................................................................................................. 26 
 
CSF ........................................................................................................................... 26 
 
Blood Collection at Lumbar Puncture Visits ............................................................... 26 
 
Genetic Samples, Storage and Future Use ............................................................... 26 
 STATISTICAL PLAN ....................................................................................................... 26 
 
Power Analyses ........................................................................................................ 26 
 POTENTIAL RISKS ........................................................................................................ 26 
 
Safety of Intranasal Insulin ........................................................................................ 27 
 
Risks associated with use of the Aptar CPS device ................................................... 28 
 
Risks associate with use of the Empatica E4 actigraphy device ................................ 28 
 
Risks associated with use of the Libre Freestyle Pro Glucose Monitoring System ..... 28 
 
Lumbar Puncture ....................................................................................................... 28 
 
Blood Draw ............................................................................................................... 28 
 PERSONNEL REQUIREMENTS ..................................................................................... 28 
 STUDY DRUGS ............................................................................................................... 29 
 
Humulin® R U-100 Insulin ......................................................................................... 29 
 
Randomization .......................................................................................................... 30 
 
Blinding ..................................................................................................................... 30 
 
Study Drug Dispensing .............................................................................................. 30 
 
Intranasal Administration ........................................................................................... 30 
 
Storage ..................................................................................................................... 30 
 
Drug Accountability ................................................................................................... 30 
 ADVERSE EVENTS ........................................................................................................ 30 
 
Definition ................................................................................................................... 30 
 
Following Up on AEs ................................................................................................. 31 
 SERIOUS ADVERSE EVENTS (SAE) ............................................................................. 31 
 
Definition ................................................................................................................... 31 
 
Reporting SAEs ......................................................................................................... 31 
 
IND Safety Reporting ................................................................................................ 31 
 ETHICS & REGULATORY CONSIDERATIONS ............................................................. 31 
 
Ethical Standard ........................................................................................................ 31 
 
Institutional Review Board (IRB) ................................................................................ 32 
 
Informed Consent & HIPAA Authorization ................................................................. 32 
 
Participant Confidentiality | HIPAA ............................................................................ 32 
 GENETIC RESEARCH & STORAGE OF GENETIC MATERIAL .................................... 33 
 
Storage of Biospecimen Samples.............................................................................. 33 
 RISKS AND BENEFITS ASSOCIATED WITH THIS STUDY .......................................... 33 
 
Potential Benefits of the Proposed Research to Human Subjects.............................. 33 
 
Inclusion of Women and Minorities ............................................................................ 33 
 
Inclusion of Children as Participants in Research Involving Human Subjects ............ 33 
 
Data and Safety Monitoring Plan ............................................................................... 33 

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 PUBLICATION POLICY .................................................................................................. 34 
 SHARING OF FINAL RESEARCH DATA ....................................................................... 34 
 TABLE 4: SCHEDULE OF PROCEDURES AND ASSESSMENTS ................................ 35 
 LITERATURE CITED ...................................................................................................... 37 
 
 

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STUDY GLOSSARY 
 
3MSE 
MODIFIED MINI-MENTAL STATUS EXAM 
AΒ 
BETA AMYLOID 
AD 
ALZHEIMER’S DISEASE 
ADAS-COG 
ALZHEIMER’S DISEASE ASSESSMENT SCALE – COGNITIVE 
SUBSCALE 
ADCS 
ALZHEIMER’S DISEASE COOPERATIVE STUDY 
ADCS-ADL 
ALZHEIMER’S DISEASE COOPERATIVE STUDY - ACTIVITIES OF 
DAILY LIVING 
ADEAR 
ALZHEIMER’S DISEASE EDUCATION AND REFERRAL CENTER 
ADNI 
ALZHEIMER’S DISEASE NEUROIMAGING INITIATIVE 
AE 
ADVERSE EVENT 
AMCI 
AMNESTIC MILD COGNITIVE IMPAIRMENT 
ANCOVA 
ANALYSIS OF COVARIANCE 
APOE/APOE4 
APOLIPOPROTEIN (APOE) EPSILON 4 (APOE4) 
BDNF 
BRAIN-DERIVED NEUROTROPHIC FACTOR 
BID | BID 
BIS IN DIE (TWICE A DAY) 
BUN 
BLOOD UREA NITROGEN 
CDR-SB 
CLINICAL DEMENTIA RATING – SUM OF BOXES 
CFR 
CODE OF FEDERAL REGULATIONS 
CMRGLC 
CEREBRAL METABOLIC RATE OF GLUCOSE UTILIZATION 
CNS 
CENTRAL NERVOUS SYSTEM 
CPD 
CONTROLLED PARTICLE DISPERSION 
CPK 
CREATINE PHOSPHOKINASE 
CREB 
CAMP RESPONSE ELEMENT-BINDING PROTEIN 
CRF/E-CRF 
CASE REPORT FORM/ELECTRONIC CASE REPORT FORM  
CSF 
CEREBRAL SPINAL FLUID 
DNA 
DEOXYRIBONUCLEIC ACID 
DSMB 
DATA & SAFETY MONITORING BOARD 
DSM-IV 
DIAGNOSTIC AND STATISTICAL MANUAL OF MENTAL 
DISORDERS, FOURTH EDITION 
DSRS 
DEMENTIA SEVERITY RATING SCALE 
EAG 
ESTIMATED AVERAGE GLUCOSE 
ECG 
ELECTROCARDIOGRAM 
EDC 
ELECTRONIC DATA CAPTURE 

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EDTA 
ETHYLENE DIAMINE TETRA ACETIC ACID 
ELISA 
ENZYME-LINKED IMMUNOSORBENT ASSAY 
EMPA 
EMPAGLIFLOZIN 
FCSRT 
FREE AND CUED SELECTIVE REMINDING TEST  
FDA 
FOOD AND DRUG ADMINISTRATION 
FDG PET 
FLUORO DEOXY GLUCOSE POSITRON EMISSION TOMOGRAPHY 
GCP 
GOOD CLINICAL PRACTICE 
GEE 
GENERALIZED ESTIMATING EQUATION 
GGT 
GAMMA GLUTAMYL TRANSPEPTIDASE 
GSK3Β 
GLYCOGEN SYNTHASE KINASE 3 BETA 
HGA1C 
HEMOGLOBIN A1C 
HC 
HOMOCYSTEINE 
HCT 
HEMATOCRIT 
HCY 
HOMOCYSTEINE 
HEENT 
HEAD | EARS | EYES | NOSE | THROAT 
HGB 
HEMOGLOBIN 
HIPAA 
HEALTH INSURANCE PORTABILITY AND ACCOUNTABILITY ACT 
HOMA-IR 
HOMEOSTATIS MODEL ASSESSMENT OF INSULIN RESISTANCE 
ICF 
INFORMED CONSENT FORM 
ICH 
INTERNATIONAL CONFERENCE ON HARMONISATION 
IDE 
INSULIN DEGRADING ENZYME 
IGF-1 
INSULIN-LIKE GROWTH FACTOR-1 
INI 
INTRANASAL INSULIN  
IRB 
INSTITUTIONAL REVIEW BOARD 
ITT 
INTENT-TO-TREAT 
LDH 
LACTATE DEHYDROGENASE 
LP 
LUMBAR PUNCTURE 
LTP 
LONG TERM POTENTIATION 
MCV 
MEAN CORPUSCULAR VOLUME 
ML 
MILLILITER 
MMA 
METHYLMALONIC ACID 
MMSE 
MINI MENTAL STATE EXAMINATION 
MPRAGE 
MAGNETIZATION PREPARED RAPID GRADIENT ECHO 
MR/MRI 
MAGNETIC RESONANCE / MAGNETIC RESONANCE IMAGING 

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NBAC 
NATIONAL BIOETHICS ADVISORY COMMISSION 
NIA 
NATIONAL INSTITUTE ON AGING 
NIH 
NATIONAL INSTITUTES OF HEALTH 
NINCDS/ADRDA 
NATIONAL INSTITUTE OF NEUROLOGICAL AND COMMUNICATIVE 
DISEASES AND STROKE / ALZHEIMER'S DISEASE AND RELATED 
DISORDERS ASSOCIATION 
NBDA 
N-METHYL – D-ASPARTATE 
NSAID 
NON-STEROIDAL ANTI-INFLAMMATORY DRUGS 
OHRP 
OFFICE FOR HUMAN RESEARCH PROTECTIONS 
PBMC 
PERIPHERAL BLOOD MONONUCLEAR CELL 
PD 
PROJECT DIRECTOR  
PCP 
PRIMARY CARE PHYSICIAN 
PET 
POSITRON EMISSION TOMOGRAPHY 
PHI 
PROTECTED HEALTH INFORMATION 
PI 
PRINCIPAL INVESTIGATOR 
PID 
PARTICIPANT ID  
RBC 
RED BLOOD CELL 
RE 
RANDOM EFFECTS 
ROI 
REGIONS OF INTEREST 
SAE 
SEVERE ADVERSE EVENT 
SD 
STANDARD DEVIATION 
SGOT 
SERUM GLUTAMIC OXALOACETIC TRANSAMINASE 
SGPT 
SERUM GLUTAMIC PYRUVIC TRANSAMINASE 
T 
TESLA 
TSH 
THYROID STIMULATING HORMONE 
U/A 
URINALYSIS 
WBC 
WHITE BLOOD COUNT 
 
 
 

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TITLE  
 
 
Study of Nasal Insulin to Fight Forgetfulness “SNIFF” – 
Combination Intranasal Insulin and Empagliflozin Trial 
PROJECT DIRECTOR 
Suzanne Craft, Ph.D. 
STUDY SPONSOR 
Wake Forest University Health Sciences (WFUHS) / 
Alzheimer’s Disease Research Center (ADRC) 
STUDY PHASE  
 
 
Phase II Pilot Study 
INDICATION 
 
 
 
Preclinical AD, amnestic mild cognitive impairment (aMCI) or 
mild Alzheimer’s disease (AD) 
AIM OF STUDY 
The proposed pilot study will provide safety and efficacy 
preliminary data regarding singular and combined effects of two 
therapeutic approaches, intranasal insulin and treatment with 
the sodium-glucose cotransporter type 2 inhibitor (SGLT2i) 
empagliflozin, to correct bioenergetic and vascular dysfunction 
in adults with preclinical AD and amnestic mild cognitive 
impairment (aMCI) or early AD 
PRIMARY OBJECTIVE 
We will test the hypothesis that 1 month of treatment with INI 
(40 IU q.i.d.), with the SGLT2i empagliflozin (empa; 10 mg 
q.d.), and with combined insulin plus empagliflozin (INI+empa) 
will result in safety profiles (number of treatment-related serious 
adverse events/primary outcome) that are similar to placebo.  
SECONDARY OBJECTIVES 
We will determine the effects of singular and combined therapy 
on AD cerebrospinal fluid biomarkers, cerebral blood flow 
(CBF) and cognition.  
PRIMARY OUTCOME MEASURE 
Number of treatment-related serious adverse events 
SECONDARY OUTCOME MEASURES  PACC5, ADAS-Cog14, CSF Aβ40, Aβ42, total tau, and 
phospho-tau 181, whole brain CBF 
STUDY DESIGN  
Single-site, double-blind study  
SAMPLE SIZE  
n=60 
SUMMARY OF KEY ELIGIBILITY 
CRITERIA  
 
• 
Cognitively normal with positive amyloid (amyloid PET 
SUVR>1.21, CSF Aβ42<600 pg/ml) 
• 
 Diagnosis of amnestic mild cognitive impairment (MCI) or 
early AD (CDR 0.5 or 1, MMSE>21)  
• 
Age: 55 to 85 years (inclusive) 
DRUG DOSAGE & FORMULATION 
40 International Units Humulin® R U-100 (Eli Lilly, Inc.) q.i.d.; 
empagliflozin (10 mg q.d.) 
DURATION OF PARTICIPATION 
8 weeks (4 weeks on active treatment) 
PLACEBO 
Insulin diluent (Eli Lilly, Inc.); cellulose capsule 

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ROUTE OF ADMINISTRATION 
 
Intranasal (insulin); oral (empagliflozin) 
PROCEDURES  
Physical and neurological exam, nasal examination, cognitive 
testing, magnetic resonance imaging (MRI), continuous glucose 
monitoring, actigraphic sleep assessment, lumbar puncture, 
vitals, clinical labs, CSF and blood analysis and banking and 
APOE genotyping. 
 
1.0       INTRODUCTION  
An urgent need exists to find effective treatments for AD that can arrest or reverse the disease at its 
earliest stages. The emotional and financial burden of AD to patients, family members, and society is 
enormous, and is predicted to grow exponentially as the median population age increases. Current 
FDA-approved therapies are modestly effective at best. The proposed study will examine singular and 
combined effects of two novel therapeutic approaches to correct bioenergetic and vascular 
dysfunction in adults with amnestic mild cognitive impairment (aMCI). Metabolic and vascular 
disorders are well-known risk factors for Alzheimer’s disease (AD); although the mechanisms through 
which they influence risk is not completely understood, accruing research has identified numerous 
potential contributors, including insulin resistance, bioenergetic defects, inflammation, impaired 
vascular function, and reduced clearance of protein aggregates, all of which have cumulative adverse 
effects on brain function with aging. Based on this evidence, it has been suggested that medications 
used to treat metabolic and vascular function could be repurposed as therapies for AD. 
Sodium-glucose co-transporter-2 inhibitors (SGLT2is) belong to a new class of agents that has 
attracted considerable interest because of concomitant effects on metabolism and vascular function in 
adults with Type 2 diabetes and/or cardiovascular disease. In the periphery, SGLT2is reduce 
hyperglycemia through off-loading of glucose into urine, and modulate sodium handling leading to 
reduced blood pressure and improved cardiovascular function. They have been tested in numerous 
large trials and shown to be safe; because they do not increase insulin, they can be administered 
without risk of hypoglycemia in non-diabetic adults. SGLT2 has been identified in brain, as well as in 
choroid plexus epithelial cells where it may play a role in cerebrospinal fluid (CSF) production and 
brain glymphatics. SGLT2 expression in brain is increased with traumatic brain injury and cerebral 
ischemia. Preclinical studies indicate effects of SGLTis in the brain that are of relevance to AD, 
including a shift from glycolysis to fatty acid metabolism, thereby providing an alternative energy 
substrate, enhanced mitochondrial function, decreased oxidative stress, and inhibition of the mTOR 
signaling pathway. In an AD mouse model, the SGLT2 inhibitor empagliflozin reduced amyloid 
plaques, inflammation, and microhemorrhages, while enhancing performance on memory measures.  
Another approach to correcting brain metabolic and vascular dysregulation in AD involves direct 
provision of insulin to the brain via intranasal insulin (INI) administration using devices specialized for 
nose-to-brain delivery. In pilot and Phase II trials this approach improved cerebral glucose utilization, 
AD CSF biomarker profiles, white matter hyperintensity volume progression, and cognition. Although 
a recent Phase II trial showed improvement with only one of two devices, in subsequent pilot studies 
we have determined that this failure may have been due in part to different methods of delivery used 
by the two devices, requiring different dosing strategies, and have validated a dosing protocol that 
verifies reliable delivery of insulin to the CNS.  
SGLT2is and insulin exert beneficial metabolic and vascular effects in brain through different 
mechanisms. SGLT2i effects are not directly related to insulin, although through promoting a switch to 
fatty acid metabolism, peripheral hyperinsulinemia is reduced with corresponding decrease in insulin 
resistance. In contrast, insulin likely works directly through its receptors which are prolific throughout 
brain to promote synaptic viability, neurogenesis, and vasoreactivity, with downstream amelioration of 
amyloid and tau pathology. Thus, although each approach could be predicted to benefit AD symptoms 
and pathology, it is possible that additive or synergistic effects would be observed by combining the 

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two approaches. In large trials, insulin and SGLT2is have been co-administered with no adverse 
events. Further, because intranasal insulin does not lower blood glucose, and SGLT2is lower blood 
glucose only in the presence of hyperglycemia, and are not associated with hypoglycemia, the 
combined regimen is likely to be safe.  This hypothesis will be tested in the proposed proof of concept 
study which will examine whether the safety profiles of 4 weeks of combined and singular 
administration of intranasal insulin and empagliflozin compared with placebo. We will also examine 
effects of treatment on cognition, CSF biomarkers, and cerebral perfusion. If successful, information 
gained from the study will inform the design of a future Phase II trial.  
 
 
Primary Aim 
We will test the hypothesis that 4 months of treatment with INI (40 IU q.i.d.), with the SGLT2i 
empagliflozin (empa; 10 mg q.d.), and with combined insulin plus empagliflozin (INI+empa) will result 
in safety profiles (number of treatment-related serious adverse events/primary outcome) that are 
similar to placebo.  
 
 
Secondary Aim 1 
We will determine the effects of singular and combined therapy on AD cerebrospinal fluid biomarkers, 
cerebral blood flow (CBF) and cognition. 
 
 
 
BACKGROUND AND SIGNIFICANCE 
 
 
Rationale for Insulin 
The rationale for the study is derived from growing evidence that insulin carries out multiple functions 
in the brain, and that insulin dysregulation may contribute to AD pathogenesis (Craft and Watson 
2004). Insulin receptors are densely localized in the hippocampus and in entorhinal, frontal, and other 
cortical areas; they are found primarily in synapses, where insulin signaling modulates 
synaptogenesis and synaptic remodeling (Chiu, Chen et al. 2008, Zhao and Townsend 2009). Insulin 
facilitates memory at optimal levels, possibly through synaptic effects and enhanced hippocampal 
glucose utilization (Grillo, Piroli et al. 2009).  
 
The importance of insulin in normal brain function is underscored by evidence that insulin 
dysregulation contributes to the pathophysiology of AD, a disorder characterized in its earliest stages 
by synaptic loss and memory impairment. Hoyer and colleagues first identified a reduction in insulin 
receptors and signaling markers in the AD brain (Frolich, Blum-Degen et al. 1998). This initial finding 
has been confirmed and extended by other investigators, who have demonstrated reduced CSF 
insulin in patients with AD and MCI (Craft, Peskind et al. 1998, Gil-Bea, Solas et al. 2010), and 
reduced insulin and IGF-I messaging with increasing AD pathology and cholinergic deficit (Rivera, 
Goldin et al. 2005). Insulin has a close relationship with β-amyloid, the toxic peptide produced by 
cleavage of the amyloid precursor protein (Zhao and Townsend 2009). In AD, insoluble Aβ peptides 
deposit in brain parenchyma and vasculature. Soluble Aβ species, particularly oligomers of the 42 
amino acid specie (Aβ42), have synaptotoxic effects, possibly resulting in synapse loss, which is the 
earliest structural defect observed in AD (Selkoe 2008). Insulin reduces oligomer formation and 
protects against Aβ-induced synaptotoxicty and LTP disruption (Gasparini, Gouras et al. 2001, De 
Felice, Vieira et al. 2009, Lee, Kuo et al. 2009). Interestingly, Aβ also regulates brain insulin signaling. 
Soluble Aβ binds to the insulin receptor and disrupts insulin signaling and LTP induction in mouse 
hippocampal slice preparations (Townsend, Mehta et al. 2007). These effects could be prevented by 
exposing tissue to insulin prior to Aβ exposure. Insulin pre-treatment also prevented synthetic soluble 
Aβ oligomers from downregulating plasma membrane insulin receptors and reducing dendritic spines 
in primary hippocampal neurons (De Felice, Vieira et al. 2009). Insulin may also modulate Aβ 
degradation by regulating expression of insulin degrading enzyme (IDE), a metalloprotease that 
catabolizes insulin (Zhao, Teter et al. 2004). Collectively, these findings suggest that soluble Aβ may 

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induce central nervous system (CNS) insulin resistance and synapse loss, and that treatment with 
insulin may prevent these pathological processes. 
 
A role for insulin has also been suggested for other AD-related mechanisms. Insulin inhibits 
phosphorylation of tau, through its regulation of glycogen synthase kinase 3β, a downstream target in 
the insulin signaling pathway (Hong and Lee 1997). Insulin dysregulation is also associated with 
oxidative stress, inflammation, and impaired neurogenesis (Craft and Watson 2004). Thus, insulin has 
been implicated in numerous processes related to AD pathophysiology, suggesting that correction of 
insulin dysregulation may be a therapeutic strategy with considerable clinical and scientific 
significance. 
 
 
Insulin as a Therapeutic Agent 
The study uses insulin as a therapeutic agent and intranasal administration focusing on nose to brain 
transport as a mode of delivery. As reviewed above, insulin has pleiotropic effects on pathways 
implicated in AD pathogenesis. As such, augmenting CNS insulin is an alternative approach to AD 
therapy, in contrast to the majority of therapeutic approaches that focus on narrowly defined 
mechanisms such as acetylcholine modulation or amyloid accumulation. Restoring normal brain 
insulin levels in persons with AD may improve cognition and AD pathologic processes. Such an 
approach is possible with an intranasal administration technique. 
 
 
Intranasal Pathways to the CNS  
Olfactory sensory neurons are directly exposed to the external environment in the upper nasal cavity 
while their axons extend through the cribriform plate to the olfactory bulb. Following intranasal 
administration, drugs can be directly transported to the CNS, bypassing the periphery. Several 
extraneuronal and intraneuronal pathways from the nasal cavity to the CNS are possible. The 
extraneuronal pathways appear to rely on bulk flow transport through perineural channels to the brain 
or CSF. In recent studies, labeled INI or a closely related peptide, insulin-like growth factor-I (IGF-I), 
were administered to rodents (Thorne, Pronk et al. 2004, Francis, Martinez et al. 2008). Within 30 
minutes, signal was detected along olfactory and trigeminal channels, as well as in the hippocampus, 
amygdala and rostral and caudal cortex. An additional extracellular pathway was identified with quick 
access to the CSF after absorption into the submucosa along the olfactory nerve and cribriform plate 
(Born, Lange et al. 2002, Frey 2002, Thorne, Pronk et al. 2004). These extracellular pathways provide 
direct access to the CNS within minutes of intranasal administration. Additionally, an intraneuronal 
pathway delivers drugs to the CNS hours or days later (Broadwell and Balin 1985, Shipley 1985, 
Baker and Spencer 1986, Balin, Broadwell et al. 1986). Viruses and microorganisms (Fairbrother and 
Hurst 1930, Faber 1938, Bodian and Howe 1941), amino acids (Weiss and Holland 1967), and 
proteins (Kristensson and Olsson 1971, Shipley 1985, Thorne, Emory et al. 1995) can also enter the 
CNS via nasal routes. In particular, substances with lower molecular weights are more likely to be 
transported to the CNS along intranasal pathways (Sakane, Akizuki et al. 1995). Insulin’s molecular 
weight of about 5800 g/mol makes it a good candidate for intranasal delivery. Animal studies show 
labeled uptake to hippocampus and rostral and caudal cortex following INI administration (Francis, 
Martinez et al. 2008). In a murine diabetes model, INI reduced brain atrophy, while increasing 
synaptic markers and activation of Akt, CREB, and GSK3β. Memory enhancement was also observed 
on Water Maze and radial arm tasks (Francis, Martinez et al. 2008). Human functional and cognitive 
studies of INI also support insulin’s transport to the CNS. INI treatment increases CSF insulin levels 
and induces changes in auditory-evoked brain potentials compared to placebo (Kern, Born et al. 
1999). INI improves verbal memory acutely in persons with AD and aMCI without affecting plasma 
insulin or glucose levels at the dose included in the study (Reger, Watson et al. 2006). Regarding 
chronic effects, several studies reported that 2 months of daily insulin administration (160 International 
Units/day) significantly improves verbal memory in young healthy adults (Benedict, Hallschmid et al. 
2004, Stockhorst, de Fries et al. 2004, Benedict, Kern et al. 2008, Hallschmid, Benedict et al. 2008, 
Stockhorst, de Fries et al. Submitted for publication). Finally, Section 3.0 presents results in a 

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preliminary study in which insulin was administered to adults with AD or aMCI for 4 months. 
 
 
Intranasal Delivery System Devices 
The intranasal delivery device to be used in this study has been developed and extensively tested by 
Aptar Pharmaceuticals, a company specializing in drug delivery. Specifications and instructions for the 
device are provided in attached materials:  
 
1) The CPS pump is a multidose device utilizing an air-filtered preservative free system, in which 
a .2 micrometer filter membrane filters the airflow and insulin will be delivered through a spring 
loaded tip. This device is currently used in the delivery of Esketamine, a Janssen product 
approved by the FDA for the treatment of refractory depression. 
 
2.5  
Rationale for Sodium-Glucose Cotransporter Type 2 Inhibitors for the Treatment of 
Alzheimer’s Disease 
 
As noted previously, metabolic and vascular disorders are well-known risk factors for Alzheimer’s 
disease (AD); although the mechanisms through which they influence risk is not completely 
understood, accruing research has identified numerous potential contributors, including insulin 
resistance, bioenergetic defects, inflammation, impaired vascular function, and reduced clearance of 
protein aggregates, all of which have cumulative adverse effects on brain function with aging. Based 
on this evidence, it has been suggested that medications used to treat metabolic and vascular 
function could be repurposed as therapies for AD. 
Sodium-glucose co-transporter-2 inhibitors (SGLT2is) belong to a new class of agents that has 
attracted considerable interest because of concomitant effects on metabolism and vascular function in 
adults with Type 2 diabetes and/or cardiovascular disease. In the periphery, SGLT2is reduce 
hyperglycemia through off-loading of glucose into urine, and modulate sodium handling leading to 
reduced blood pressure and improved cardiovascular function. They have been tested in numerous 
large trials and shown to be safe; because they do not increase insulin or lower glucose except in the 
context of hyperglycemia, they can be administered without risk of hypoglycemia in non-diabetic 
adults. SGLT2 has been identified in brain, as well as in choroid plexus epithelial cells where it may 
play a role in cerebrospinal fluid (CSF) production and brain glymphatics. SGLT2 expression in brain 
is increased with traumatic brain injury and cerebral ischemia. Preclinical studies indicate effects of 
SGLTis in the brain that are of relevance to AD, including a shift from glycolysis to fatty acid 
metabolism, thereby providing an alternative energy substrate, enhanced mitochondrial function, 
decreased oxidative stress, and inhibition of the mTOR signaling pathway. In an AD mouse model, 
the SGLT2 inhibitor empagliflozin reduced amyloid plaques, inflammation, and microhemorrhages, 
while enhancing performance on memory measures. 
 
Empagliflozin has been tested in large trials of patients with Type 2 diabetes and cardiovascular 
disease (i.e., EMPA-REG OUTCOME) and like other SGLT2is has been shown to have an excellent 
safety profile (as reviewed in Scheen, Nature, 2020), improving glucose metabolism and reducing the 
incidence of major cardiovascular events. Adverse events that occur infrequently include diabetic 
ketoacidosis and genital infection. Medical monitoring for the study will be carried out by an 
experienced diabetologist, Don McClain, MD, and by a neurologist expert in care of adults with 
Alzheimer’s disease, James Trey Bateman, MD, MPH.  
 
 
PRELIMINARY STUDIES 
A preliminary study examined the impact of 4-month INI administration using the ViaNase device (10 
or 20 International Units bid vs. placebo) on the primary outcome measures of delayed story recall 
and the Dementia Severity Rating Scale (DSRS) as well as on measures of global cognition and 
function used in traditional AD clinical trials. In a subset of participants, effects on CSF AD biomarkers 

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(Aβ42 and tau/Aβ42 ratio), and on cerebral metabolic rate of glucose utilization (CMRglc) assessed 
by F18FDG PET were also examined (Craft, Baker et al. 2012). 
 
 
Participants 
A total of 111 older adults were 
randomized in the study (Fig. 1). Data 
from 104 participants were analyzed. 
 
Sample composition (aMCI, n=64; AD 
with MMSE >15, n=40), size, and 
diagnostic criteria were based on a 
previous study (Reger, Watson et al. 
2008). Forty participants (15 placebo, 
13 low dose insulin and 12 high dose 
insulin) completed the PET sub-study, 
and 23 participants (n=8 placebo and 
15 insulin) completed the LP sub-study. 
Diagnoses were determined by expert 
physician and neuropsychologist 
consensus. Participants, caregivers, 
and all personnel involved in data 
collection were blinded to treatment 
assignment. Groups did not differ in education, body mass index, MMSE, gender, diagnosis, 
cholinesterase inhibitor treatment, or apolipoprotein E (APOE) ε4 allele carriage (Table 1).  
 
Table 1 | Participant Characteristics 
 
 
Participants in the high dose insulin group were younger than placebo-assigned participants (p=0.02), 
whereas no differences were observed between placebo and low dose insulin groups. Age was 
included as a covariate in all analyses. 
Figure 1 | Trial Enrollment Flow 

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Procedures 
Participants were randomized to receive 10 International Units INI bid for a total daily dose of 20 
International Units INI (n=36), 20 International Units INI bid for a total dose of 40 International Units 
(n=38) or placebo (saline bid, n=30) for 4 months. Participants were stratified by APOE-ε4 carriage. 
Saline or insulin (Novolin R, Novo) was administered after breakfast and dinner with ViaNase™, an 
intranasal delivery system (Craft, Baker et al. 2012). Parallel versions of the cognitive protocol were 
administered at baseline, and months 2 and 4 of treatment. Co-primary outcome measures were 
delayed story recall and the DSRS which had previously demonstrated beneficial effects of insulin 
(Reger, Watson et al. 2008). Secondary measures included the AD Assessment Scale for Cognition 
(ADAS-Cog) (Reger, Watson et al. 2008), a test comprised of measures of memory, orientation, and 
language, with higher scores reflecting impairment ranging from 0 (best) to 70 (worst), and the ADCS-
Activities of Daily Living scale (ADCS-ADL) (Galasko, Bennett et al. 1997). Baseline and post-
treatment fasting CSF was analyzed for Aβ42 and tau with multi-parameter bead-based immunoassay 
INNO-BIA AlzBio3 (Innogenetics NV). Resting PET images were obtained using a GE Advance PET 
scanner (GE Medical Systems, Milwaukee, WI) using a previously described protocol (Baker, Cross et 
al. 2011). 
 
 
Safety and Compliance 
Support persons supervised participants’ intranasal administration. Blood glucose was measured daily 
for the first week and then weekly; no group changes were observed over the course of the study 
(Craft, Baker et al. 2012). Compliance was monitored by quantifying unused drug. Safety data were 
reviewed semi-annually by a Data and Safety Monitoring Board. Adverse event reporting followed 
standard guidelines. 
 
 
Statistical Analyses 
For the intent-to-treat (ITT) sample, co-primary (delayed story recall and DSRS) and secondary 
(ADAS-Cog and ADAS-ADL) scores were log transformed. Scores were first subjected to mixed 
model repeated ANCOVA including all treatment groups (placebo, low dose insulin or high dose 
insulin) as the between subjects factor, and time (baseline, month 2, month 4) as the repeated factor 
using the SAS v9.2 General Linear Models procedure. After a significant (p<0.05) time by treatment 
group interaction reflecting a different pattern of change, each of the two insulin groups was compared 
separately with the placebo group using repeated measures ANCOVAs. Effect sizes (Cohen’s f2) 
were calculated for all significant effects. Age was included as a covariate in all analyses. Diagnosis 
(aMCI or AD), gender, APOE-ε4 carriage status (yes or no), baseline 3MSE score, and years of 
education were also included as covariates. Non-significant covariates were dropped from the model. 
Significant relationships with covariates were explored with Pearson correlation (continuous variables) 
or follow-up ANOVAs (class variables). Missing values were treated with multiple imputation (Rubin 
1987). For exploratory CSF biomarker analyses, because only a subset of participants elected to 
undergo LP and no differences were observed between the two insulin dose arms, the groups were 
combined into a single insulin-treated group to maximize power. Biomarkers were analyzed with the 
repeated ANCOVA strategy described above and, due to the small sample size, exploratory 
Spearman correlations were conducted to examine relationships among changes in biomarkers and 
outcome measures. Only study completers underwent post-treatment FDG-PET. Pre and post 
treatment scans were co-registered within subject and anatomically standardized to Talairach and 
Tournoux stereotactic coordinates (Talairach and Tournoux 1988, Minoshima, Koeppe et al. 1994). 
Pixel intensity was normalized to pontine values (Minoshima, Frey et al. 1995). Interval regional 
CMRglc changes within groups were assessed using voxel-wise one-sample t statistics (pre-/post-
treatment pair) and probability integral conversion to z scores (Worsley, Evans et al. 1992). Interval 
changes in regional CMRglc were then compared between 1) low insulin vs. placebo groups, and 2) 
high insulin vs. placebo groups. Based on the number of voxels and smoothness of the statistical 
map, a Type I error rate was controlled at 0.05 to account for multiple comparisons (Worsley, Evans 
et al. 1992). The resulting statistical maps were visualized in three-dimensional stereotactic surface 

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projections. 
 
 
Results: Cognitive and Functional Outcome Measures 
The three groups did not differ at baseline on any outcome measure; change from baseline is 
represented in figures for ease of interpretation. A significant overall treatment group by time 
interaction was observed for primary outcome delayed story recall (p=0.005). Compared to placebo, 
the low dose group had improved delayed recall (Fig. 2A; treatment by time p=0.02, 
Cohen’s f2=0.36), whereas no effect was observed for the high dose group. Exploratory post-hoc 
analyses were then conducted to more closely examine the relationship of insulin dose to story recall, 
as this was a primary goal of this pilot clinical trial. Given findings that delayed recall may not be a 
sensitive measure for AD subjects due to increased variability and floor effects (Sano, Raman et al. 
2011) we constructed a total story recall score (immediate and delayed), which showed improvement 
for the high-dose group (time by treatment interaction p<0.05, mean log total story recall change score 
with SEM = -.15(.1) for placebo vs.12 (.09) for the high dose group). A significant overall treatment by 
time interaction was observed for the other primary outcome measure, the DSRS (p=0.008). 
Compared with placebo, DSRS scores were preserved for both low and high dose groups (Fig.2B; 
treatment by time ps=0.01 and 0.01, Cohen’s f2=0.38 and 0.41). For secondary measures, significant 
effects were observed for the ADAS-Cog (overall treatment by time interaction p=0.004). Both low and 
high insulin groups had less decline in cognition compared with placebo (Fig. 2C; treatment by time 
ps=0.04 and p=0.002, Cohen’s f2=0.27 and .40). Treatment effects on the ADAS-Cog interacted with 
age; for the high dose insulin group, greater improvement (lowered score) tended to be associated 
with younger age (r=.31, p=0.06). For the ADCS-ADL, no overall effects of treatment on daily function 
were observed. However, a significant interaction with diagnosis was observed for this measure 
(overall treatment by time by diagnosis interaction p=0.02). Participants with AD receiving either dose 
of insulin had preserved function compared with placebo-assigned participants with AD who showed 
slight decline, whereas participants with aMCI showed no change regardless of treatment assignment 
(interactions for the participants with AD in low and high dose groups compared with placebo, ps=0.01 
and 0.02, Cohen’s f=0.45 and 0.43; Fig. 2D). Adjustment for APOE-ε4 status, baseline MMSE score, 
cholinesterase inhibitor treatment, gender, and education did not affect the pattern of any result. 
Figure 2 | Change (Month 4-baseline) in log scores for (A) delayed story recall, (B) DSRS, (C) 
ADAS-Cog and (D) ADCS-ADL. 
 
 
 
 

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Results: AD Biomarkers  
CSF Aβ42, Aβ40 and tau did not change for the placebo or insulin-treated groups as a whole. In 
exploratory analyses, however, for insulin-treated participants, increased CSF Aβ42 concentrations 
were associated with improved delayed story recall and ADCS-ADL scores, whereas decreased Aβ42 
was associated with worse performance (Spearman rhos=.59, p=0.02 and .60, p=0.02). Similarly, 
decreased tau/Aβ42 ratios over the 4-month study period correlated with improved delayed story 
recall and better daily function on both ADAS-ADL and DSRS for insulin-treated participants 
(Spearman rhos=-.52, p=0.05, -.50, p=0.07, and .53, p=0.05). No significant correlations were 
observed for the placebo group. 
 
 
Results: FDG-PET CMRglc 
Compared with placebo-assigned participants, the lower dose insulin group showed reduced 
progression of hypometabolism in bilateral frontal, right temporal, bilateral occipital, and right 
precuneus and cuneus regions over the 4-month treatment period (Fig. 3 and Table 2). The higher 
dose insulin group showed even greater treatment effects (higher Z scores) indicating less 
hypometabolism progression in most regions and in left parietal cortex. 
 
Figure 3 | Areas of hypometabolism at baseline (scan 1) and month 4 (scan 2), along with 
changes in hypometabolism (time 2-time 1) within each group and differences in change 
between placebo and low or high insulin groups (nasal insulin-placebo).  Hotter floors indicate 
areas of greater hypometabolism from time 1 to time 2, and from placebo to insulin groups. 
 
 
Table 2 | Z scores and stereotactic coordinates for areas of reduced progression for low and 
high does insulin groups compared to placebo 
 

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Safety and Compliance 
No treatment-related serious adverse events (SAEs) occurred during the study; most adverse events 
(AEs) were minor, such as mild rhinitis. AEs with an occurrence of >5% in any group are listed in 
Table 3. The total AE mean was higher for the low dose group compared with placebo (low dose 
mean total AEs with standard error=1.44±0.20, placebo =0.80±.22, p=0.04), with a similar trend for 
the high dose and placebo group comparison (high dose mean total AEs =1.21±0.16, placebo 
=0.80±.22, p=0.10). Mean compliance (number of completed doses) ranged from 95-97% and did not 
differ across groups. 
Table 3 | Total number of adverse events and percent of sample for all events occurring for at 
least 5% of the participants in any treatment group. 
 
 
 
 Implications 
These results suggest that adults with aMCI or AD may benefit from INI treatment. Compared with 
placebo, the lower dose of insulin improved delayed memory, and both insulin doses preserved 
caregiver-rated ability to carry out daily functions. General cognition as assessed with the ADAS-Cog, 
the primary outcome measure for the current trial, was also preserved by both doses of INI. In 
exploratory analyses, changes in CSF Aβ42 and tau/Aβ42 ratios were associated with cognitive and 
functional changes for insulin-treated participants. Placebo-assigned participants showed decreased 
CMRglc values in frontal, temporal, and parietal cortices as well as precuneus and cuneus over the 4-
month period, whereas insulin-treated participants showed no decline. The longstanding FDG PET 
finding of posterior cingulate, precuneus and cuneus hypometabolism in AD has been hypothesized to 
be due to functional disconnection of the hippocampal formation, so enhanced metabolism and 
memory with INI may reflect enhanced hippocampal input to this region. Similarly there are strong 
connections between the posterior cingulate, precuneus, cuneus and prefrontal and superior temporal 
cortex (Cavanna and Trimble 2006), which may also be affected by INI. Finally, no treatment-related 
SAEs occurred. These promising results provide a strong rationale for the longer, larger, multi-site trial 
proposed in this application. 
 
 
PRELIMINARY STUDY 2 
A follow-up study has recently concluded and results have been published (Craft et al. JAMA 
Neurology, 2020). 
 
Objectives: This study tested the effects of 40 International Units of intranasal insulin administered 
daily for 12 months, compared with placebo, on cognition, daily function and safety in adults with MCI 
or mild AD. Longer-term effects were examined in a six-month open-label extension offered to all 
participants. Safety and feasibility issues relating to the use of intranasal delivery devices were also 
evaluated. The trial is nearing completion; all participants have concluded the blinded phase. 
 
Methods: Twenty-six sites enrolled 289 participants with MCI or mild AD in this randomized, double-
blind, Phase II/III trial (NCT01767909). Adults 45 to 85 years of age with diagnoses of amnestic MCI 

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or AD (National Institute on Aging-Alzheimer’s Association criteria) with Mini-Mental State Exam 
(MMSE) scores >19, Clinical Dementia Ratings (CDR) of 0.5 or 1, and delayed Logical Memory 
scores within a specified education-adjusted range were eligible. Participants with diabetes requiring 
medication were excluded, as were participants who had used insulin within one year of the screening 
visit. Participants were randomized on a 1:1 basis using a covariate-adaptive algorithm that weighted 
MMSE, apolipoprotein E-ε4 (APOE-ε4) allele carriage, study site, sex, and age based on previous 
work indicating these factors may impact treatment response. Participants received 40 International 
Units of insulin or insulin diluent placebo (Humulin R U-100 or insulin diluent, Eli Lilly, Indianapolis, 
USA) daily for 12 months. At the end of the 12-month blinded phase, all participants were offered 
open-label insulin treatment for 6 months.  The primary outcome (Alzheimer’s Disease Assessment 
Scale for Cognition-12/ADAS-Cog12) was administered at baseline and then at 3 month intervals. 
Secondary functional outcomes (Alzheimer’s Disease Cooperative Study-Activities of Daily Living 
Scale for MCI; CDR Sum of Boxes) were assessed at 6 month intervals, as was a memory composite 
(Free and Cued Selective Reminding Test and Story Recall). Cerebrospinal fluid biomarkers (Aβ42 
and Aβ42/tau ratio) and magnetic resonance imaging hippocampal and entorhinal cortex volumes 
were measured at baseline and after 12 months.  
 
Device issues. Intranasal delivery device monitoring revealed no safety issues. However, for the first 
49 participants, the delivery device had frequent malfunctions (i.e. failure to turn on) that impacted 
dosing reliability. At that time, a newly available device was introduced (Precision Olfactory 
Device/POD, Impel NeuroPharma, Seattle, USA) which was used by the remaining 240 participants 
with good reliability.  
 
Results: Demographic characteristics of enrolled participants are 
presented in Table 1. Retention was excellent, with only 25 
participants discontinuing treatment during the blinded phase, 
and 15 also discontinuing study visits during the blinded phase. 
Quarterly DSMB reviews did not detect any safety issues and 
approved unmodified continuation of the trial.  
 
Data from the subgroup of participants who used the POD were 
analyzed separately from the ViaNase group. The primary 
analysis of the POD group showed was negative; no difference in 
ADAS-Cog12 scores were observed after 12 months of INI 
treatment compared with placebo (Fig.1). In contrast, analysis of 
the ViaNase group showed beneficial effects of insulin compared 
with placebo; the effect was significant at 6 months (p<0.01), and 
persisted at 12 months (p=0.09; Fig. 2). 
 
 
Table 1. Baseline Participant 
Characteristics 
N (F/M) 
289 (134 / 
155) 
Age (years) 
70.95 ± 7.1 
Diagnosis 
(MCI/AD) 
   105 / 184 
MMSE 
  24.8 ± 2.7 
Logical 
Memory 
    2.1 ± 2.7 
APOE (ε4+/ε4-
) 
    193 / 96 

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Implication: These results suggest that the two delivery devices may be providing different doses of 
insulin to the CNS. The ViaNase device results are consistent with multiple previous studies. The 
POD had not been used previously in clinical trials with insulin, and thus was relatively untested in 
terms of delivery efficacy, although modeling in organoid nasal cavities was conducted to verify that 
the target amount of insulin was dispersed to the target location in the nasal cavity. These divergent 
results raise the possibility that specific characteristics of the device can affect delivery. Any future 
studies of intranasal insulin will need to utilize devices that have been demonstrated to effectively 
deliver insulin to the CNS. A study currently in progress compared delivery of two doses of insulin with 
2 devices in order to provide supportive evidence for future trials.  
 
 
PRELIMINARY STUDY 3 
 
Objectives: The Device Delivery Study compared whether delivery of 20 or 40 IU of insulin with the 
Aptar VP7 or CPS devices raised insulin acutely in CSF.  
 
Methods: The study consists of a single site, randomized, double-blind trial comparing the acute 
effects of INI (20 or 40 International Units) delivered with the Aptar CPS or VP7 devices on CSF 
insulin levels, AD biomarkers and memory. At study entry, participants were randomized to the CPS 
or VP7 device. They were then randomized to receive either a 20 or 40 IU dose of insulin first, and the 
other dose on a second visit. Participants who were cognitively normal or who had aMCI (n=10) were 
enrolled. The primary outcome measure consisted of CSF insulin levels. Secondary measures 
included a memory test and CSF biomarker levels.  
 
Two lumbar punctures were performed in the morning after a minimum 8-hour overnight fast at least a 
week apart. After insertion of the spinal catheter, blood and CSF was collected immediately. Insulin 
(20 or 40 IU) was then be administered, and additional CSF and blood was collected at 10 and 20 
minutes following administration, after which the spinal catheter was withdrawn. 
 
Results: Although the study is ongoing, preliminary analyses have been conducted to inform the 
design of the current trial. Change in CSF insulin from baseline values are shown below for individual 
participants (left) using the VP7 and CPS devices to administer 20 IU (Dose 1) and 40 IU (Dose 2). 
Mean change for the groups is shown on the right. 
 
 
 
Results showed that the VP7 device did not reliably increase CSF insulin levels, with only one 
participant in the 40 IU dose showing elevations after administration. More reliable elevations were 
demonstrated with the CPS device, particularly for the 40 IU dose. All five participants showed 
increased CSF insulin after administration, and the average change was significant. Based on these 
Fig. 1 
Fig. 2 

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data, the CPS device has been selected for this pilot trial, as has the 40 IU dose.  
 
 
Rationale for Dosage Selection 
The dosage of insulin selected (40 International Units INI q.i.d.) has been used in multiple prior 
studies with positive results. The dosage of empa (10 mg q.d.) is also standard and has been used in 
previous large trials (EMPA-REG-OUTCOME). 
 
 
Rationale for Primary and Secondary Outcome Measures  
Because no published study has examined the safety of empagliflozin in adults with Alzheimer’s 
disease or in combination with intranasal insulin, the proposed study specifies safety, defined as 
number of treatment-related adverse events, as the primary outcome..  
 
Regarding secondary measures, in previous studies intranasal insulin acutely improved cognitive 
performance and affected CSF levels of AD biomarkers as well as MRI measures of vascular integrity 
(Reger et al. 2008; Craft et al. 2012; Kellar et al. 2021). Thus examining effects on these measures 
will provide information to be used in the design of a future Phase II trial. 
 
 
Rationale for Design of Trial  
The proposed pilot study will provide safety and efficacy preliminary data regarding singular and 
combined effects of two therapeutic approaches, intranasal insulin and treatment with the SGLT2i 
empagliflozin, to correct bioenergetic and vascular dysfunction in adults with preclinical AD and 
amnestic mild cognitive impairment (aMCI) or early AD. 
 
 
Rationale for Biofluids 
The CSF biomarkers insulin, Aβ42, Aβ40, total tau and phospho-tau will be measured, as well as CSF 
markers of vascular function. Plasma levels of insulin and glucose will be also assessed. Plasma and 
CSF samples obtained in this study will be banked so that other putative biomarkers may be 
measured by qualified investigators in the future given adequate rationale and feasibility.  
 
 
STUDY OVERVIEW 
The study will consist of a single site, randomized, double-blind trial comparing the effects of 4 weeks 
of INI (40 International Units q.i.d.), empa (10 mg q.d.) and combined INI and empa compared with 
placebo on CSF biomarkers and cognition. At study entry, participants will be randomized to one of 4 
conditions: INI, empa, INI+empa, placebo. Participants who are cognitively normal but have abnormal 
elevations of brain amyloid (n=20) or who have MCI or early AD (n=40) will be enrolled. The primary 
outcome measure will consist of safety (treatment-related serious adverse events). Secondary 
measures will consist of CSF biomarkers, cognition, and cerebral blood flow. Exploratory measures 
will include continuous glucose levels and sleep quality as assessed with actigraphy.  
 
 
Study Population 
A total of 60 adults who are 1) cognitively normal/amyloid positive (n=20) or 2) diagnosed with aMCI 
or early AD (n=40) will be enrolled in this trial. To determine eligibility, all participants will undergo 
cognitive assessment, physical and neurological examination, ECG, clinical/safety laboratory 
assessment, and interviews of the participant and study partner conducted by the investigators and 
staff of the Clinical Core of the Wake Forest Alzheimer’s Disease Research Center (ADRC). 
 
 
Diagnosis Criteria 
Diagnoses will be assigned by consensus of investigators from the Clinical Core of the Wake Forest 
ADRC using criteria specified by the NIA and Alzheimer’s Association workgroups (Petersen, Doody 
et al. 2001, Albert, Dekosky et al. 2011). 
 
Criteria for cognitively normal/amyloid positive adults: 

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a) No evidence of significant cognitive impairment on objective testing 
b) Amyloid positivity as determined by amyloid PET (amyloid SUVR>1.21 or CSF Aβ42<600 
pg/ml) 
 
Diagnosis of aMCI or AD dementia requires:  
a) Diagnosis of amnestic mild cognitive impairment (MCI) or early AD according to NIA-
Alzheimer’s Association Criteria (CDR 0.5 or 1, MMSE>21)  
b) Amyloid positivity as determined by amyloid PET (amyloid SUVR>1.21 or CSF Aβ42<600 
pg/ml) 
 
 
Inclusion Criteria 
The following inclusion criteria will be used:  
1. 
Age 55 to 85 (inclusive)  
2. 
Fluent in English  
3. 
Cognitively normal or diagnosis of aMCI or mild AD 
4. 
Amyloid positive by PET or CSF criteria 
5. 
Stable medical condition for 3 months prior to screening visit 
6. 
Stable medications for 4 weeks prior to the screening and study visits (exceptions may be 
made on a case by case basis by study physician)  
7. 
Clinical laboratory values must be within normal limits or, if abnormal, must be judged to be 
clinically insignificant by the study physician  
 
 
Exclusion Criteria 
The following exclusion criteria will be used:  
1. A diagnosis of dementia other than AD 
 
2. History of a clinically significant stroke 
 
3. Current evidence or history in past two years of epilepsy, head injury with loss of 
consciousness, any major psychiatric disorder including psychosis, major depression, bipolar 
disorder 
 
4. Diabetes (type I or type II) insulin dependent and non-insulin dependent diabetes mellitus 
 
5. Current or past regular use of insulin or any other anti-diabetic medication within 2 months of 
screening visit 
 
6. History of seizure within past five years 
 
7. Pregnancy or possible pregnancy 
 
8. Use of anticoagulants, unless documentation received from prescribing clinician that 
anticoagulant medication can be held before LP, and approved by study clinician 
 
9. Residence in a skilled nursing facility at screening 
 
10. Use of an investigational agent within two months of screening visit 

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11. Regular use of alcohol, narcotics, anticonvulsants, anti-parkinsonian medications, or any other 
exclusionary medications (exceptions may be made on a case by case basis by study 
physician)  
 
 
Recruitment and Retention Strategies 
Recruitment will occur primarily from the Clinical Core of the Wake Forest ADRC. Some participants 
may also be identified from community recruitment efforts or from recently completed studies.  
 
 
STUDY TIMELINE  
The approximate timeline for this 18-month study is projected as follows: 1) approximately two months 
for study startup activities; 2) study visits will occur over a ten-month period; 3) CSF and blood 
analyses, data analyses and study dissemination will occur over the final 6 months.  
 
 
DESCRIPTION OF STUDY VISITS 
The “Schedule of Study Procedures and Assessments” in Table 4 (located in section 23.0) provides 
an overview of study visit activities.  Genotyping will occur at Screening for participants who have not 
previously received APOE genotypes through the ADRC.  
 
 
Screening (Visit 1) 
The purpose of this visit is to determine study eligibility.  Potential participants must sign an informed 
consent form and HIPAA Authorization prior to administration of any study-related procedures. After 
consent is obtained, participants will be given the MMSE, the CDR and Story Recall to determine 
study eligibility. Screening may be waived for participants who have received evaluations from the 
ADRC within the past 12 months. In addition, all cognitively normal participants must have amyloid 
PET or CSF Aβ42 results available from the ADRC or affiliated studies prior to screening. 
 
Information regarding demographics, concurrent medications, medical history and adverse events will 
be gathered from the participant. Vital signs, height and weight will be measured. A brief physical and 
neurological examination (which include a nasal examination) and a resting ECG will be performed. 
The ECG report will be reviewed, signed, and dated by the investigator or a medically qualified staff 
member as delegated by the Principal Investigator. Those with clinically significant ECG findings will 
be referred for follow-up as deemed appropriate by the investigator. These procedures may be waived 
for participants who have received evaluations from the ADRC within the past 12-months. 
 
Fasting blood will be drawn for routine clinical laboratory evaluations. If values are outside of the 
laboratory’s normal range and determined clinically significant by the medically qualified study 
investigators, lab tests may need to be repeated and may be considered exclusionary for participation 
in the study. Blood samples will also be collected for ApoE genotyping and DNA storage.  
 
 
Baseline Visit 1 (Visit 2) 
Results from all screening procedures must be reviewed and all inclusion/exclusion criteria must be 
met prior to proceeding to baseline. For participants whose screening visit was waived because they 
had received ADRC evaluations within the past 12-months will sign an informed consent form and 
HIPAA Authorization prior to administration of any study-related procedures. 
 
According to the randomization schedule, the participant will be assigned to the insulin, empa, 
insulin+empa or placebo arm. On their first baseline visit they will receive the following:  
 
1) Cognitive assessment  
2) MRI (ADRC MRI may be used if done within 6 months of enrollment)  
3) Insertion of the Libre Freestyle Pro continuous glucose monitor 

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4) Training on use of the Libre Freestyle Pro continuous glucose monitor 
5) Training on how to use the intranasal delivery device and provision of saline to use with the 
device for the 1 week practice period (if applicable) 
6) Training on how to record compliance 
7) Training on use of the Empatica E4 actigraph for sleep assessment 
8) PROMIS Sleep Disturbance and Sleep-Related Impairment Questionnaires 
9) Patient Health Questionnaire-9 (PHQ-9) 
10) General Anxiety Disorder Scale (GAD-7) 
11) Nasal examination 
12) Vital signs and weight 
13) Collection of AEs and Con Meds 
 
 
Baseline Visit 2 (Visit 3; one week after Baseline Visit 1) 
For the 2nd baseline visit which occurs one week after Baseline Visit 1, the following procedures will 
be administered: 
 
1) Lumbar puncture will be performed in the morning after a minimum 8-hour overnight fast. After 
insertion of the spinal catheter, CSF will be collected immediately. Insulin (40 IU) will then be 
administered, additional CSF will be collected following administration, and then the spinal 
catheter will be withdrawn.  Blood will be drawn immediately before and following the LP under 
fasting conditions. Participants will then receive a snack and instructions about post-lumbar 
puncture care. Site staff will call the participant within 24 hours from the lumbar puncture 
procedure to inquire about the participant’s well-being and possible adverse events. 
2) The ADCS-ADL-MCI and qDRS will be administered 
3) The Libre Freestyle Pro continuous glucose monitor will be removed 
4) The Empatica E4 actigraph and PROMIS sleep questionnaires will be collected 
5) Device use and feasibility issues during the run-in period will be reviewed  
6) Distribution of study drug and placebos (if applicable) 
7) Nasal examination 
8) Vital signs and weight 
9) Collection of AEs and Con Meds 
 
 
Re-supply Visit and Safety/Compliance Assessment 1 (Visit 4; one week after 
Baseline Visit 2) 
For the re-supply visit, the following procedures will be administered: 
 
1) Device use and instructions will be reviewed  
2) Collection of nasal drug vial and pill count 
3) Distribution of study drug and placebos (if applicable) 
4) Vital signs and weight 
5) Collection of AEs and Con Meds 
 
 
Phone Safety/Compliance Assessment 2 
Participants will receive a call from study staff 2 weeks after initiating treatment to monitor for adverse 
events and compliance. 
 
 
Post-treatment Visit 1 (Visit 5) 
On their first post-treatment visit they will receive the following:  
1) Cognitive assessment  
2) MRI 
3) Insertion of the Libre Freestyle Pro continuous glucose monitor 
4) Provision of the Empatica E4 actigraph for sleep assessment 

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5) PROMIS Sleep Disturbance Questionnaires 
6) Patient Health Questionnaire-9 (PHQ-9) 
7) General Anxiety Disorder Scale (GAD-7) 
8) Nasal examination 
9) Vital signs and weight 
10) Collection of AEs and Con Meds 
 
 
Post-treatment Visit 2 (Visit 6; one week after Post-treatment Visit 1) 
On their second post-treatment visit which occurs 1 week after Post-treatment Visit 1 they will receive 
the following:  
 
1) Lumbar puncture will be performed in the morning after a minimum 8-hour overnight fast. After 
insertion of the spinal catheter, CSF will be collected immediately. Insulin (40 IU) will then be 
administered, additional CSF will be collected following administration, and then the spinal 
catheter will be withdrawn.  Blood will be drawn immediately before and following the LP under 
fasting conditions, including routine clinical laboratory evaluations. Participants will then 
receive a snack and instructions about post-lumbar puncture care. Site staff will call the 
participant within 24 hours from the lumbar puncture procedure to inquire about the 
participant’s well-being and possible adverse events. 
2) The ADCS-ADL-MCI and qDRS will be administered 
3) The Libre Freestyle Pro continuous glucose monitor will be removed 
4) The Empatica E4 actigraph and sleep questionnaire will be collected 
5) Nasal examination  
6) Vital signs and weight 
7) Collection of AEs and Con Meds 
8) Collection of nasal drug vials and pill count 
 
Order of administration of procedures may occasionally vary due to scheduling constraints, as 
approved by the study clinicians. 
  
 
STUDY-SPECIFIC PROCEDURES 
 
 
Cognitive Evaluation Instruments Administered to the Participant 
Objective tests of cognitive function will include tests comprising the PACC5 (MMSE, Story Recall, the 
Rey Auditory Verbal Learning Test, Verbal Fluency), the CDR, and the ADAS-Cog14. 
 
 
Clinical and Functional Evaluations  
 
 Quick Dementia Rating System (QDRS) 
The QDRS (Galvin, 2015) is a clinical scale that rates the severity of dementia as absent, 
questionable, mild, moderate, or severe (CDR score of 0, 0.5, 1, 2, or 3, respectively). The score 
assesses six domains: memory, orientation, judgment and problem solving, community affairs, home 
and hobbies, and personal care.  At Screening the QDRS global score will be used for eligibility 
purposes. For all other administrations, the 6 domain scores will be summed to get the QDRS Sum of 
Boxes (SB) score.  
 
 Activities of Daily Living Scale for MCI/ADL-MCI 
The ADL-MCI is an activities of daily living questionnaire aimed at detecting functional decline. In a 
structured interview format, informants are queried as to whether participants attempted each item in 
the inventory during the prior 4 weeks and their level of performance. The ADL-MCI scale 
discriminates well between normal controls and patients with mild AD or MCI. It has good test-retest 

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reliability. The questions focus predominantly on instrumental activities of daily living scales (e.g. 
shopping, preparing meals, using household appliances, keeping appointments, reading). 
 
9.2.3. PROMIS Sleep Disturbance and Sleep-Related Impairment Questionnaires (Short Form). 
These 8-item forms ask participants to rate quality of sleep on a 5-point scale.  
 
9.2.4 Patient Health Questionnaire-9 (PHQ-9).   
This widely-used 9 item questionnaire assesses depression. 
 
9.2.5 General Anxiety Disorder Scale (GAD-7) 
This widely-used 7-item questionnaire assesses anxiety. 
 
 
STUDY METHODS 
 
 
Safety Assessments 
At each study visit, all participants will undergo a nasal examination and any occurrence of adverse 
events will be reviewed and documented; concomitant medications will be recorded as well. In 
addition, 24 hours after the Lumbar Puncture, each study participant, or a person designated to speak 
for them will be contacted by phone to confirm the participant’s well-being and queried about any new 
adverse events. All adverse events will be reported to the study MD for review.  
 
 
Physical and Neurological Examination 
A brief physical examination will be performed by a medically qualified professional at the screening 
visit. A review of the major body systems will be performed for example: skin, 
head/ears/eyes/nose/throat (HEENT), cardiovascular, pulmonary, abdomen, musculoskeletal, 
neurological, and gastrointestinal. Assessments of height (Screening visit only), weight, and vital signs 
(systolic and diastolic blood pressure, pulse, temperature, and respiration) are included. Neurological 
examination will include an assessment of cranial nerves, strength, coordination, reflexes, sensation, 
tremor and gait at every study visit. A nasal examination will also be performed. The examination will 
assess irritation or other abnormalities of the nares.  
 
 
Electrocardiogram (ECG) 
A resting ECG will be performed at Screening visit. The ECG report will be reviewed, signed, and 
dated by the study clinician.  Those with clinically significant ECG findings will be referred for follow-up 
as deemed appropriate by the investigator and may be excluded from the study. 
  
 
Clinical Laboratory Evaluations 
All routine laboratory samples will be analyzed by Laboratory Corporation of America (Labcorp). Lab 
reports will be reviewed, signed and dated by the study clinician. If a value is outside of the 
laboratory’s normal range, the clinician will indicate if it is clinically significant or not. If clinically 
significant, lab tests may need to be repeated and follow up with the participant’s PCP should occur.    
 
 
Actigraphy 
The Empatica E4 device (https://www.empatica.com/en-eu/research/e4/) will be used to assess sleep 
quality. It uses an accelerometer to assess physical activity and heart rate variability from which a 
sleep quality index is calculated. 
 
 
Continuous Glucose Monitoring 
The Abbott Freestyle Libre Pro is a continuous glucose monitor (CGM) system; a thin filament sensor 
the size of a quarter covered with a waterproof patch will be placed by a clinician or qualified study 
team member on the back of the participant’s upper arm and worn for 7 days, then removed by the 
participant. The sensor records and uploads glucose data every 15 min. A report of average glucose 

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levels and number of hyperglycemic/hypoglycemic episodes can be linked to events such as meals, 
sleep, and activity. Participants will be assessed over a 1-week period. 
 
 
BIOMARKER STUDIES 
 
 
CSF 
All CSF samples will be collected in the morning before breakfast and after an overnight fast.  
Participants who are taking anticoagulants cannot be enrolled unless documentation is received from 
the prescribing clinician that the anticoagulant medication can be held before the LP visits, and is 
approved by a study clinician. Based on clinician judgment and depending on the clinical indication, it 
may be suitable to discontinue participants from their anti-platelet agent (e.g., aspirin, Plavix, NSAIDs) 
for 5-7 days prior to lumbar puncture and until at least 24 hours after lumbar puncture.  It is not 
required that participants be discontinued from their anti-platelet agent in order to screen and enroll in 
the study.  
 
A minimal total volume of CSF (~25 ml) will be required for this study.  To clear any blood from minor 
trauma associated with needle insertion, the first 1-2 mL of CSF are discarded (or more if 
needed).  Collected CSF is aliquoted into sterile microtubes. Approximately 2ml of CSF or volume per 
local laboratory requirements will be sent at ambient temperature to the CRU laboratory for protein, 
glucose and cell count. CSF will be immediately frozen upright on dry ice for at least 20 minutes then 
stored at -70 until analysis.  
 
CSF samples will be used to measure levels of insulin, Aβ42, Aβ40, total tau, and phospho-tau181. 
Assays will be performed by the Wake Forest ADRC Biomarker Service. CSF samples will also be 
frozen and stored for future analysis of putative biomarkers. 
 
 
Blood Collection at Lumbar Puncture Visits 
All samples will be collected in the morning before breakfast and after a minimum 8-hour overnight 
fast. Blood samples (26 mL) will be collected before and after insulin administration. Blood will be 
processed for fasting plasma insulin and glucose. Additional blood for plasma and serum will be 
processed and banked. 
 
 
Genetic Samples, Storage and Future Use 
DNA will be extracted from participant blood samples and will be analyzed for ApoE genotyping. ApoE 
genotyping will be used as a weighting factor for the minimization strategy during randomization.  This 
will allow secondary analyses of data on the impact of the ApoE genotype on putative biomarkers of 
AD, clinical outcome measures, and adverse events. ApoE genotyping will be performed by Dr. Tim 
Howard under the auspices of the ADRC Biomarker service using established protocols.  
 
 
STATISTICAL PLAN  
Statistical analyses will be conducted by the ADRC Biostatistics Core. To address the Primary Aim, 
number of treatment-related SAEs will be subjected to a repeated measures analysis of variance with 
group and time as the repeated measures. Secondary analyses will examine the effect of diagnostic 
status (cognitively normal vs. MCI/AD). Secondary analyses will also examine treatment-related 
differences for other CSF and plasma biomarkers, cognitive scores, and whole brain cerebral blood 
flow.  
 
 
Power Analyses 
As this study is designed as a proof of concept study power calculations have not been conducted.  
 
 
POTENTIAL RISKS 
 

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Safety of Intranasal Insulin 
Safety issues pertaining to INI administration for the treatment of diabetes have been extensively 
explored for over two decades (Pontiroli, Alberetto et al. 1982). For diabetes treatment, absorption 
enhancers must be used to increase the transport of insulin across the nasal membrane to the 
periphery due to the fact that peripheral bioavailablity of insulin without absorption enhancers is less 
than 1% (Illum 2002). A recent safety study of INI administration without absorption enhancers 
demonstrated no treatment induced changes in blood glucose levels, nasal airway patency, or 
transnasal pressure gradient (Kupila, Sipila et al. 2003). There are no known serious risks associated 
with INI without enhancers. A recent industry report raised the issue of rare but significant increases in 
lung cancer in smokers treated with inhaled insulin; six of 4740 patients taking inhaled insulin 
developed lung cancer compared with one of 4292 patients who received an active comparator 
(incidence per 100 patient years exposure, 0.13 vs 0.02). However, the inhaled insulin protocol used 
for diabetes treatment in this report included absorption enhancers to maximize delivery to lungs, 
whereas the nose-to-brain delivery device to be used in this study greatly minimizes lung delivery.  
 
Regarding the risk of hypoglycemia, at least five peer reviewed human studies (Kern, Born et al. 
1999, Born, Lange et al. 2002, Kupila, Sipila et al. 2003, Benedict, Hallschmid et al. 2004, Stockhorst, 
de Fries et al. Submitted for publication)  and four preliminary studies (Reger, Watson et al. 2006, 
Reger, Watson et al. 2008, Reger, Watson et al. 2008) (Craft, Baker et al. 2012) revealed no change 
in blood glucose levels following intranasal insulin administration with doses that included 40 
International Units 4 times daily for two months. There was one exception with the case of a single 
participant who experienced mild hypoglycemia (52 mg/dl) after skipping a meal and engaging in 
sustained vigorous exercise. In addition, a recent safety study (Kupila, Sipila et al. 2003) examined 
intranasal insulin administration of 60 International Units once a day for three weeks in 21 healthy 
adults. This randomized, double-blind, placebo-controlled crossover trial measured blood glucose 
levels six times a day during the first two and the last two days of treatment. Pre- and post-treatment 
blood laboratory tests and nasal examinations were performed. The nasal studies included rhinoscopy 
to detect local irritation, a saccharin particle test to analyze mucocilary clearance, and 
rhinomanometry to evaluate nasal airway patency and transnasal pressure gradient. Results indicated 
no change in blood glucose values with insulin, and no change in the frequency of glucose values 
above 3.0 mmol/L.  
 
The only symptomatic hypoglycemic value occurred during placebo treatment. Insulin treatment had 
no effect on other laboratory values (C-peptide, total cholesterol, HDL, LDL, triglycerides, creatinine, 
glutamyl transferase), blood pressure, or body weight. In addition, nasal examinations revealed no 
adverse effects or functional disturbances following intranasal insulin administration. No serious 
adverse effects of treatment were observed in the preliminary studies (Reger, Watson et al. 2006, 
Reger, Watson et al. 2008, Reger, Watson et al. 2008, Craft, Baker et al. 2012). 
 
13.1 Safety of empagliflozin 
SGLT2is including empagliflozin has been studied in multiple large trials of adults with Type 2 
diabetes, as well as in non-diabetics with cardiovascular disease, and generally found to be safe (for a 
review, see Scheen, Nature, 2020). They are now considered standard of care for Type 2 diabetic 
patients with cardiovascular disease. Adverse effects that have been reported include: genital yeast 
infections especially in females, volume depletion, and diabetic ketoacidosis in the subset of Type 2 
diabetic patients who have reduced insulin secretory capacity. Other effects may include frequent 
urination. Adults with seriously compromised kidney function (eGFR<45 mL/min/1.73m2) are not 
advised to take empa; kidney function will be assessed at the screening visit. Hypotension may also 
be observed rarely in patients with low systolic blood pressure, or who are taking diuretics. 
Participants will be monitored for hypotension and/or associate symptoms at each visit and during the 
two phone safety/compliance calls.  
 

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13.2  Safety of combined insulin and empagliflozin 
Combined therapy of insulin and SGLT2is, including empa, was associated with rare but increased 
risk of ketoacidosis in the subset of Type 2 diabetic patients who have reduced insulin secretory 
capacity. Hypoglycemia has also been noted with combined administration, thought to be due to 
reduced hyperglycemia and related improvement in insulin sensitivity without concomitant reduction in 
insulin dosing. The proposed study will exclude participants with Type 2 diabetes, and will monitor 
blood glucose at all visits, as well as with continuous glucose monitoring during the last week of 
treatment. No other adverse interactions have been noted and SGLT2is are commonly prescribed for 
insulin-treated diabetics because of the multiple benefits on glycemic control and vascular function 
(Scheen, Nature, 2020). 
 
 
Risks associated with use of the Aptar CPS device  
The Aptar CPS device has been extensively characterized and found to be safe for use. Participants 
could experience some discomfort to their eyes or face if they do not hold the device to their nose as 
directed; however, again, the study nurse will carefully instruct the participant regarding device use so 
that any errors can be immediately corrected.  
 
 
Risks associate with use of the Empatica E4 actigraphy device 
Rarely, skin irritation may occur in reaction to the wristband or sensors. 
 
 
Risks associated with use of the Libre Freestyle Pro Glucose Monitoring System 
The CGM is FDA-approved and widely used for assessing glucose levels in diabetes. The following 
are possible adverse effects of inserting a sensor and wearing the adhesive patch: local erythema 
(redness), local infection, inflammation, pain or discomfort, bleeding at the glucose sensor insertion 
site, bruising, itching, scarring or skin discoloration, hematoma, and adhesive irritation. There is a 
remote risk of sensor or needle fracture during insertion, wear or removal, with fragments retained 
under the skin. 
 
 
Lumbar Puncture 
Lumbar puncture may be associated with pain during the performance of the procedure. This is 
usually temporary and confined to the lower back. Headache may occur in about 5% of elderly people 
who undergo lumbar puncture. Less commonly, in about 1-4% of participants, a persistent low-
pressure headache may develop, probably due to leakage of CSF. Lower rates of post-LP headache 
have been noted in elderly patients, and when atraumatic (Sprotte) needles are used. If a post-LP 
headache persists it may need additional treatment, e.g. with fluids and analgesics. Uncommonly a 
blood patch (injection of some of the participant’s blood to patch the CSF leak) may be needed. 
Potential but rare risks of lumbar puncture include infection, damage to nerves in the back, and 
bleeding into the CSF space. The risk of these is much less than 1%.   
 
 
Blood Draw 
The risks of blood draw include pain from the needle, bruising or infection at the site of venipuncture, 
or fainting as a response to blood draw.  Approximately 100 mLs of blood will be drawn for routine and 
biomarker laboratory assessments over the course of this study.   
 
 
PERSONNEL REQUIREMENTS 
The following staff-member roles will be required to conduct the protocol.   
 
 
Principal Investigator: The Principal Investigator (PI; Suzanne Craft, PhD) is responsible for the 
overall conduct of the study. The PI will perform or supervise clinical evaluation of all participants 
and ensure protocol adherence. The PI will supervise project personnel and ensure that clinical 
raters maintain a high level of skill and accuracy in conducting assessments.  

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 
Study Physicians: Don McClain, MD, PhD and James Tray Bateman, MD, MHS will serve as 
Study Physicians for the trial. Dr. McClain is a diabetologist with extensive experience in 
prescribing and monitoring SGLT2is and review all safety data for the trial as well as provide 
consultation as needed. Dr. Bateman is a Behavioral Neurologist with extensive experience in the 
assessment and care of older adults with Alzheimer’s disease. He will be responsible for 
conducting and supervising the medical evaluation (nasal examination, physical and neurological 
examinations), reviewing adverse events, interpreting laboratory results, and supervising clinical 
care provided to the participant during the study. He will also supervise, and on some occasions 
may perform the lumbar punctures. 
 
Study Clinician: Samantha Rogers, PA-C, will conduct the majority of the lumbar punctures. She 
has conducted more than 300 lumbar punctures for the ADRC and affiliated studies over the past 
5 years. 
 
Study Nurse/Coordinator: Deborah Dahl, RN will serve as Study Nurse/Coordinator for the trial. 
She will be responsible for managing the day-to-day conduct of the trial.  Duties may include 
tracking recruitment, ensuring accurate administration of all instruments at the site, maintaining 
case report forms, processing of laboratory samples, and coordinating clinic visits. She will also 
oversee the use and maintenance of the devices, and coordinate with the Pharmacy regarding 
ordering and storage of saline and insulin.  
 
Interviewer/Psychometrician:  This person will be responsible for administering the memory 
assessments.   
 
QDRS Rater: This person will render the QDRS rating based on clinical assessment of participant 
and study participant.  
 
Regulatory Affairs: Sarah Bohlman, MSL will be responsible for managing all regulatory related 
documents for the duration of the trial. 
 
 
STUDY DRUGS 
Participants will take empa or placebo 30 minutes before breakfast daily. Participants will also take 
four doses of INI (40 International Units per dose) or placebo 30 minutes before each meal and 30 
minutes before bedtime.  
 
 
Humulin® R U-100 Insulin 
Humulin® R U-100 (NDC: 0002-8215, Eli Lilly & Company) is a polypeptide hormone structurally 
identical to human insulin synthesized through rDNA technology in a special non-disease-producing 
laboratory strain of Escherichia coli bacteria. Humulin R U-100 has the empirical formula 
C257H383N65O77S6 and a molecular weight of 5808 Da. Humulin R U-100 is a sterile, clear, aqueous, 
and colorless solution that contains human insulin (rDNA origin) 100 units/mL, glycerin 16 mg/mL and 
metacresol 2.5 mg/mL, endogenous zinc (approximately 0.015 mg/100 units) and water for injection. 
The pH is 7.0 to 7.8. Sodium hydroxide and/or hydrochloric acid may be added during manufacture to 
adjust the pH. 
 
More information about the Humulin® R U-100 insulin, including risks, contraindication and adverse 
reactions can be found in the the US package insert (USPI).  
 
15.2  Empagliflozin  
Empagliflozin (Jardiance, Boehringer-Ingelheim) is a sodium glucose co-transporter2 inhibitor. The 
chemical name of empagliflozin is D-Glucitol,1,5-anhydro-1-C-[4-chloro-3-[[4-[[(3S)-tetrahydro-3- 
furanyl]oxy]phenyl]methyl]phenyl]-, (1S). Its molecular formula is C23H27ClO7 and the molecular 
weight is 450.91. Empagliflozin is a white to yellowish, non-hygroscopic powder. It is very slightly 
soluble in water, sparingly soluble in methanol, slightly soluble in ethanol and acetonitrile; soluble in 
50% acetonitrile/water; and practically insoluble in toluene. Each film-coated tablet of JARDIANCE 

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contains 10 mg empagliflozin (free base) and the following inactive ingredients: lactose monohydrate, 
microcrystalline cellulose, hydroxypropyl cellulose, croscarmellose sodium, colloidal silicon dioxide 
and magnesium stearate. In addition, the film coating contains the following inactive ingredients: 
hypromellose, titanium dioxide, talc, polyethylene glycol, and yellow ferric oxide.  
 
More information about empagliflozin can be found the prescribing information included on the 
Jardiance/Boehringer-Ingelheim website: 
https://docs.boehringeringelheim.com/Prescribing%20Information/PIs/Jardiance/jardiance.pdf 
 
 
Randomization 
Eligible participants will be randomized on a 1:1 schedule to placebo (insulin diluent; placebo 
capsule), INI, empa, or INI+empa. Randomization will be stratified by diagnosis (normal, MCI or AD) 
 
 
Blinding 
Neither participants nor site personnel involved in assessing participants will know which dose of 
insulin is being administered. Exceptions will be the study nurse who is directly involved in preparing 
the insulin. 
 
 
 
Study Drug Dispensing 
Study drug will be dispensed by the study nurse. 
 
 
Intranasal Administration 
Insulin will be administered with the Aptar CPS delivery device. This device release a metered insulin 
dose into the participant’s nose, allowing the administration of smaller particle sizes to increase 
deposition in the upper nasal cavity while minimizing transport to the lungs. A volume of about 0.7 mL 
of insulin will be administered in each nostril, for a total volume of 1.4 mL per dose, 4 times daily.  
 
 
Storage   
Insulin and empa will be maintained at a controlled temperature. 
 
 
Drug Accountability   
The study nurse coordinator will maintain a log of study drug usage.  
 
 
ADVERSE EVENTS 
 
 
Definition 
An adverse event (AE) is defined as per the Code of Federal Regulation Title 21 Part 312. 
 
http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/cfrsearch.cfm?fr=312.32 
 
Adverse events which occur after informed consent is signed include but are not limited to: (1) 
worsening or change in nature, severity, or frequency of conditions or symptoms present at the start 
of the study; (2) participant deterioration due to primary illness; (3) intercurrent illness; and (4) drug 
interaction. An abnormal laboratory value will only be reported as an AE if the investigator considers it 
to be an AE, or if it leads to the participant being withdrawn from the study. 
 
The investigator should attempt to establish a diagnosis of the event based on signs, symptoms, and 
or other clinical information. In such cases, the diagnosis should be documented as the AE and not 
the individual signs or symptoms.  Symptoms and conditions present at the beginning of the study will 
be characterized, so that AEs can be defined as any new symptom, or any increase in frequency or 
severity of an existing symptom. 
 

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Following questioning and evaluation, all AEs, whether determined to be related or unrelated to the 
study drug by a medically qualified site PI or clinician (MD, DO, NP or PA), must be documented in 
the participant’s medical records, in accordance with the investigator’s normal clinical practice, and on 
the AE e-CRF. Each AE is evaluated for duration, severity, seriousness, and causal relationship to the 
study drug. 
 
 
Following Up on AEs 
The investigator is obliged to follow participants with AEs until the events have subsided, the 
conditions are considered medically stable, or the participants are no longer available for follow up. 
Participants who discontinue due to adverse experiences will be treated and followed according to 
established medical practice. Adverse events will be rated as mild, moderate or severe.  This will also 
pertain to abnormal laboratory values deemed clinically significant by the site clinician.  
 
 
SERIOUS ADVERSE EVENTS (SAE) 
 
 
Definition  
A serious adverse event is defined as per the Code of Federal Regulation Title 21 Part 312  
 
http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/cfrsearch.cfm?fr=312.32 
 
 
Reporting SAEs 
Any serious and adverse event due to any cause, which occurs during the course of the investigation 
(i.e. any time after informed consent, regardless of study drug exposure), will be reported to the PI 
and study physician within 24 hours of learning of the event. All serious adverse events will be 
reported to the Wake Forest IRB within 7 days of study personnel learning of the event. 
 
 
IND Safety Reporting 
As the Sponsor of the IND related to this study, any unexpected fatal or life-threatening suspected 
adverse reactions will be reported to the FDA no later than 7 calendar days after the initial receipt of 
the information per 21 CFR 312.32(c)(2). 
 
Reporting of any (1) serious, unexpected suspected adverse reactions (2) findings from other clinical, 
animal, or in-vitro studies that suggest significant human risk, and (3) a clinically important increase in 
the rate of a serious suspected adverse reaction no later than 15 calendar days after determining that 
the information qualifies for reporting per 21 CFR 312.32(c)(1). 
 
 
ETHICS & REGULATORY CONSIDERATIONS 
 
 
Ethical Standard 
Study investigators are charged with conducting this study in full conformity with: 
1. Good Clinical Practice (GCP) guidelines, as defined by the International Conference on 
Harmonisation (ICH) Guideline, Topic E6 
2. The United States Code of Federal Regulations, Title 21, Part 50 (21CFR50) – Protection of 
Human Subjects 
3. 21CFR56 – Institutional Review Boards (IRBs) 
4. HIPAA 
5. State and Federal regulations and all other applicable local regulatory requirements and laws.    
 
Study personnel involved in conducting this study will be qualified by education, training and 
experience to perform their respective task(s) in accordance with GCP.   

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Institutional Review Board (IRB) 
This protocol and the associated informed consent documents and recruitment material will be 
approved by the Wake Forest IRB which is registered with the Office for Human Research Protections 
(OHRP). Any amendments to the protocol or consent materials must also be approved before they 
are placed into use. The investigator shall notify the IRB of deviations from the protocol or serious 
adverse events occurring at the site, in accordance with local procedures.  
 
 
Informed Consent & HIPAA Authorization 
In obtaining and documenting informed consent, the investigator should comply with the applicable 
regulatory requirement(s), and should adhere to GCP and to the ethical principles that have their 
origin in the Declaration of Helsinki.  Prior to the beginning of the trial, the investigator should have the 
IRB’s written approval of the written informed consent form (ICF) and any other written information to 
be provided to participants.  Participants, their relatives, guardians, or authorized representatives and 
study partners will be given ample opportunity to inquire about the details of the study. Prior to a 
subject’s participation in the trial, the written informed consent form and HIPAA Authorization should 
be signed and personally dated by the subject and/or the subject’s legally authorized representative, 
the study partner and by the person who conducted the informed consent discussion.  Participants 
should be provided a copy of the signed ICF. 
 
The informed consent will not only cover consent for the trial itself, but for the genetic research, 
biomarker studies and biological sample storage. The consent for storage will include consent to 
access stored data and biological samples for secondary analyses. Consent forms will specify that 
DNA and biomarker samples are for research purposes only; the tests on the DNA and biomarker 
samples are not diagnostic in nature and participants will never receive results.  
 
 
Participant Confidentiality | HIPAA 
Participant confidentiality is strictly held in trust by the participating investigators, research staff, and 
the sponsoring institution and their agents.  This confidentiality is extended to cover testing of 
biological samples and genetic tests in addition to the clinical information relating to participants. The 
study protocol, documentation, data and all other information generated will be held in strict 
confidence.  No information concerning the study or the data will be released to any unauthorized 
third party, without prior written approval. Any data, specimens, forms, reports, and other records that 
leave the site will be identified only by a subject identification number to maintain confidentiality.  All 
records will be kept in a locked file cabinet.  All computer entry and networking programs will be done 
using subject IDs only. Information will not be released without written permission of the subject, 
except as necessary for monitoring by the IRB, FDA, NIA, and the OHRP. 
 
Information about subjects will be kept confidential and managed according to the requirements of the 
Health Insurance Portability and Accountability Act of 1996 (HIPAA).  Those regulations require a 
signed HIPAA Authorization informing the subject of the following:  
 
• What protected health information (PHI) will be collected from participants in this study 
• Who will have access to that information and why 
• Who will use or disclose that information 
• The rights of a research participant to revoke their authorization for use of their PHI.  
 
In the event that a subject revokes authorization to collect or use PHI, the investigator, by regulation, 
retains the ability to use all information collected prior to the revocation of subject authorization. Each 
site PI, under the guidance of his/her IRB, is responsible for ensuring that all applicable HIPAA 
regulations and State laws are met. 
 

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GENETIC RESEARCH & STORAGE OF GENETIC MATERIAL  
The DNA is banked in locked freezers in the ADRC Biomarker Service.  Sample tubes are bar-coded 
and linked to participant ID number only and banked without personal identifiers.  
 
Only DNA from consenting participants will be banked and used to facilitate future research on aging 
and dementia, particularly in the discovery of genetic polymorphisms that may influence risk of 
developing AD.  Collection of DNA will permit investigators to probe candidate genetic polymorphisms 
as predictors of outcome in future studies.  The samples will be stored by the ADRC.  
 
 
Storage of Biospecimen Samples  
All biospecimens being banked for future AD biomarker research will be stored by the ADRC 
Biomarker Service. Participant will be asked to consent to allow samples to be used for future 
research.  
 
 
RISKS AND BENEFITS ASSOCIATED WITH THIS STUDY 
 
 
Potential Benefits of the Proposed Research to Human Subjects 
There is an urgent need to identify promising treatments for patients with AD and its prodrome aMCI. 
In a previous trial (Craft, Baker et al. 2012), intranasal insulin safely improved delayed memory and 
function in these patients. In a recent trial, these results were replicated with the subgroup of 
participants who used the ViaNase device, but not with the POD cohort. The proposed trial is 
designed to answer the important question of whether the Aptar CPS device is effective in delivering 
intranasal insulin into the CNS. This knowledge will enable the conduct of future Phase III trials of 
intranasal insulin.  
 
There are no significant potential clinical benefits for the participants in this study. Rather, there is a 
clear scientific benefit for the field as a whole. The relatively minor risks posed by the intranasal 
administration, cognitive testing, and LP are outweighed by the value of the scientific investigations 
outlined in this study. 
 
 
Inclusion of Women and Minorities 
There are currently no studies that definitively support or negate the existence of significant 
differences in response to intranasal insulin in subgroups defined by gender or ethnic background. A 
specific goal percentage for women and minority enrollment is not set for this study. However, we will 
monitor minority enrollment throughout the study and make special effort to encourage minority 
enrollment. Minority enrollment will be facilitated through minority outreach effort coordinated by the 
Recruitment Core at the ADRC. No participant will be excluded due to his or her sex, race, or ethnic 
group.  
 
 
Inclusion of Children as Participants in Research Involving Human Subjects 
Children will not be included. 
 
 
Data and Safety Monitoring Plan  
The principal investigator and study clinician(s) will be responsible for the overall monitoring of the 
data and safety of study participants, with assistance by members of the study staff. Participants will 
be screened at the beginning of the study and will be monitored carefully at each study visit.  
Participants will be queried for serious adverse events (SAEs) and selected AEs (those temporally 
related to study procedures) at each study visit. All SAEs and selected AEs will be recorded on an AE 
case report form. Based on the nature of the AE, study clinicians will determine the severity of the 
event and association with the study.  All serious adverse events will be reported. Serious adverse 
events are defined as events that are (1) life-threatening or fatal, (2) result in severe or permanent 
disability, or (3) require hospitalization. SAEs will be reported to the IRB within 7 days of investigator 

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knowledge of the event if they are related and unanticipated (i.e. if they qualify as an unanticipated 
problem). All serious adverse events will be followed until resolution, or 90 days after study 
participation has been terminated. 
 
"Severity" of events will be graded according to the following guidelines: 
 
1. 
Mild: The participant is aware of, but can easily tolerate, the event 
 
2. 
Moderate: The discomfort of the event is severe enough to interfere with some usual activities 
 
3. 
Severe: The participant is incapacitated, and unable to perform most or all usual activities 
 
The "study-relatedness" will be assigned according to the following guidelines: 
 
1. Definitely Related: The reaction follows a reasonable temporal sequence and is known 
    to be an effect of participation or a procedure 
2. Possibly Related: The reaction follows a reasonable temporal sequence, and current  
medical knowledge does not preclude a relationship between participation or procedure  
and the event. 
 
3. Unlikely: Little evidence to suggest a causal relationship (e.g. the event did not occur within a 
reasonable time after administration of the trial medication) or another reasonable explanation for the 
event (e.g. another clinical condition or other concomitant treatment). 
 
4. Definitely Unrelated: Current medical knowledge precludes a causal relationship between 
participation or procedure and the event or the cause of the reaction is known to be other than 
participation or procedure. 
 
 
PUBLICATION POLICY 
The results of this study may be published, depending on the results. 
 
 
SHARING OF FINAL RESEARCH DATA 
Data from this research will be shared with other researchers pursuant to the 02/26/2003 “NIH Final 
Statement on Sharing Research Data”. NIH believes that data sharing is important for further 
translation of research results into knowledge, products, and procedures to improve human health.  
The NIH endorses the sharing of final research data to serve these and other important scientific 
goals.  To protect subjects’ rights and confidentiality, identifiers will be removed from the data before 
they are shared. 
 
 
 
 

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TABLE 4: SCHEDULE OF PROCEDURES AND ASSESSMENTS 
 
Visit # 
1 
2 
3 
4 
 
5 
6 
Visit Name 
Screen2 
Baseline 
V1 
Baseline 
V2 
Re-
supply 
Phone  
Post-
Treatment 
V1 
Post-
Treatment 
V2 
 
 
 
 
 
 
 
 
Informed 
Consent 
X 
X3 
 
 
 
 
 
Demographics 
X 
X3 
 
 
 
 
 
Medical History 
X 
X3 
 
 
 
 
 
Concomitant 
Meds 
X 
X 
X 
X 
X 
X 
X 
Adverse Events 
X 
X 
X 
X 
X 
X 
X 
Nasal Exam 
X 
X 
X 
 
 
X 
X 
Physical and 
Neurological 
Exam 
X 
 
 
 
 
 
 
Vital Signs 
X 
X 
X 
X 
 
X 
X 
Height 
X 
 
 
 
 
 
 
Weight 
X 
X 
X 
X 
 
X 
X 
ECG 
X 
 
 
 
 
 
 
Cognitive 
Assessment 
X 
X 
X 
 
 
X 
X 
Questionnaires 
X 
X 
X 
 
 
X 
X 
Aptar CPS 
Device & 
Compliance 
Training 
 
X 
 
 
 
 
 
Compliance 
Assessment 
 
 
X 
X 
X 
X 
X 
Study Drug 
Dispensed 
 
 
X4 
X 
 
 
 
Actigraphy 
 
X 
 
 
 
X 
 
Continuous 
Glucose 
Monitor (CGM) 
Placement  
 
X 
 
 
 
X 
 
Blood Draw 
X 
 
X 
 
 
 
X 
- Clinical 
Labs 
X 
 
X 
 
 
 
X 
- ApoE 
Genotyping 
| DNA 
Banking 
X 
 
 
 
 
 
 
- Biomarkers 
| Plasma | 
Serum | 
Sample 
Banking 
 
 
X 
 
 
 
X 
LP 
 
 
X 
 
 
 
X 

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- CSF 
Biomarkers 
| Banking 
 
 
X 
 
 
 
X 
- Post-
Procedure 
Safety 
Telephone 
Check 
 
 
X 
 
 
 
X 
MRI 
 
X1 
 
 
 
X 
 
1MRI can be excluded if done within 6 months of enrollment 
2Screening may be waived if evaluations have been performed within the last 12 months 
3Will be performed if participant was eligible to skip Screening 
4Study drug may be re-dispensed as needed (if applicable) 

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LITERATURE CITED 
 
Albert, M. S., S. T. Dekosky, D. Dickson, B. Dubois, H. H. Feldman, N. C. Fox, A. Gamst, D. M. 
Holtzman, W. J. Jagust, R. C. Petersen, P. J. Snyder, M. C. Carrillo, B. Thies and C. H. Phelps 
(2011). "The diagnosis of mild cognitive impairment due to Alzheimer's disease: Recommendations 
from the National Institute on Aging and Alzheimer's Association workgroup." Alzheimers Dement. 
Baker, H. and R. F. Spencer (1986). "Transneuronal transport of peroxidase-conjugated wheat germ 
agglutinin (WGA-HRP) from the olfactory epithelium to the brain of the adult rat." Exp Brain Res 63(3): 
461-473. 
Baker, L. D., D. J. Cross, S. Minoshima, D. Belongia, G. S. Watson and S. Craft (2011). "Insulin 
resistance and Alzheimer-like reductions in regional cerebral glucose metabolism for cognitively 
normal adults with prediabetes or early type 2 diabetes." Arch Neurol 68(1): 51-57. 
Balin, B. J., R. D. Broadwell, M. Salcman and M. el-Kalliny (1986). "Avenues for entry of peripherally 
administered protein to the central nervous system in mouse, rat, and squirrel monkey." J Comp 
Neurol 251(2): 260-280. 
Benedict, C., M. Hallschmid, A. Hatke, B. Schultes, H. L. Fehm, J. Born and W. Kern (2004). 
"Intranasal insulin improves memory in humans." Psychoneuroendocrinology 29(10): 1326-1334. 
Benedict, C., W. Kern, B. Schultes, J. Born and M. Hallschmid (2008). "Differential sensitivity of men 
and women to anorexigenic and memory-improving effects of intranasal insulin." J Clin Endocrinol 
Metab 93(4): 1339-1344. 
Bodian, D. and H. A. Howe (1941). "Experimental studies on intraneuronal spread of poliomyelitis 
virus." Bull Johns Hopkins Hosp 68: 248-267. 
Born, J., T. Lange, W. Kern, G. P. McGregor, U. Bickel and H. L. Fehm (2002). "Sniffing 
neuropeptides: a transnasal approach to the human brain." Nat Neurosci 5(6): 514-516. 
Broadwell, R. D. and B. J. Balin (1985). "Endocytic and exocytic pathways of the neuronal secretory 
process and trans-synaptic transfer of wheat germ agglutinin-horseradish peroxidase in vivo." J Comp 
Neurol 242(4): 632-650. 
Cavanna, A. E. and M. R. Trimble (2006). "The precuneus: a review of its functional anatomy and 
behavioural correlates." Brain 129(Pt 3): 564-583. 
Chiu, S. L., C. M. Chen and H. T. Cline (2008). "Insulin receptor signaling regulates synapse number, 
dendritic plasticity, and circuit function in vivo." Neuron 58(5): 708-719. 
Craft, S., L. D. Baker, T. J. Montine, S. Minoshima, G. S. Watson, A. Claxton, M. Arbuckle, M. 
Callaghan, E. Tsai, S. R. Plymate, P. S. Green, J. Leverenz, D. Cross and B. Gerton (2012). 
"Intranasal insulin therapy for Alzheimer disease and amnestic mild cognitive impairment: a pilot 
clinical trial." Arch Neurol 69(1): 29-38. 
Craft, S., L. D. Baker, T. J. Montine, S. Minoshima, G. S. Watson, A. Claxton, M. Arbuckle, M. 
Callaghan, E. Tsai, S. R. Plymate, P. S. Green, J. Leverenz, D. J. Cross and B. Gerton (2012). 
"Intranasal Insulin Therapy for AD and MCI: A Pilot Clinical Trial." Arch Neurol 69: 29-38. 
Craft, S., E. Peskind, M. W. Schwartz, G. D. Schellenberg, M. Raskind and D. Porte, Jr. (1998). 
"Cerebrospinal fluid and plasma insulin levels in Alzheimer's disease: relationship to severity of 
dementia and apolipoprotein E genotype." Neurology 50(1): 164-168. 
Craft S, Raman R, Chow TW, Rafii MS, Sun CK, Rissman RA, Donohue MC, Brewer JB, Jenkins C, 
Harless K, Gessert D, Aisen PS. Safety, Efficacy, and Feasibility of Intranasal Insulin for the 
Treatment of Mild Cognitive Impairment and Alzheimer Disease Dementia: A Randomized Clinical 
Trial. JAMA Neurol. 2020 Sep 1;77(9):1099-1109. doi: 10.1001/jamaneurol.2020.1840. PMID: 
32568367; PMCID: PMC7309571. 
Craft, S. and G. S. Watson (2004). "Insulin and neurodegenerative disease: shared and specific 
mechanisms." Lancet Neurol 3(3): 169-178. 
De Felice, F. G., M. N. Vieira, T. R. Bomfim, H. Decker, P. T. Velasco, M. P. Lambert, K. L. Viola, W. 
Q. Zhao, S. T. Ferreira and W. L. Klein (2009). "Protection of synapses against Alzheimer's-linked 
toxins: insulin signaling prevents the pathogenic binding of Abeta oligomers." Proc Natl Acad Sci U S 
A 106(6): 1971-1976. 

14-JUL-2023 
IND #: 119232 
Page 38 of 40 
Faber, H. K. (1938). "The early lesions of poliomyelitis after intranasal inoculation." J Pediat 13(1): 10-
37. 
Fairbrother, R. W. and E. W. Hurst (1930). "The pathogenesis of, and propagation of the virus in, 
experimental poliomyelitis." J Path Bact 33: 17-45. 
Fishel MA, Watson GS, Montine TJ, Wang Q, Green PS, Kulstad JJ, Cook DG, Peskind ER, Baker 
LD, Goldgaber D, Nie W, Asthana S, Plymate SR, Schwartz MW, Craft S. 
Arch Neurol. 2005 Oct;62(10):1539-44. "Hyperinsulinemia provokes synchronous increases in central 
inflammation and beta-amyloid in normal adults." 
Francis, G. J., J. A. Martinez, W. Q. Liu, K. Xu, A. Ayer, J. Fine, U. I. Tuor, G. Glazner, L. R. Hanson, 
W. H. Frey, 2nd and C. Toth (2008). "Intranasal insulin prevents cognitive decline, cerebral atrophy 
and white matter changes in murine type I diabetic encephalopathy." Brain 131(Pt 12): 3311-3334. 
Frey, W. H. (2002). "Intranasal delivery: Bypassing the blood-brain barrier to deliver therapeutic 
agents to the brain and spinal cord." Drug Delivery Technology 2(5): 46-49. 
Frolich, L., D. Blum-Degen, H. G. Bernstein, S. Engelsberger, J. Humrich, S. Laufer, D. Muschner, A. 
Thalheimer, A. Turk, S. Hoyer, R. Zochling, K. W. Boissl, K. Jellinger and P. Riederer (1998). "Brain 
insulin and insulin receptors in aging and sporadic Alzheimer's disease." J Neural Transm 105(4-5): 
423-438. 
Galasko, D., D. Bennett, M. Sano, C. Ernesto, R. Thomas, M. Grundman and S. Ferris (1997). "An 
inventory to assess activities of daily living for clinical trials in Alzheimer's disease. The Alzheimer's 
Disease Cooperative Study." Alzheimer Dis Assoc Disord 11 Suppl 2: S33-39. 
Gasparini, L., G. K. Gouras, R. Wang, R. S. Gross, M. F. Beal, P. Greengard and H. Xu (2001). 
"Stimulation of beta-amyloid precursor protein trafficking by insulin reduces intraneuronal beta-
amyloid and requires mitogen-activated protein kinase signaling." J Neurosci 21(8): 2561-2570. 
Gil-Bea, F. J., M. Solas, A. Solomon, C. Mugueta, B. Winblad, M. Kivipelto, M. J. Ramirez and A. 
Cedazo-Minguez (2010). "Insulin levels are decreased in the cerebrospinal fluid of women with 
prodomal Alzheimer's disease." J Alzheimers Dis 22(2): 405-413. 
Grillo, C. A., G. G. Piroli, R. M. Hendry and L. P. Reagan (2009). "Insulin-stimulated translocation of 
GLUT4 to the plasma membrane in rat hippocampus is PI3-kinase dependent." Brain Res 1296: 35-
45. 
Hallschmid, M., C. Benedict, B. Schultes, J. Born and W. Kern (2008). "Obese men respond to 
cognitive but not to catabolic brain insulin signaling." Int J Obes (Lond) 32(2): 275-282. 
Hong, M. and V. M. Lee (1997). "Insulin and insulin-like growth factor-1 regulate tau phosphorylation 
in cultured human neurons." J Biol Chem 272(31): 19547-19553. 
Hughes, C. P., L. Berg, W. L. Danziger, L. A. Coben and R. L. Martin (1982). "A new clinical scale for 
the staging of dementia." Br J Psychiatry 140: 566-572. 
Illum, L. (2002). "Nasal drug delivery: new developments and strategies." Drug Discov Today 7(23): 
1184-1189. 
Kern, W., J. Born, H. Schreiber and H. L. Fehm (1999). "Central nervous system effects of intranasally 
administered insulin during euglycemia in men." Diabetes 48(3): 557-563. 
Kristensson, K. and Y. Olsson (1971). "Uptake of exogenous proteins in mouse olfactory cells." Acta 
Neuropathol 19(2): 145-154. 
Kupila, A., J. Sipila, P. Keskinen, T. Simell, M. Knip, K. Pulkki and O. Simell (2003). "Intranasally 
administered insulin intended for prevention of type 1 diabetes--a safety study in healthy adults." 
Diabetes Metab Res Rev 19(5): 415-420. 
Lee, C. C., Y. M. Kuo, C. C. Huang and K. S. Hsu (2009). "Insulin rescues amyloid beta-induced 
impairment of hippocampal long-term potentiation." Neurobiol Aging 30(3): 377-387. 
Minoshima, S., K. A. Frey, N. L. Foster and D. E. Kuhl (1995). "Preserved pontine glucose metabolism 
in Alzheimer disease: a reference region for functional brain image (PET) analysis." J Comput Assist 
Tomogr 19(4): 541-547. 
Minoshima, S., R. A. Koeppe, K. A. Frey and D. E. Kuhl (1994). "Anatomic standardization: linear 
scaling and nonlinear warping of functional brain images." J Nucl Med 35(9): 1528-1537. 

14-JUL-2023 
IND #: 119232 
Page 39 of 40 
Morris, J. C. (1993). "The Clinical Dementia Rating (CDR): current version and scoring rules." 
Neurology 43(11): 2412-2414. 
Morris, J. C., C. Ernesto, K. Schafer, M. Coats, S. Leon, M. Sano, L. J. Thal and P. Woodbury (1997). 
"Clinical dementia rating training and reliability in multicenter studies: the Alzheimer's Disease 
Cooperative Study experience." Neurology 48(6): 1508-1510. 
Petersen, R. C., R. Doody, A. Kurz, R. C. Mohs, J. C. Morris, P. V. Rabins, K. Ritchie, M. Rossor, L. 
Thal and B. Winblad (2001). "Current concepts in mild cognitive impairment." Arch Neurol 58(12): 
1985-1992. 
Pontiroli, A. E., M. Alberetto, A. Secchi, G. Dossi, I. Bosi and G. Pozza (1982). "Insulin given 
intranasally induces hypoglycaemia in normal and diabetic subjects." Br Med J (Clin Res Ed) 
284(6312): 303-306. 
Reger, M. A., G. S. Watson, W. H. Frey, 2nd, L. D. Baker, B. Cholerton, M. L. Keeling, D. A. Belongia, 
M. A. Fishel, S. R. Plymate, G. D. Schellenberg, M. M. Cherrier and S. Craft (2006). "Effects of 
intranasal insulin on cognition in memory-impaired older adults: modulation by APOE genotype." 
Neurobiol Aging 27(3): 451-458. 
Reger, M. A., G. S. Watson, P. S. Green, L. D. Baker, B. Cholerton, M. A. Fishel, S. R. Plymate, M. M. 
Cherrier, G. D. Schellenberg, W. H. Frey and S. Craft (2008). "Intranasal insulin administration dose-
dependently modulates verbal memory and plasma amyloid-beta in memory-impaired older adults." 
Journal of Alzheimers Disease 13(3): 323-331. 
Reger, M. A., G. S. Watson, P. S. Green, C. W. Wilkinson, L. D. Baker, B. Cholerton, M. A. Fishel, S. 
R. Plymate, J. C. Breitner, W. DeGroodt, P. Mehta and S. Craft (2008). "Intranasal insulin improves 
cognition and modulates beta-amyloid in early AD." Neurology 70(6): 440-448. 
Rivera, E. J., A. Goldin, N. Fulmer, R. Tavares, J. R. Wands and S. M. de la Monte (2005). "Insulin 
and insulin-like growth factor expression and function deteriorate with progression of Alzheimer's 
disease: link to brain reductions in acetylcholine." J Alzheimers Dis 8(3): 247-268. 
Rubin, D. B. (1987). Multiple Imputation for Nonresponse in Surveys. Hoboken, New Jersey, John 
Wiley & Sons, Inc. 
Sakane, T., M. Akizuki, Y. Taki, S. Yamashita, H. Sezaki and T. Nadai (1995). "Direct drug transport 
from the rat nasal cavity to the cerebrospinal fluid: the relation to the molecular weight of drugs." J 
Pharm Pharmacol 47(5): 379-381. 
Sano, M., R. Raman, J. Emond, R. G. Thomas, R. Petersen, L. S. Schneider and P. S. Aisen (2011). 
"Adding Delayed Recall to the Alzheimer Disease Assessment Scale is Useful in Studies of Mild 
Cognitive Impairment But Not Alzheimer Disease." Alzheimer Dis Assoc Disord 25(2): 122-127. 
Scheen AJ. Sodium-glucose cotransporter type 2 inhibitors for the treatment of type 2 diabetes 
mellitus. Nat Rev Endocrinol. 2020 Oct;16(10):556-577. doi: 10.1038/s41574-020-0392-2. Epub 2020 
Aug 27. PMID: 32855502. 
Selkoe, D. J. (2008). "Soluble oligomers of the amyloid beta-protein impair synaptic plasticity and 
behavior." Behav Brain Res 192(1): 106-113. 
Shipley, M. T. (1985). "Transport of molecules from nose to brain: transneuronal anterograde and 
retrograde labeling in the rat olfactory system by wheat germ agglutinin-horseradish peroxidase 
applied to the nasal epithelium." Brain Res Bull 15(2): 129-142. 
Stockhorst, U., D. de Fries, Y. Schottenfeld-Naor, A. Huebinger, H. J. Steingrueber and W. A. 
Scherbaum (Submitted for publication). "Intranasally administered insulin and its CNS effects in 
healthy humans: unconditioned and conditioned responses." 
Stockhorst, U., D. de Fries, H. J. Steingrueber and W. A. Scherbaum (2004). "Insulin and the CNS: 
effects on food intake, memory, and endocrine parameters and the role of intranasal insulin 
administration in humans." Physiol Behav 83(1): 47-54. 
Talairach, J. and P. Tournoux (1988). Co-planar stereotaxic atlas of the human brain: 3-dimensional 
proportional system: an aproach to cerebral imaging. New York, New York, Thieme Medical 
Publishers. 
Thorne, R. G., C. R. Emory, T. A. Ala and W. H. Frey, 2nd (1995). "Quantitative analysis of the 
olfactory pathway for drug delivery to the brain." Brain Res 692(1-2): 278-282. 

14-JUL-2023 
IND #: 119232 
Page 40 of 40 
Thorne, R. G., G. J. Pronk, V. Padmanabhan and W. H. Frey, 2nd (2004). "Delivery of insulin-like 
growth factor-I to the rat brain and spinal cord along olfactory and trigeminal pathways following 
intranasal administration." Neuroscience 127(2): 481-496. 
Townsend, M., T. Mehta and D. J. Selkoe (2007). "Soluble Abeta inhibits specific signal transduction 
cascades common to the insulin receptor pathway." J Biol Chem 282(46): 33305-33312. 
Weiss, P. and Y. Holland (1967). "Neuronal dynamics and axonal flow, ii. The olfactory nerve as 
model test object." Proc Natl Acad Sci U S A 57(2): 258-264. 
Worsley, K. J., A. C. Evans, S. Marrett and P. Neelin (1992). "A three-dimensional statistical analysis 
for CBF activation studies in human brain." J Cereb Blood Flow Metab 12(6): 900-918. 
Zhao, L., B. Teter, T. Morihara, G. P. Lim, S. S. Ambegaokar, O. J. Ubeda, S. A. Frautschy and G. M. 
Cole (2004). "Insulin-degrading enzyme as a downstream target of insulin receptor signaling cascade: 
implications for Alzheimer's disease intervention." J Neurosci 24(49): 11120-11126. 
Zhao, W. Q. and M. Townsend (2009). "Insulin resistance and amyloidogenesis as common molecular 
foundation for type 2 diabetes and Alzheimer's disease." Biochim Biophys Acta 1792(5): 482-496. 
Yu L, Buysse DJ, Germain A, Moul DE, Stover A, Dodds NE, Johnston KL, Pilkonis PA. Development 
of short forms from the PROMIS™ sleep disturbance and Sleep-Related Impairment item banks. 
Behav Sleep Med. 2011 Dec 28;10(1):6-24. doi: 10.1080/15402002.2012.636266. PMID: 22250775; 
PMCID: PMC3261577. 
Kroenke K, Spitzer RL, Williams JB. The PHQ-9: validity of a brief depression severity measure. J 
Gen Intern Med. 2001 Sep;16(9):606-13. doi: 10.1046/j.1525-1497.2001.016009606.x. PMID: 
11556941; PMCID: PMC1495268. 
Spitzer RL, Kroenke K, Williams JB, Löwe B. A brief measure for assessing generalized anxiety 
disorder: the GAD-7. Arch Intern Med. 2006 May 22;166(10):1092-7. doi: 
10.1001/archinte.166.10.1092. PMID: 16717171.