Conditions: Amnestic Mild Cognitive Impairment Alzheimer's Disease
Keywords: Intranasal insulin Alzheimer's disease Amnestic mild cognitive impairment Dementia Brain diseases Memory problems Mental disorders Cognitive disorders
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 240
Healthy volunteers: no

Brief summary:
An urgent need exists to find effective treatments for Alzheimer's disease (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. This study will examine a novel therapeutic approach using intranasal insulin (INI) that has shown promise in short-term clinical trials. If successful, information gained from the study has the potential to move INI forward rapidly as a therapy for AD. The study will also provide evidence for the mechanisms through which INI may produce benefits by examining key cerebral spinal fluid (CSF) biomarkers and hippocampal/entorhinal atrophy. These results will have considerable clinical and scientific significance, and provide therapeutically-relevant knowledge about insulin's effects on AD pathophysiology. Growing evidence has shown that insulin carries out multiple functions in the brain, and that insulin dysregulation may contribute to AD pathogenesis.

This study will examine the effects of intranasally-administered insulin on cognition, entorhinal cortex and hippocampal atrophy, and cerebrospinal fluid (CSF) biomarkers in amnestic mild cognitive impairment (aMCI) or mild AD. It is hypothesized that after 12 months of treatment with INI compared to placebo, subjects will improve performance on a global measure of cognition, on a memory composite and on daily function. In addition to the examination of CSF biomarkers and hippocampal and entorhinal atrophy, the study aims to examine whether baseline AD biomarker profile, gender, or Apolipoprotein epsilon 4 (APOE-ε4) allele carriage predict treatment response.

In this study, 240 people with aMCI or AD will be given either INI or placebo for 12 months, following an open-label period of 6 months where all participants will be given active drug. The study uses insulin as a therapeutic agent and intranasal administration focusing on nose to brain transport as a mode of delivery.

Detailed description:

Arms:
arm 1: 120 subjects will take two daily doses of INI (20 IU bid for a total daily dose of 40 IU) approximately 30 minutes after breakfast and dinner for 12 months. A 6-month open label period will follow in which all participants will receive INI. arm 2: 120 subjects will take two daily doses of placebo approximately 30 minutes after breakfast and dinner for 12 months. A 6-month open label period will follow in which all participants will receive INI.

Interventions: intervention 1: Insulin (Humulin® R U-100) intervention 2: Placebo
Intervention descriptions: intervention 1: 20 IU bid taken twice daily (approximately 30 minutes after breakfast and dinner) for a total of 40 IU daily, which will be administered intranasally. The device used to administer insulin releases a metered dose into a chamber covering the participant's nose. The insulin is then inhaled by breathing evenly over a specified period. intervention 2: Placebo taken twice daily (approximately 30 minutes after breakfast and dinner), which will be administered intranasally. The device used to administer placebo releases a metered dose into a chamber covering the participant's nose. The placebo is then inhaled by breathing evenly over a specified period.

Protocol document:
Prot_000.pdf: 
ADC-046-INI 
IND #: 119,232
NCT01767909 
Page 1 of 55 
Therapeutic effects of intranasally-administered insulin (INI)  
in adults with amnestic mild cognitive impairment (aMCI) or mild 
Alzheimer’s disease (AD) 
Protocol #: ADC-046-INI 
IND #: 119,232 
PROJECT DIRECTOR / 
IND SPONSOR 
COORDINATION & DATA 
MANAGEMENT CENTER 
Suzanne Craft, Ph.D. 
Wake Forest University 
School of Medicine 
Medical Center Boulevard 
Winston-Salem, NC  27157-1207 
Paul Aisen, M.D. 
ATRI Director 
Alzheimer’s Therapeutic Research Institute 
(ATRI) 
University of Southern California 
PROTOCOL VERSION 
VERSION DATE 
Final Protocol v5.0 
05DEC2016 


 
 
  
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TABLE OF CONTENTS 
1.0 
INTRODUCTION ............................................................................................................... 11 
1.1 
Primary Aim ................................................................................................................... 11 
1.2 
Secondary Aims ............................................................................................................. 11 
1.2.1 
Secondary Aim 1 ..................................................................................................... 11 
1.2.2 
Secondary Aim 2 ..................................................................................................... 11 
1.2.3 
Secondary Aim 3 ..................................................................................................... 11 
1.2.4 
Secondary Aim 4 ..................................................................................................... 11 
1.2.5 
Secondary Aim 5 ..................................................................................................... 11 
2.0 
BACKGROUND AND SIGNIFICANCE ............................................................................. 12 
2.1 
Rationale for Insulin ....................................................................................................... 12 
2.2 
Insulin as a Therapeutic Agent ...................................................................................... 12 
2.2.1 
Intranasal Pathways to the CNS ............................................................................. 13 
2.2.2 
Intranasal Delivery System Device ......................................................................... 13 
3.0 
PRELIMINARY STUDY ..................................................................................................... 15 
3.1 
Participants .................................................................................................................... 15 
3.2 
Procedures ..................................................................................................................... 16 
3.3 
Safety and Compliance .................................................................................................. 16 
3.4 
Statistical Analyses ........................................................................................................ 16 
3.5 
Results: Cognitive and Functional Outcome Measures ................................................. 17 
3.6 
Results: AD Biomarkers ................................................................................................. 18 
3.7 
Results: FDG-PET CMRglc ........................................................................................... 18 
3.8 
Safety and Compliance .................................................................................................. 19 
3.9 
Implications .................................................................................................................... 20 
4.0 
STUDY DESIGN RATIONALE .......................................................................................... 20 
4.1 
Rationale for Population Selection ................................................................................. 20 
4.2 
Rationale for Dosage Selection ..................................................................................... 20 
4.3 
Rationale for Primary and Secondary Outcome Measures ............................................ 21 
4.4 
Rationale for Length and Design of Trial ....................................................................... 21 
4.5 
Rationale for Magnetic Resonance Imaging .................................................................. 21 
4.6 
Rationale for Biofluids .................................................................................................... 22 
5.0 
STUDY OVERVIEW .......................................................................................................... 22 
5.1 
Study Population ............................................................................................................ 23 
5.2 
Diagnosis Criteria ........................................................................................................... 23 
5.3 
Inclusion Criteria ............................................................................................................ 24 
5.4 
Exclusion Criteria ........................................................................................................... 24 
5.5 
Recruitment and Retention Strategies ........................................................................... 25 
6.0 
STUDY TIMELINE ............................................................................................................. 26 
7.0 
DESCRIPTION OF STUDY VISITS ................................................................................... 26 
7.1 
Pre-Screening ................................................................................................................ 26 
7.2 
Screening (Visit 1) .......................................................................................................... 26 
7.3 
Baseline (Visit 2) ............................................................................................................ 27 
7.4 
Month 3 (Visit 3) ............................................................................................................. 28 
7.5 
Month 6 (Visit 4) ............................................................................................................. 28 
7.6 
Month 9 (Visit 5) ............................................................................................................. 28 
7.7 
Month 12 (Visit 6) ........................................................................................................... 29 


 
 
  
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7.8 
Month 15 (Visit 7, Open Label) ...................................................................................... 29 
7.9 
Month 18 (Visit 8, Open Label) ...................................................................................... 30 
8.0 
EARLY TREATMENT/STUDY DISCONTINUATION ........................................................ 30 
8.1 
Reasons for Early Discontinuation ................................................................................. 30 
9.0 
STUDY-SPECIFIC PROCEDURES ................................................................................... 31 
9.1 
Cognitive Evaluations .................................................................................................... 31 
9.1.1 
Alzheimer’s Disease Assessment Scale-Cognitive (ADAS-Cog12) ........................ 31 
9.1.2 
Mini-Mental State Examinations (MMSE) ............................................................... 31 
9.1.3 
Memory Composite (Story Recall and Free and Cued Selective Reminding Test) 31 
9.1.4 
Logical Memory Test (Wechsler Memory Scale-Revised) ...................................... 32 
9.1.5 
Trail-making Test (Part A and B) ............................................................................ 32 
9.2 
Clinical and Functional Evaluations ............................................................................... 32 
9.2.1 
Clinical Dementia Rating Scale – Sum of Boxes (CDR-SB) ................................... 32 
9.2.2 
ADCS-MCI Activities of Daily Living (ADCS-ADL-MCI) .......................................... 32 
9.2.3 
Neuropsychiatric Inventory (NPI) ............................................................................ 33 
9.2.4 
Modified Hachinski .................................................................................................. 33 
9.3 
Research Satisfaction Survey ........................................................................................ 33 
9.4 
Treatment Blinding Questionnaire ................................................................................. 33 
10.0 STUDY METHODS ............................................................................................................ 33 
10.1 
Safety Assessments .................................................................................................... 33 
10.2 
Physical and Neurological Examination ....................................................................... 34 
10.3 
Electrocardiogram (ECG) ............................................................................................. 34 
10.4 
Clinical Laboratory Evaluations .................................................................................... 34 
11.0 BIOMARKER STUDIES .................................................................................................... 34 
11.1 
CSF .............................................................................................................................. 34 
11.2 
Blood Collection ........................................................................................................... 35 
11.3 
Insulin & Glucose (HgA1c Test) ................................................................................... 35 
11.4 
Magnetic Resonance Imaging (MRI) ........................................................................... 36 
11.5 
Genetic Samples, Storage and Future Use ................................................................. 36 
12.0 STATISTICAL PLAN ......................................................................................................... 36 
12.1 
Power Analyses ........................................................................................................... 37 
13.0 POTENTIAL RISKS .......................................................................................................... 38 
13.1 
Safety of Intranasal Insulin ........................................................................................... 38 
13.2 
Risks associated with use of the POD device .............................................................. 39 
13.3 
Lumbar Puncture ......................................................................................................... 39 
13.4 
MRI .............................................................................................................................. 39 
13.5 
Blood Draw .................................................................................................................. 40 
14.0 PERSONNEL REQUIREMENTS ...................................................................................... 40 
15.0 STUDY DRUG ................................................................................................................... 41 
15.1 
Humulin® R U-100 Insulin ............................................................................................ 41 
15.2 
Placebo (Sterile Diluent) .............................................................................................. 41 
15.3 
Randomization ............................................................................................................. 41 
15.4 
Blinding ........................................................................................................................ 41 
15.5 
Study Drug Dispensing ................................................................................................ 41 
15.6 
Intranasal Administration ............................................................................................. 42 
15.7 
Compliance .................................................................................................................. 42 
15.8 
Dose Adjustments ........................................................................................................ 42 


 
 
  
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15.9 
Breaking the Blind ........................................................................................................ 42 
15.10 
Storage ...................................................................................................................... 42 
15.11 
Drug Accountability .................................................................................................... 43 
16.0 ADVERSE EVENTS .......................................................................................................... 43 
16.1 
Definition ...................................................................................................................... 43 
16.2 
Following Up on AEs .................................................................................................... 43 
17.0 SERIOUS ADVERSE EVENTS (SAE) .............................................................................. 43 
17.1 
Definition ...................................................................................................................... 43 
17.2 
Reporting SAEs ........................................................................................................... 44 
18.0 ETHICS & REGULATORY CONSIDERATIONS .............................................................. 44 
18.1 
Ethical Standard ........................................................................................................... 44 
18.2 
Institutional Review Board (IRB) .................................................................................. 44 
18.3 
Informed Consent & HIPAA Authorization ................................................................... 45 
18.4 
Participant Confidentiality | HIPAA ............................................................................... 45 
19.0 GENETIC RESEARCH & STORAGE OF GENETIC MATERIAL ..................................... 46 
19.1 
Storage of Biospecimen Samples ................................................................................ 46 
20.0 RISKS AND BENEFITS ASSOCIATED WITH THIS STUDY ........................................... 46 
20.1 
Potential Benefits of the Proposed Research to Human Subjects ............................... 46 
20.2 
Inclusion of Women and Minorities .............................................................................. 47 
20.3 
Inclusion of Children as Participants in Research Involving Human Subjects ............. 47 
21.0 STUDY MONITORING & DATA COLLECTION ............................................................... 47 
21.1 
Communication Plan & Site Monitoring ....................................................................... 48 
22.0 DATA AND SAFETY MONITORING PLAN AND BOARD ............................................... 48 
23.0 PUBLICATION POLICY .................................................................................................... 49 
24.0 CLINICAL TRIAL REGISTRATION & SHARING OF FINAL RESEARCH DATA ........... 49 
25.0 TABLE 4: SCHEDULE OF PROCEDURES AND ASSESSMENTS ................................ 50 
26.0 LITERATURE CITED ........................................................................................................ 51 


 
 
  
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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) 
ATRI 
ALZHEIMER’S THERAPEUTIC RESEARCH INSTITUTE 
BDNF 
BRAIN-DERIVED NEUROTROPHIC FACTOR 
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 


 
 
  
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EAG 
ESTIMATED AVERAGE GLUCOSE 
ECG 
ELECTROCARDIOGRAM 
EDC 
ELECTRONIC DATA CAPTURE 
EDTA 
ETHYLENE DIAMINE TETRA ACETIC ACID 
ELISA 
ENZYME-LINKED IMMUNOSORBENT ASSAY 
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 
IU 
INTERNATIONAL UNIT 
LDH 
LACTATE DEHYDROGENASE 
LP 
LUMBAR PUNCTURE 
LTP 
LONG TERM POTENTIATION 
MCV 
MEAN CORPUSCULAR VOLUME 
ML 
MILLILITER 


 
 
  
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MMA 
METHYLMALONIC ACID 
MMSE 
MINI MENTAL STATE EXAMINATION 
MPRAGE 
MAGNETIZATION PREPARED RAPID GRADIENT ECHO 
MR/MRI 
MAGNETIC RESONANCE / MAGNETIC RESONANCE IMAGING 
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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SIGNATURE PAGE 
 
Protocol #: ADC-046-INI 
IND #: 119,232 
Final Protocol v. 5.0 
05-December-2016 
 
(SIGNATURES ON FILE AT ATRI) 
 
 
 
 
 
 
 
 
_______________________________________ 
 
___________________ 
Tiffany Chow, M.D.  
 
 
 
 
Date (MM/DD/YYYY) 
Medical Monitor 
Alzheimer’s Therapeutic Research Institute  
 
 
 
_______________________________________ 
 
___________________ 
Suzanne Craft, Ph.D.  
 
 
 
 
Date (MM/DD/YYYY) 
Project Director 
Wake Forest University 
 
 
 
 
_______________________________________ 
 
___________________ 
Paul Aisen, M.D. 
 
 
 
 
 
Date (MM/DD/YYYY) 
Director 
Coordination & Data Management Center 
Alzheimer’s Therapeutic Research Institute 


 
 
  
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PROTOCOL SYNOPSIS 
 
TITLE  
 
 
Therapeutic effects of intranasally-administered insulin (INI) 
in adults with amnestic mild cognitive impairment (aMCI) or 
mild Alzheimer’s disease (AD) 
PROJECT DIRECTOR 
Suzanne Craft, Ph.D. 
STUDY SPONSOR 
National Institute on Aging 
STUDY PHASE  
 
 
Phase II/III 
INDICATION 
 
 
 
Amnestic mild cognitive impairment (aMCI) or probable mild 
Alzheimer’s disease (AD) 
AIM OF STUDY 
To examine the effects of intranasally-administered insulin 
(INI) on cognition, entorhinal cortex and hippocampal 
atrophy, and cerebrospinal fluid biomarkers in amnestic mild 
cognitive impairment (aMCI) or probable mild Alzheimer’s 
disease (AD)  
PRIMARY OBJECTIVE 
To test the hypothesis that 12 months of treatment with INI 
(compared to placebo) in adults with aMCI and probable 
mild AD will improve performance on a global measure of 
cognition  
SECONDARY OBJECTIVES 
1. To test the hypothesis that 12 months of treatment with 
INI (compared to placebo) in adults with aMCI and 
probable mild AD will improve performance on a 
memory composite (Story Recall and FCSRT) and daily 
functioning. 
 
2. To test the hypothesis that INI treatment reduces the 
rate of hippocampal and entorhinal atrophy as 
measured by MRI, and to conduct exploratory analyses 
of other brain regions. 
 
3. To test the hypothesis that INI will favorably alter CSF 
Aβ and the CSF Aβ/tau ratio, and will modulate 
inflammatory markers. 
 
4. To examine whether baseline AD biomarker profile, 
APOE-ε4 allele carriage and gender predict treatment 
response. 
 
5. To determine whether further improvement occurs after 
18 months of treatment. 
PRIMARY OUTCOME MEASURE 
Alzheimer’s 
Disease 
Assessment 
Scale-Cognition 
12 
(ADAS-Cog12) 


 
 
  
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SECONDARY OUTCOME 
MEASURES  
Memory composite, executive function test, CDR-SB, 
MMSE, ADCS-ADL-MCI, rate of hippocampal and entorhinal 
atrophy, CSF total tau, Aβ40, Aβ42, phospho-tau 181 
STUDY DESIGN  
Multi-site, double-blind, placebo-controlled study in 240-300 
participants with aMCI or probable mild AD for 12 months, 
followed by a 6-month open-label period in which all 
participants will receive INI.  
SAMPLE SIZE  
• 
n=240-300 
• 
50% (n=120-150) assigned to active INI (20 IU bid); 50% 
(n=120-150) assigned to placebo 
SUMMARY OF KEY ELIGIBILITY 
CRITERIA  
 
• 
Diagnosis of aMCI or probable mild AD according to the 
core clinical criteria updated in the NIA and Alzheimer’s 
Association guidelines.   
• 
Age: 55 to 85 yrs (inclusive) 
• 
MMSE > 20 at screening  
• 
Clinical Dementia Rating 0.5-1 at screening 
• 
For aMCI only: Logical Memory ≤10 for 16 or more years 
of education, ≤6 for 8-15 years of education, ≤4 for 0-7 
years of education (Delayed Paragraph Recall of 
Wechsler 
Memory 
Scale–Revised). 
Measured 
at 
screening 
• 
Modified Hachinski score ≤ 4 
DRUG DOSAGE & FORMULATION 
20 IU Humulin® R U-100 or matching placebo twice daily 
(bid) for a total of 40 IU daily 
DURATION OF PARTICIPATION 
The approximate timeline for this four-year study is 
projected as: 1) approximately six months for study startup 
activities including FDA and IND submission, site IRB and 
regulatory approval, and training meeting; 2) approximately 
18 months for recruitment; 3) 18 months of follow up (last 6 
months are open label) 
PLACEBO 
A matching placebo (sterile diluent) will be used 
ROUTE OF ADMINISTRATION 
 
Intranasal 
PROCEDURES  
Physical and neurological exam, nasal examination, optional 
lumbar puncture, MRI, ECG, Modified Hachinski, ADAS-
Cog12, CDR-SB, ADCS-ADL-MCI, memory composite 
(Story Recall and FCSRT), Trail-making test, MMSE, NPI, 
Vitals, Clinical Labs, CSF analysis & banking, Peripheral 
Blood Mononuclear Cell (PBMC) isolation & banking, 
plasma biomarkers and genotyping. 


 
 
  
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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. This study will 
examine a novel therapeutic approach using intranasally administered insulin (INI) that has 
shown promise in short-term clinical trials. If successful, information gained from the study has 
the potential to move INI forward rapidly as a therapy for AD. The study will also provide 
evidence for the mechanisms through which INI may produce benefits by examining key CSF 
biomarkers and hippocampal/entorhinal atrophy. These results will have considerable clinical 
and scientific significance, and provide therapeutically-relevant knowledge about insulin’s 
effects on AD pathophysiology. 
 
1.1 
Primary Aim 
 
To test the hypothesis that 12 months of treatment with INI (compared to placebo) in adults with 
aMCI or probable mild AD will improve performance on a global measure of cognition (ADAS-
Cog12).  
 
1.2 
Secondary Aims 
 
1.2.1 Secondary Aim 1 
 
To test the hypothesis that 12 months of treatment with INI (compared to placebo) in adults with 
aMCI or probable mild AD will improve performance on a memory composite (Story Recall and 
FCSRT) and on daily function in adults with aMCI and mild AD.  
 
1.2.2 Secondary Aim 2 
 
To test the hypothesis that INI treatment reduces the rate of hippocampal and entorhinal 
atrophy as measured by MRI, and conduct exploratory analyses of other brain regions. 
 
1.2.3 Secondary Aim 3 
 
To test the hypothesis that INI will favorably alter CSF Aβ and the CSF Aβ/tau ratio, and will 
modulate inflammatory markers. 
 
1.2.4 Secondary Aim 4 
 
To examine whether baseline AD biomarker profile, gender or APOE-ε4 allele carriage predict 
treatment response.  
 
1.2.5 Secondary Aim 5 
 
To determine whether further improvement occurs after 18 months of treatment. 
 
 
 


 
 
  
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2.0 
BACKGROUND AND SIGNIFICANCE 
 
2.1 
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 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. 
 
2.2 
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 


 
 
  
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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. 
 
2.2.1 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 IU/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 preliminary study in which 
insulin was administered to adults with AD or aMCI for 4 months. 
 
2.2.2 Intranasal Delivery System Devices 
 
The Precision Olfactory Delivery (POD) Device developed by Impel NeuroPharma (Seattle, WA) 
will be provided to study participants. Typical spray bottle administration results in large droplets 
that penetrate only within the first 20% of the lower nasal cavity.  Due to gravity and insufficient 
airflow, approximately 90% of the droplets end up in the stomach. The POD device  delivers a 
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maximizing access to nose-to-brain channels.   
 
The POD device reliably delivers drug to the upper nasal cavity by employing a commercially 
available metered dose inhaler canister, an actuator containing a diffuser and a nasal tip.  The 
canister contains liquid hydrofluoralkane (HFA), a propellant used whose use in metered dose 
inhalers was required by the FDA in 2012 as a replacement to chlorofluorocarbons. HFA 
propels the study drug to the nasal cavity.  The HFA travels through the POD actuator body and 
enters the POD tip, where it mixes with the study drug formulation.  The study drug formulation 
and HFA are then sprayed from the device. 
 
A drug-device compatibility and functionality verification of the Impel NeuroPharma clinical 
Precision Olfactory Delivery device and the Novolog® insulin formulation was conducted (Impel 
NeuroPharma, 2015) to identify the optimal dose volume of Novolog® insulin required to 
achieve the target dose and confirm that the POD device consistently delivered the desired 
amount of both insulin and placebo. A qualitative assessment using an anatomically 
representative human nasal cavity model (derived from Liu, Johnson et al, 2008) confirmed that 
the insulin deposition was concentrated in the upper nasal cavity region.  


 
 
  
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3.0 
PRELIMINARY STUDY 
 
A preliminary study using the Kurve ViaNaseTM device – a device similar to the Impel POD 
device in terms of drug delivery action -- examined the impact of 4-month INI administration (10 
or 20 IU 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 (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).  Given the 
similar drug delivery action and equivalent dosing between the Kurve ViaNaseTM and Impel 
NeuroPharma POD devices, the preliminary study is applicable to the Impel POD device used in 
this study. 
 
3.1 
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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3.2 
Procedures 
 
Participants were randomized to receive 10 IU INI bid for a total daily dose of 20 IU INI (n=36), 
20 IU INI bid for a total dose of 40 IU (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). 
 
3.3 
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. 
 
3.4 
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 


 
 
  
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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 projections. 
 
3.5 
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. 
 


 
 
  
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Figure 2 | Change (Month 4-baseline) in log scores for (A) delayed story recall, (B) DSRS, (C) 
ADAS-Cog and (D) ADCS-ADL. 
 
 
3.6 
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. 
 
3.7 
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. 
 


 
 
  
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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 
 
 
3.8 
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. 


 
 
  
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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. 
 
 
3.9 
 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. 
 
4.0 
STUDY DESIGN RATIONALE 
 
4.1 
Rationale for Population Selection 
 
Similar to previous pilot trials that yielded positive results (Craft, Baker et al. 2012), participants 
with aMCI or probable mild AD will be enrolled in the current study.  The rationale for this 
approach is based on the lack of clear clinical or neuropathologic borders between the two 
conditions, such that recent reviews on trial design have suggested a combined group approach 
(Aisen, Andrieu et al. 2011). Enrollment will not be restricted to biomarker-positive patients.  The 
trial will collect CSF biomarkers and MRI measures of atrophy.  Thus, secondary analyses will 
explore the relationship of biomarkers to treatment response. Inclusionary criteria parallel the 
Alzheimer’s Disease Neuroimaging Initiative (ADNI) criteria, which will allow comparisons of 
imaging and biomarker data to the large ADNI cohort. 
 
4.2 
Rationale for Dosage Selection 
  
The dosage selected (20 IU INI bid for a total of 40 IU INI daily) showed greater effect sizes for 


 
 
  
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the ADAS-Cog and greater ability to preserve cerebral glucose metabolism with PET with a 
good safety profile than did a lower 10 IU dose (Craft, Baker et al. 2012). Although the delayed 
story recall did not change with 20 IU INI bid, the trial used different inclusion criteria than the 
current study (MMSE ≥15 compared with MMSE ≥ 20) and did not sum immediate and delayed 
story recall. Exploratory analyses were conducted comparing the 20 IU bid dose with placebo in 
only participants with MMSE ≥ 20, and summed immediate and delayed recall. A significant time 
by treatment interaction was observed (p<0.05), reflecting improved performance for the 20 IU 
bid INI-treated group and decline for the placebo group (mean logical memory total story recall 
change score with standard error = .15 (.1) for placebo vs .12 (.09) for the 20 bid IU INI group) 
(Craft, Baker et al. 2012). 
 
4.3 
Rationale for Primary and Secondary Outcome Measures  
 
Recent results from ADNI have shown that the ADAS-Cog changes reliably over 12 months in 
both aMCI and AD groups; these results served as the basis for the study power analyses. A 
version of the ADAS-Cog that includes a delayed list recall will be used as primary outcome 
measure. Although adding delayed recall to ADAS-Cog does not improve ability to detect 
change over time in AD patients because of increased variability, it does provide added 
sensitivity to detect change in aMCI (Petersen, Aisen et al. 2010). ADAS-Cog12 will be 
administered at 3 month intervals to increase power and optimize imputation of missing values. 
Several secondary outcome measures in cognitive and functional domains were chosen as they 
have previously shown to be sensitive to INI and have demonstrated significant decline over a 
12-month period for aMCI and mild AD (Petersen, Aisen et al. 2010).  In particular, a memory 
composite that combines immediate and delayed story recall scores and the Free and Cued 
Selective Reminding Test (FCSRT) will be used. The two measures will be combined with equal 
weighting in order to be sensitive to improved memory for both aMCI and AD. With respect to 
MRI measures, both hippocampal and entorhinal volume changes occurred over 12 months in 
ADNI (Petersen, Aisen et al. 2010). 
 
4.4 
Rationale for Length and Design of Trial  
 
In the pilot study (Craft, Baker et al. 2012), beneficial effects of insulin on cognition, function and 
cerebral glucose metabolism were detected in a 4-month period. The current study design will 
extend the treatment period to a year for primary analyses, which should be sufficient to detect 
clear changes in memory and imaging endpoints for both aMCI and AD groups. The additional 
6–month, open-label extension used in this semi-crossover design may incentivize participants 
to enroll and remain in the trial, and also provide added power for safety analyses (Jarjoura 
2003). Exploratory efficacy analyses may also provide useful information, although these results 
will need to be interpreted with caution in light of their unblinded status. 
 
4.5 
Rationale for Magnetic Resonance Imaging 
 
Recent data from ADNI demonstrate that structural MRI can be a credible predictor of future 
cognitive and functional decline, and it is sensitive to volume reductions over a 12-month period 
(Vemuri, Wiste et al. 2009). Jack, et al, examined the role of structural MRI as an endpoint for 
disease progression in therapeutic trials for AD and reported that multi-site consistency is 
feasible (Jack, Slomkowski et al. 2003). They also report hippocampal atrophy as one of the 
most reliable correlates of AD progression, such that mitigation of progression would be 
detectable over a year, given adequate power. Recent studies have also shown that entorhinal 
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(Holland, Brewer et al. 2009). Given this finding, we will evaluate INI effects on both 
hippocampal and entorhinal volumes as our primary MRI outcome measures. However, we will 
also conduct exploratory analyses on other regions such as the precuneus and cuneus regions 
that have shown INI effects with FDG PET in a previous study (Craft, Baker et al. 2012).  
 
Each site’s scanner will have an identifying number, which will be appended to the image 
header of all MRI data. Each scanner will be subjected to a qualifying process that includes an 
evaluation for excessive vibration or other image artifact revealed through submitted MR images 
obtained through a standard protocol of an American College of Radiology phantom. In addition, 
3D T1 MPRAGE or IR-FSPGR images of a human brain obtained through a standardized 
imaging protocol of a volunteer at each site will be submitted for automated segmentation and 
the resulting segmentations will be examined centrally for anatomical accuracy.  Once the 
phantom and volunteer scan pass quality control, the scanner will be considered certified for the 
INI study. Because longitudinal analysis of brain volumetry will be used in this study, sites will 
be required to use the same scanner for both scans of a particular participant. Each site will be 
required to procure a clinical read of each participant’s MRI data by a board certified radiologist. 
In addition, the image-derived inclusion and exclusion criteria will be evaluated centrally. All 
imaging data will be de-identified through a numerical coding system. 
 
4.6 
Rationale for Biofluids 
 
CSF biomarkers such as CSF Aβ42, CSF Aβ40 and CSF total tau will be measured in 
participants agreeing to the optional lumbar puncture. Plasma biomarkers including Aβ42, and 
Aβ40 will be also assessed. Plasma and CSF samples obtained in this study will be banked so 
that other putative biomarkers (such as CSF insulin, isoprostane 8,12-iso-iPF2alpha-VI) may be 
measured by qualified investigators in the future given adequate rationale and feasibility. These 
putative biomarkers are among the leading candidates for AD biomarker development (Turner 
2003; Grossman, Farmer et al. 2005).  For example, Gilman et al, report a highly significant (p < 
0.001) effect of Aβ immunization (with AN1792) on CSF tau with a total sample size of only 21 
(N = 11 antibody responders and 10 placebo control participants; Gilman, Koller et al. 2005).  
 
Several previous studies suggest that response to insulin may differ according to APOE 
genotype. In dose response studies that acutely elevated insulin through intravenous or 
intranasal administration, the greatest cognitive benefit was observed for adults with AD who 
were not ε4 carriers (Reger, Watson et al. 2008). The current study will examine ε4 carriage as 
a treatment response predictor.  
 
Although Alzheimer's disease (AD) is a degenerative brain disorder, it may also affect peripheral 
tissue such as peripheral blood mononuclear cells (PBMCs). It has been suggested that PBMC-
associated biomarkers may provide insights into the pathogenesis of AD and be used to monitor 
disease diagnosis and progression (Maes, Schipper et al. 2009). Thus biochemical analysis of 
the effects of intranasal insulin on PBMCs may provide some insights into therapeutic 
mechanisms. In addition, we will be able to compare treatment-related biomarker profiles in 
CSF and PBMCs. 
 
5.0 
STUDY OVERVIEW 
 
The study will consist of a multisite, randomized, double-blind trial comparing the effects of INI 
(20 IU bid for total daily dose of 40 IU) and placebo for 12 months, followed by a 6-month open-
label period in which all participants will receive INI. Participants with aMCI or mild AD (n=240-


 
 
  
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300) will be enrolled. The primary outcome measure will consist of the ADAS-Cog12. Secondary 
measures will include a memory composite, an executive function test, CDR Sum of Boxes 
(CDR-SB), and the ADCS-ADL-MCI. The ADAS-Cog12 will be administered at 3 month intervals 
to optimize imputation of missing data. Other cognitive and functional measures will be 
administered at 6 month intervals. MRI measures of entorhinal cortex and hippocampal atrophy 
will be obtained at screening and 12 months. CSF and plasma biomarkers, as well as PBMCs 
and APOE-ε4 allele carriage will also be assessed. The trial will be conducted in collaboration 
with the Alzheimer Therapeutic Research Institute (ATRI).   
 
5.1 
Study Population 
 
A total of 240-300 adults diagnosed with aMCI or probable  mild AD will be enrolled in this trial. 
We expect to enroll no more than 50% of participants with probable mild AD and no more than 
60% participants with aMCI diagnosis. 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.  
 
5.2 
Diagnosis Criteria 
 
Diagnoses will be assigned by an expert clinician, which will be ensured by the fact that the 
majority of sites are affiliated with NIH-funded Alzheimer’s Disease Centers and all sites are 
affiliated with reknowned Alzheimer’s programs; thus all sites are staffed by clinicians expert in 
the diagnosis of AD and aMCI.  
 
Participants with a MCI or probable mild AD will be diagnosed according to core clinical criteria 
updated in the NIA and Alzheimer’s Association. 
 
Diagnosis of aMCI requires:  
a) Evidence of a decline in episodic memory  
b) General preservation of independence in functional abilities 
c) Absence of dementia  
 
Biomarker evidence consistent with AD will not be required for entry for either aMCI or probable 
mild AD, given that knowledge in this area is evolving. Also, recent evidence indicates that 
requiring an AD-positive biomarker does not uniformly improve the efficiency of clinical trials 
(Schneider, Kennedy et al. 2010).  


 
 
  
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5.3 
Inclusion Criteria 
 
The following inclusion criteria* will be used:  
1. Age 55 to 85 (inclusive)  
2. Fluent in English or Spanish 
3. Diagnosis of aMCI or probable mild AD according to the core clinical criteria outlined in 
the NIA and Alzheimer’s Association Guidelines. 
4. MMSE ≥ 20 at screening. Approval for exceptions can be requested to the Project 
Director for minority or low education participants with MMSE scores of 18 or 19  
5. CDR 0.5-1 (inclusive) at screening 
6. For aMCI group only: Logical Memory ≤10 for 16 or more years of education, ≤6 for 8-15 
years of education, ≤4 for 0-7 years of education. Scores measured at screening on 
Delayed Paragraph Recall from the Wechsler Memory Scale–Revised  
7. Modified Hachinski score ≤ 4 
8. A study partner able to accompany the participant to most visits and answer questions 
about the participant. The study partner must have direct contact with the participant > 
2 days/week (minimum of 10 hrs a week)  
9. A study partner able to participate in study drug administration or able to assure that 
another person (such a family member or friend or other) will be able to assist the 
participant in the study drug administration 
10. Stable medical condition for 3 months prior to screening visit 
11. Stable medications for 4 weeks prior to the screening and baseline visits. However, 
cholinesterase inhibitors and memantine are allowable if stable for 12 weeks prior to 
screening and baseline visits. 
12. Able to complete baseline assessments 
13. At least six years of education or a work history sufficient to exclude mental retardation 
14. Clinical laboratory values must be within normal limits or, if abnormal, must be judged to 
be not clinically  significant by the investigator  
15. Visual and auditory acuity adequate for neuropsychological testing 
 
*Exceptions to these criteria may be considered on a case-by-case basis at the discretion of the 
Project Director. 
 
5.4 
Exclusion Criteria 
 
The following exclusion criteria* will be used:  
1. A diagnosis of dementia other than probable AD 
2. Probable AD with Down syndrome  
3. History of a clinically significant stroke 
4. Current evidence or history in past two years of epilepsy, focal brain lesion, head injury 


 
 
  
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with loss of consciousness or DSM IV criteria for any major psychiatric disorder 
including psychosis, major depression, bipolar disorder, alcohol or substance abuse  
5. Sensory impairment that would preclude the participant from participating in or 
cooperating with the protocol 
6. Diabetes (type I or type II) requiring pharmacologic treatment (including both insulin 
dependent and non-insulin dependent diabetes mellitus) 
7. Current or past use of insulin or any other anti-diabetic medication within one (1) year of 
Screening visit.. 
8. Evidence of any significant clinical disorder or laboratory finding that renders the 
participant unsuitable for receiving an investigational drug including clinically significant 
or unstable hematologic, hepatic, cardiovascular, pulmonary, gastrointestinal, 
endocrine, metabolic, renal or other systemic disease or laboratory abnormality  
9. Active neoplastic disease, history of cancer within five years prior to screening (history of  
stable prostate cancer or non-melanoma skin cancers are not exclusionary) 
10. History of seizure within past five years 
11. Pregnancy or possible pregnancy. Participant is not pregnant, lactating, or of 
childbearing potential (i.e. women must be two years post-menopausal or surgically 
sterile) 
12. For participants undergoing optional lumbar puncture, contraindications to LP: prior 
lumbosacral spine surgery, severe degenerative joint disease or deformity of the spine, 
platelets <100,000 or history of a bleeding disorder 
13. For participants undergoing optional lumbar puncture, use of anticoagulants warfarin 
(Coumadin) and dabigatran (Pradaxa)  
14. Contraindications for MRI (claustrophobia, craniofacial metal implants of any kind, 
pacemakers)  
15. Residence in a skilled nursing facility at screening 
16. Use of an investigational agent within two months of screening visit 
17. Regular use of narcotics, anticonvulsants, medications with significant anticholinergic 
activity, antiparkinsonian medications, or any other exclusionary medications 
 
*Exceptions to these criteria may be considered on a case-by-case basis at the discretion of the 
Project Director. 
 
5.5 
Recruitment and Retention Strategies 
 
Recruitment will occur through a variety of mechanisms. The trial design includes a 6-month, 
open-label extension period in which all participants will receive active drug, which will facilitate 
recruitment and retention. Recruitment efforts will also be overseen by the Coordinating Center. 
The Coordinating Center has developed a coordinated recruitment plan to ensure that 
enrollment occurs in a timely fashion. The overall goals of the plan are to raise awareness of 
this trial among targeted populations to ensure adequate enrollment. The Coordinating Center 
partners with NIA and coordinates with its ADEAR Center to leverage existing resources. The 
Coordinating Center will oversee the development of materials specific to the INI trial for use by 
the sites, provide ongoing recruitment assistance and support, and develop tracking procedures 


 
 
  
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to monitor effectiveness of recruitment efforts. The recruitment plan for the INI trial will include 
both the utilization of participants existing at clinical trial centers and the recruitment of 
additional populations by methods of outreach.  
 
6.0 
STUDY TIMELINE  
 
The approximate timeline for this four-year study is projected as follows: 1) approximately six 
months for study startup activities including FDA/IND submission, site IRB and regulatory 
approvals and the Investigator training meeting; 2) recruitment will last for approximately 18 
months (for a total of 240-300 participants across about 30 sites); and 3) 18 months of follow up 
(last 6 months are open label) 
 
7.0 
DESCRIPTION OF STUDY VISITS 
 
The “Schedule of Study Procedures and Assessments” in Table 4 (section 21.0) provides an 
overview of study visit activities.  The primary outcome measure (ADAS-Cog12) will be 
measured at Baseline and all subsequent visits at three month intervals to optimize imputation 
of missing data. Secondary outcome measures, plasma biomarkers, and PBMCs will be 
measured every six months: Baseline (with the exception of MMSE and CDR conducted at 
Screening), Month 6, Month 12 and Month 18. MRI measures of entorhinal cortex and 
hippocampal atrophy will be obtained at Screening and Month 12. CSF biomarkers will be 
obtained at Baseline and Month 12.  Genotyping will occur at Screening.  
 
7.1 
Pre-Screening 
 
Prior to in-person screening evaluation, the investigator through routine clinical contact or 
through a pre-screening process will identify potential participants.  Prospective participants will 
have a diagnosis of aMCI or probable mild AD and will be known to meet as many of the 
inclusion/exclusion criteria as possible. Potential participants and study partners will not be 
considered study participants until they sign the consent form.  
 
7.2 
Screening (Visit 1) 
 
The purpose of this visit is to determine study eligibility and may be conducted over multiple 
days.  Potential participants and their study partners 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 Logical Memory (Delayed 
Paragraph Recall from the Wechsler Memory Scale–Revised) to determine study eligibility. 
 
In addition, information regarding demographics, concurrent medications, medical history and 
adverse events will be gathered from the participant and study partner. Vital signs, height and 
weight will be measured. A brief physical and neurological examination (which include a nasal 
examination), Modified Hachinski scale and a standard 12-lead 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.  
 
Blood will be drawn and a urine sample obtained for routine clinical laboratory evaluations. If 
values are outside of the laboratory’s normal range and determined clinically significant by the 
investigator, lab tests may need to be repeated and may be considered exclusionary for 


 
 
  
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participation in the study. Blood samples will also be collected for ApoE genotyping and optional 
DNA storage.  
 
A screening MRI scan will be conducted for participants who met eligibility criteria (as 
determined by the site investigator). A local clinical read of the MRI will be reviewed by the site 
principal investigator (or appropriately delegated staff member) for eligibility. If the participant 
continues to meet all inclusion and exclusion criteria and no safety concerns are identified from 
the MRI, the participant can proceed to the baseline visit after approval by a clinical monitor. 
 
Prior to randomization, the Project Director must approve any exceptions or questions regarding 
possible exclusionary medications, medical conditions, or laboratory tests. Re-screen is 
allowed, should the original screen be a failure.  The re-screen should typically occur more than 
3 months after the original screen failure. 
 
7.3 
Baseline (Visit 2) 
 
Results from all screening procedures must be reviewed and all inclusion/exclusion criteria must 
be met prior to proceeding to baseline. Baseline visit will be scheduled 2-4 weeks after the 
screening visit. If the baseline visit is scheduled out-of-window (more than 4 weeks from 
screening), approval to proceed to the baseline visit should be obtained from the project director 
and a rescreen may be required. The baseline procedures may be completed over multiple 
days, but all assessments should be completed within seven days. 
 
Vital signs and weight will be obtained, a nasal examination will be performed and concurrent 
medications and adverse events will be documented.  
 
Blood for plasma biomarkers will be collected immediately before participants undergo the 
optional lumbar puncture. Plasma insulin, glucose, Aβ40 and Aβ42 should be measured in the 
morning after an overnight fast, if possible. Additional plasma, along with serum and peripheral 
blood mononuclear cells (PBMCs) will be collected and banked. 
 
Cognitive and behavioral assessments will be performed and study procedures will be 
standardized such that the ADAS-Cog12 will be given first. Additional assessments include a 
memory composite (Story Recall and FCSRT), an executive function test (Trail-making Test), 
the ADCS-MCI-ADL and the Neuropsychiatric Inventory (NPI). A Research Satisfaction Survey 
will also be administered. Cognitive and behavioral assessments should not be administered 
while participants are fasting or immediately after the LP procedure.   At study visits during 
which fasting samples are collected on the same day that cognitive assessments are 
administered, a break and food should be given to participants in between the fasting procedure 
and cognitive and behavioral assessments.  
 
The optional lumbar puncture should be performed in the morning after an overnight fast, if 
possible. CSF samples will be used to measure levels of Abeta42, Abeta40, total tau, phospho-
tau181, insulin, F2-Isoprostanes, cytokines, and BDNF. Additionally, CSF sample will be banked 
for future exploratory analysis. CSF will be collected and immediately frozen upright on dry ice 
for at least 20 minutes before being packaged along with the frozen plasma to be shipped 
overnight frozen on dry ice to the central biomarker laboratory.. Site staff will call the participant 
within approximately 24 hours from the lumbar puncture procedure to inquire about the 
participant’s well being and possible adverse events. 
 


 
 
  
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Participants who continue to meet all protocol inclusion and exclusion criteria will be randomized 
to receive either insulin or placebo for the next 12 months, plus 6 month of open-label.  All 
Baseline study procedures should occur before the start of the administration of any study drug.  
 
Participants and study partners will be trained on how to appropriately use the intranasal device 
utilizing a test dose of placebo and shown how to record information in the study diary. The 
participant and study partner will be instructed to bring any unused study drug and their study 
diary to the clinic at each study visit (Month 3, 6, 9, 12, 15, 18) so that study staff can assess 
study drug compliance.  
  
7.4 
Month 3 (Visit 3) 
 
The Month 3 visit is scheduled three months (+/- 2 weeks) from day one of the Baseline visit. At 
the Month 3 visit, the ADAS-Cog12 will be administered. The Research Satisfaction Survey will 
also be administered Additionally, vital signs and weight will be obtained, a nasal examination 
will be conducted, adverse events and concurrent medications will be recorded. Participants will 
bring their device, study diary and any used and unused study drug vials to the site.  Study drug 
compliance will be assessed.  POD components (actuators, vial adapters, canisters and POD 
tips) will be dispensed.. 
. 
 
7.5 
Month 6 (Visit 4) 
 
The Month 6 visit is scheduled six months (+/- 2 weeks) from day one of the Baseline visit. At 
the Month 6 visit, blood for plasma biomarkers will be collected. Plasma insulin, glucose, Aβ40 
and Aβ42 should be measured in the morning after an overnight fast, if possible.. Additional 
plasma, along with serum and peripheral blood mononuclear cells (PBMCs) will be collected 
and banked. Samples will be shipped frozen to the central biomarker laboratory where the 
samples will be analyzed and banked. Additional blood for clinical laboratory evaluations will be 
conducted to monitor safety and tolerability.  
 
Cognitive and behavioral assessments will be performed (during non fasting session). The study 
procedures will be standardized such that the ADAS-Cog12 will be the first cognitive 
assessment administered. Additional assessments include the MMSE, memory composite 
(Story Recall and FCSRT), an executive function test (Trail-making Test), CDR-SB, the ADCS-
ADL-MCI and NPI. The Research Satisfaction Survey will also be administered.  
 
In addition, vital signs and weight will be obtained, physical, neurological and nasal 
examinations will be conducted, adverse events and concurrent medications will be recorded.. 
Participants will bring their device, study diary and any used and unused study drug vials to the 
site.  Study drug compliance will be assessed.  POD components (actuators, vial adapters, 
canisters and POD tips) will be dispensed.   
 
7.6 
Month 9 (Visit 5) 
 
The Month 9 visit is scheduled nine months (+/- 2 weeks) from day one of the Baseline visit.  At 
the Month 9 visit, the ADAS-Cog12 will be administered. In addition, vital signs and weight will 
be obtained, a nasal examination will be conducted, adverse events and concurrent medications 
will be recorded. Participants will bring their device, study diary and any used and unused study 


 
 
  
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drug vials to the site . Study drug compliance will be assessed.  POD components (actuators, 
vial adapters, canisters and POD tips) will be dispensed.   
 
7.7 
 Month 12 (Visit 6) 
 
The Month 12 visit is scheduled 12 months (+/- 2 weeks) from day one of the Baseline visit. 
Month 12 visit is the last visit before the 6-month open-label period and the visit may occur over 
multiple days. The MRI should be conducted prior to the LP.  
 
Blood for plasma biomarkers will be collected immediately before participants undergo the 
optional lumbar puncture. Plasma insulin, glucose, Aβ40 and Aβ42 should be measured in the 
morning after an overnight fast, if possible. Additional plasma, along with serum and peripheral 
blood mononuclear cells (PBMCs) will be collected and banked. Clinical laboratory evaluations 
will be conducted to monitor safety and tolerability.  
 
The optional lumbar puncture should be performed in the morning after an overnight fast, if 
possible. Additionally, CSF sample will be banked for future exploratory analysis. CSF will be 
collected and immediately frozen upright on dry ice for at least 20 minutes before being 
packaged along with the frozen plasma to be shipped overnight frozen on dry ice to the central 
biomarker laboratory. Site staff will call the participant within approximately 24 hours from the 
LP procedure to inquire about the participant’s well being and possible adverse events. 
 
Cognitive and behavioral assessments will be performed. The study procedures will be 
standardized such that the ADAS-Cog12 will be the first cognitive test administered. Additional 
assessments include the MMSE, a memory composite (Story Recall and FCSRT), Trail-making 
Test A and B, the CDR-SB, the ADCS-ADL-MCI and the NPI. The Treatment Blinding 
Questionnaire and the Research Satisfaction Survey will also be administered.  Cognitive and 
behavioral assessments should not be administered while participants are fasting or 
immediately after the LP procedure. 
 
In addition, vital signs and weight will be obtained, physical, neurological and nasal 
examinations will be conducted, adverse events and concurrent medications will be recorded. 
Participants will bring their device components, study diary and any used and unused study 
drug vials to the site.  Study drug compliance will be assessed. POD components (actuators, 
vial adapters, canisters and POD tips) will be dispensed. 
. 
Participants who were previously taking placebo will cross over to active study drug.  All 
participants will receive the active study drug during the six-month open-label period. 
 
 
 
7.8 
Month 15 (Visit 7, Open Label) 
 
The Month 15 visit is scheduled 15 months (+/- 2 weeks) from day one of the Baseline visit. At 
the Month 15 visit, the ADAS-Cog12 will be administered. In addition, vital signs and weight will 
be obtained, a nasal examination will be conducted, adverse events and concurrent medications 
will be recorded. Participants will bring their device, study diary and any used and unused study 
drug vials to the site.   Study drug compliance will be assessed. POD components (actuators, 
vial adapters, canisters and POD tips) will be dispensed. 
 


 
 
  
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7.9 
Month 18 (Visit 8, Open Label) 
 
The Month 18 visit is scheduled 18 months (+/- 2 weeks) from day one of the Baseline visit. At 
the final Month 18 visit, plasma insulin, glucose, Aβ40 and Aβ42 should be measured in the 
morning after an overnight fast, if possible.  Additional plasma, along with serum and peripheral 
blood mononuclear cells (PBMCs) will be collected and banked. Clinical laboratory evaluations 
will be conducted to monitor safety and tolerability. 
 
Cognitive and behavioral assessments will be performed (during a non- fasting session). The 
study procedures will be standardized such that the ADAS-Cog12 will be the first cognitive test 
administered. Additional assessments include the MMSE, memory composite (Story Recall and 
FCSRT), Trail-making Test part A and B, CDR-SB, the ADCS-ADL-MCI and NPI. The Research 
Satisfaction Survey will also be administered. 
 
In addition, vital signs and weight will be obtained, physical, neurological and nasal 
examinations will be conducted, adverse events and concurrent medications will be recorded 
and study drug compliance will be assessed. Participants will return their study diary, their Impel 
POD device components, and any used and unused study drug vials to the clinic. Study drug 
compliance will be assessed during this final visit. 
 
8.0 
EARLY TREATMENT/STUDY DISCONTINUATION  
 
The investigators at each site will make every reasonable effort to maximize participant 
retention. However, if an investigator removes a participant from treatment or study, or if a 
participant declines further treatment or study participation, an Early Discontinuation Visit will be 
completed as soon as possible following discontinuation of the study drug and or at time of 
study discontinuation. The Early Discontinuation Visit will contain the same assessments as the 
Month 12 visit, to allow collection of the main outcome measures.  Depending on when the last 
LP or MRI was conducted, certain procedures may not be required at the Early Discontinuation 
Visit. If an in-person visit is not possible, site personnel will complete as much of the Early 
Discontinuation Visit as possible by telephone. All early treatment discontinuation participants 
will be strongly encouraged to complete all remaining visits through Month 12 visit.  
 
8.1 
Reasons for Early Discontinuation 
 
All reason(s) for treatment/study discontinuation will be collected using categories such as: 
• 
Adverse experience:  The participant has experienced an adverse event that, in the 
opinion of the investigator, requires early termination. This may include abnormal laboratory 
values. 
• 
Death. 
• 
Safety risk: Any participant who becomes a safety risk during the trial will be withdrawn.   
• 
Protocol violation:  The participant fails to meet protocol entry criteria or did not adhere to 
protocol requirements. 
• 
Non-compliance: The participant is non-compliant with completion of study-related 
evaluations and or intake of study drugs. 


 
 
  
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• 
In the investigator’s judgment, it is in the participant’s best interest to discontinue 
participation in the treatment/study. 
• 
Consent is withdrawn.  The participant wishes to withdraw from the study, or the legally 
authorized representative wishes the participant to be withdrawn. 
• 
The study is terminated by the Coordinating Center alone or at the recommendation of the 
Data Safety Monitoring Board. 
• 
Lost to follow up.  Participant could not be recalled back to conduct follow up visits. 
 
• 
Loss of informed study partner.  The participant no longer has a responsible study partner 
to oversee participant visits and administration of study drug. 
 
• 
Coordinating Center Request.  The Coordinating Center determines it is in the 
participant’s best interest to discontinue participation from the treatment and or study. 
 
9.0 
STUDY-SPECIFIC PROCEDURES 
 
9.1 
Cognitive Evaluations  
 
9.1.1 Alzheimer’s Disease Assessment Scale-Cognitive (ADAS-Cog12) 
 
The ADAS-Cog (Rosen, Mohs et al. 1984) is a psychometric instrument that evaluates memory, 
attention, reasoning, language, orientation, and praxis. A higher score indicates more 
impairment.  Scores from the original portion of the test range from 0 (best) to 70 (worse) and 
then number of items not recalled ranging from 0-10 is added for a maximum score of 80.  A 
positive change indicates cognitive worsening. An ADAS-Cog12 version will be used in the 
current study which includes Delayed Word Recall - a measure of episodic memory (Mohs, 
Knopman et al. 1997). The ADAS-Cog has been the primary cognitive instrument in previous 
and ongoing Alzheimer’s Disease therapeutic trials. Neuropsychometrists at each site are 
required to complete an ADAScog training and certification process. The person administering 
this test should not also administer the CDR. 
 
9.1.2 Mini-Mental State Examinations (MMSE) 
 
The MMSE (Folstein, Folstein et al. 1975) is a brief, frequently used screening instrument for 
AD drug studies.  The MMSE evaluates orientation, memory, attention, concentration, naming, 
repetition, comprehension, and ability to create a sentence and to copy two overlapping 
pentagons.  A lower score indicates more cognitive impairment.  The highest score is 30.   
 
9.1.3 Memory Composite (Story Recall and Free and Cued Selective Reminding Test) 
 
A memory composite measure combines two episodic memory measures, immediate and 
delayed recall. Story Recall is a test of contextual verbal recall, in which participants listen to a 
story containing 44 informational bits that is read once. Participants will be asked to recall the 
story immediately after the reading and after approximately 20-minute delay. Credit is awarded 
for each bit recalled verbatim or accurately paraphrased. The previous results demonstrate the 
sensitivity of this task to changes in plasma insulin levels and intranasal insulin administration in 
adults with AD/MCI (Craft, Newcomer et al. 1996; Craft, Asthana et al. 1999; Craft, Asthana et 
al. 1999).  


 
 
  
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The FCSRT (Grober et al. 1988, 2008; Ferris et al. 2006) is a 16-item word list with visual and 
auditory presentation that uses semantic cueing to facilitate encoding and retrieval. Sixteen 
items, from 16 different semantic categories, are learned by identifying and naming a picture of 
each item when its category cue is provided.  After the learning phase, a 20-second interference 
period is introduced to prevent rehearsal  Retrieval from long-term memory is then tested by 
three trials of Free and Cued Recall, each preceded by a period of interference. 
 
9.1.4 Logical Memory Test (Wechsler Memory Scale-Revised) 
 
Logical Memory Test I and II (Delayed Paragraph Recall) used in this study is a modification of 
the episodic memory measure from the Wechsler Memory Scale-Revised (WMS-R) (Wechsler 
1987). In this modified version, free recall of one short story that consists of 25 bits of 
information will be elicited immediately after it is read aloud to the participant and again after a 
thirty-minute delay.  The total bits of information from the story that are recalled immediately 
(maximum score = 25) and after the delay interval (maximum score = 25) are recorded. A 
retention or “savings” score can be computed by dividing the score achieved during delayed 
recall by the score achieved during immediate recall. 
 
9.1.5 Trail-making Test (Part A and B) 
 
Part A of the Trail-making tests (Reitan 1958) consists of 25 circles numbered 1 through 25 
distributed over a sheet paper. The participant draws a line to connect the circles in order as 
quickly as possible (150 second maximum). Part B consists of 25 circles, but these circles are 
either numbered (1 through 13) or contain letters (A through L). Now the participant must 
alternate between numbers and letters in an ascending order (e.g., 1 to A; 2 to B). The time to 
complete Part B (300 second maximum), adjusted for the time taken to complete Part A to 
control for sensorimotor aspects of the task, is a sensitive measure of executive function and 
working memory.  
 
9.2 
Clinical and Functional Evaluations  
 
9.2.1 Clinical Dementia Rating Scale – Sum of Boxes (CDR-SB) 
 
The CDR (Hughes, Berg et al. 1982; Morris 1993) 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 is based on interviews with the participant and study partner, using a 
structured interview that assesses six domains: memory, orientation, judgment and problem 
solving, community affairs, home and hobbies, and personal care.   
 
At Screening the CDR global score will be used for eligibility purposes. For all other 
administrations, the 6 domain scores will be summed to get the Sum of Boxes (SB) score. 
Training on the use of the CDR will be conducted to standardize its administration across sites. 
The CDR online training tool resides on the Washington University, St. Louis website, with 
oversight provided by Dr. John C. Morris (Morris, Ernesto et al. 1997). The person administering 
the CDR should not also administer the ADAS-Cog12.  
 
9.2.2 ADCS-MCI Activities of Daily Living (ADCS-ADL-MCI) 
 
The Alzheimer’s Disease Cooperative Study - Activities of Daily Living Scale (ADCS-ADL) is an 


 
 
  
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activities of daily living questionnaire aimed at detecting functional decline in people with Mild 
Cognitive Impairment (MCI) (Grundman, Petersen et al. 2004). It was adapted from an inventory 
developed by the ADCS to assess functional performance in subjects with Alzheimer’s disease 
(Galasko, Bennett et al. 1997). 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 ADCS-ADL-MCI scale discriminates well between normal controls 
and patients with mild AD or MCI.  It has good test-retest 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 Neuropsychiatric Inventory (NPI) 
 
The NPI is a well-validated, reliable, multi-item instrument to assess psychopathology in AD 
based on interview with the study partner (Cummings 1997). The NPI evaluates both the 
frequency and severity of 10 neuropsychiatric disturbances.  Frequency assessments range 
from 1 (occasionally, less than once per week) to 4 (very frequently, once or more per day or 
continuously) as well as severity (1=mild, 2=moderate, 3=severe).  The overall score and the 
score for each subscale are the product of severity and frequency.  
 
9.2.4 Modified Hachinski 
 
This brief questionnaire incorporates questions about medical history, cognitive symptoms and 
features of stroke, reported by a study partner as well as the neurological examination and 
neuroimaging studies (Rosen, Terry et al. 1980). 
 
9.3 
Research Satisfaction Survey 
 
A Research Satisfaction Survey will be administered in order to evaluate the participant’s 
satisfaction with the current study. The survey will also reveal specific aspects of the study that 
participants may dislike, thus improvements can be made in the design of future studies. Past 
studies in multiple fields have also shown that consumer input and feedback is an important 
element in increasing retention (e.g., in psychotherapy) (Duncan, Miller et al. 2010) (Miller, 
Duncan et al. 2005).  
 
9.4 
Treatment Blinding Questionnaire 
 
The Treatment Blinding Questionnaire assesses the perception of blind being maintained until 
the end of the study.  The questionnaire is to be completed by the study participant, study 
partner, principal investigator and ADAS rater at the end of the blinded phase of the study. 
 
10.0 
STUDY METHODS 
 
10.1 
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. Physical and neurological examination and routine laboratory testing will occur at 
Screening, Month 6, Month 12 and Month 18 visits. An MRI will be conducted at Screening and 
repeated at Month 12 for safety assessment.  Study staff will telephone each study participant 
who undergoes the optional lumbar puncture, or a person designated to speak for him/her, 


 
 
  
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approximately 24 hours after the procedure to confirm the participant’s well being and query 
about any new adverse events.  All adverse events will be reported to the DSMB. Safety reports 
will be prepared and submitted to the DSMB for periodic review.  
 
10.2 
Physical and Neurological Examination 
 
A brief physical examination will be performed by a medically qualified professional every six 
months. 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 be performed every three months at each visit. The examination will 
assess irritation or other abnormalities of the nares.  
 
10.3 
Electrocardiogram (ECG) 
 
A standard 12-lead resting ECG will be performed at Screening visit.  The ECG report will be 
reviewed, signed, and dated by the local site Principal Investigator (or a medically qualified 
individual delegated by the PI).  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. 
  
10.4 
Clinical Laboratory Evaluations 
 
All routine laboratory samples will be analyzed by a central laboratory, which will provide a 
procedures manual and supplies. Lab reports will be reviewed, signed and dated by the local 
site Principal Investigator (or a medically-qualified individual delegated by the PI).  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.    
 
Clinical laboratory assessments include: 
 
• 
Chemistry Panel 
• 
TSH 
• 
CBC | Differential 
• 
Vitamin B12 (Screening visit only) 
• 
HCY | MMA (Note: HCY & MMA reflex tests are only run if the B12 level is <200 pg/mL) 
• 
HgA1c  
• 
U/A with microscopic (Screening visit only) 
 
11.0 
BIOMARKER STUDIES 
 
11.1 
CSF 
 
It is expected that many participants will receive optional lumbar punctures pre and post-
treatment. All samples should be collected in the morning before breakfast and after an 
overnight fast, if possible.  Participants who have contraindications to lumbar puncture including 


 
 
  
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prior lumbosacral spine surgery, severe degenerative joint disease or deformity of the spine, 
platelets <100,000 or a history of a bleeding disorder cannot undergo a lumbar puncture in this 
trial, nor can participants who are taking anticoagulants, warfarin (Coumadin) and dabigatran 
(Pradaxa). 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 if the 
participant voluntarily agrees to the lumbar puncture component of this study.  It is not required 
that participants be discontinued from their anti-platelet agent in order to screen and enroll in the 
study.  
 
At baseline, the optional lumbar puncture must be conducted prior to initiation of study drug.  At 
Month 12, the optional lumbar puncture should be performed after (or more than 72 hours 
before) the MRI scan while the participant is still actively taking study drug. Additionally, the time 
of the last INI dosage and the time of CSF collection will be recorded.  LP under fluoroscopy is 
allowed, if needed or required per local regulations.  A post lumbar puncture telephone follow-up 
call will be completed approximately 24 hours after participant has undergone the procedure to 
assess safety. 
 
A minimal total volume of CSF (20 ml) will be collected from participants undergoing the optional  
lumbar puncture 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  collection tubes. Approximately 2ml of CSF or volume per local laboratory 
requirements will be sent at ambient temperature to the local laboratory for protein, glucose and 
cell count. The remaining CSF will be shipped to the central biomarker laboratory.  CSF must be 
immediately frozen upright on dry ice for at least 20 minutes before being packaged and 
shipped overnight frozen on dry ice to the central biomarker laboratory. 
 
CSF samples will be used to measure levels of Abeta42, Abeta40, total tau, phospho-tau181, 
insulin, F2-Isoprostanes, cytokines, and BDNF. Assays will be performed by the central 
biomarker laboratory. CSF samples will also be frozen and stored for future analysis of putative 
biomarkers. 
 
11.2 
Blood Collection  
 
Non-fasting blood samples will be collected at screening.  Fasting blood samples (Baseline, 
Month 6, Month 12, and Month 18) should be collected in the morning before breakfast and after 
an overnight fast, if possible. 
 
At Baseline and Month 12 visits, fasting blood samples should be collected before CSF 
collection, which would be processed for fasting plasma, insulin, glucose, Abeta42, and 
Abeta40. Additional blood for plasma, serum and peripheral blood mononuclear cells (PBMCs) 
will be collected. The samples will shipped to the central biomarker laboratory for processing 
and banking. 
 
11.3 
Insulin & Glucose (HgA1c Test) 
 
HgA1c test will be performed and fasting insulin and glucose values will be used to calculate the 
homeostasis model assessment of insulin resistance (HOMA-IR) for participants (Matthews, 
Hosker et al. 1985). A central laboratory will analyze the HgA1c test. 
 


 
 
  
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11.4 
Magnetic Resonance Imaging (MRI) 
 
Screening and Month 12 MRI will use the same imaging protocol, which will include a localizer 
scan, followed by a high-resolution 3D T1 structural series (MPRAGE or IR-SPGR), a T2-
weighted series (FLAIR), a diffusion weighted scan and a gradient recalled echo scan. The 
volumetric analysis procedure will include corrections for gradient nonlinearities and intensity 
non-uniformity (Sled, Zijdenbos et al. 1998; Arnold, Liow et al. 2001; Jovicich, Czanner et al. 
2006).  
 
For clinical assessment of MR images and meeting of MRI screening criteria, images will be 
read by the site radiologist and submitted without patient identifiable information to the study 
EDC database, where quality checks will be performed. Images will be checked for image 
quality and adherence to scanning protocols. 3D T1-weighted datasets passing quality checks 
will be corrected for spatial distortion and for intensity variation. Baseline and follow-up datasets 
for each participant will be spatially registered to one another using rigid-body registration 
followed by nonlinear registration and neuroanatomic parcellation to quantify whole-brain and 
subregional volumetric change on a participant-by participant basis.  
 
MRI should be conducted prior to the LP. 1.5 Tesla (1.5T) or 3.0 Tesla (3T) scanners that have 
passed the study’s qualification procedures will be used. Participants must be scanned by the 
same scanner throughout the study. 
 
11.5 
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. Participants will 
be asked to consent to optional DNA banking for future research studies. ApoE genotyping will 
be performed and the DNA will be banked at the ADCS Biomarker Core at UCSD using 
established protocols.  
 
12.0 
STATISTICAL PLAN  
 
The intent-to-treat (ITT) data set will include all eligible individuals who are randomized. The 
per-protocol (PP) data set will include all eligible participants who complete 12-month 
assessments for the primary analysis and receive 80% of prescribed treatment doses. To 
address the Primary Aim, the longitudinal scores for the first 12 months on the primary endpoint 
(ADAS-Cog12) as well as Secondary Aims (memory composite, Trails, CDR-SB, ADCS-ADL-
MCI), will be analyzed using random effects (RE) models to assess the differences in the rate of 
change across treatment groups (insulin-treated vs. placebo). Fixed effects for time, treatment 
and time-by-treatment interaction will be included. The following covariates will be included in 
efficacy analyses if they meet the criteria described below: baseline MMSE, baseline age, years 
of education, gender, APOE-ε4 carrier status, and diagnosis. Each covariate will be included in 
RE, ANCOVA, and GEE models only if the following two conditions are satisfied: 1) dissimilarity 
at baseline as measured by Fisher‘s exact test P < .1; 2) a bivariate association exists between 
the covariate and the response (without regard to treatment) as measured by Spearman’s 
correlation coefficient P< 0.15. 
 
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volumes (hippocampus, entorhinal cortex) will be analyzed with a similar strategy except that 
only baseline and month 12 values will be used. Other CSF biomarkers will be treated similarly 
in exploratory analyses. To address Secondary Aims 3 and , the predictive ability of APOE-ε4 
allele carriage and CSF biomarkers will be evaluated using RE models. A model will be 
developed predicting longitudinal ADAS-Cog12 response. CSF Aβ42 levels and APOE-ε4 allele 
carriage will be included as fixed main effects along with treatment, time, and treatment-by-time 
interaction. A fixed effect for the interaction of Aβ42 and APOE-ε4 will also be included. 
Potential confounders evaluated as described above will also be included as main effects. RE 
models make use of all observed data, and the assumption is made that any missing data is 
missing at random. Responses will be modeled as multivariate normal. Time will be treated as 
continuous and coded as months from baseline visit, (i.e. months = (exam date minus baseline 
date)/30.5]. The correlation structure for the model will be first order autoregessive. As 
sensitivity analyses, both GEE (generalized estimating equations) and ANCOVA modeling will 
be performed. As an additional secondary (sensitivity) analysis, the above RE analysis will be 
performed treating time as categorical; differences between the active group and placebo group 
at 12 months will be assessed via contrasts in the least squares means; this analysis does not 
assume linearity over time. Associated p-values, standard errors of the differences, and 95 
percent confidence intervals for each model will be presented for all analyses.  
 
The application of the ITT principle in a change score analysis requires the imputation of any 
missing “post” observations. A multiple imputation strategy will be utilized for missing endpoints. 
Missing post-baseline data will be imputed via multiple imputations from the general location 
model (Little and Rubin 1987). Point estimates, standard errors, confidence intervals, and p-
values will be computed using standard inference procedures for multiple imputations (Little and 
Rubin 1987). Safety data will be analyzed using exact contingency table methods by study 
arms. AEs will also be subcategorized by relationship to study drug (unrelated/possibly 
related/probably related).  Details of the analytic models will be stated in a formal statistical 
analysis plan (SAP) that will be generated and finalized within six months of anticipated data 
lock. 
 
12.1 
Power Analyses 
 
Power calculations to estimate the sample sizes required to detect a difference between the rate 
of change in two groups were based on the two-sample t-test using the formula in Statistical 
Methods in Medical Research (Armitage and Berry 1994). Sample sizes per arm were estimated 
using ADNI 12-month change scores for the ADAS-Cog12 for the combined MCI and AD 
population, targeting power=.80, Type 1 error level=0.05, SD=5.72. Table 5 shows the range of 
estimates needed to detect absolute change score differences between groups ranging from 
1.6-2.6 for dropout rates between 0.15-0.25. These estimates were derived with 6 month 
intervals between testing, and thus more frequent administration of the ADAS-Cog12 in this trial 
should provide even greater power. Similarly, a 4-month pilot trial (Craft, Baker et al. 2012) 
dropout rate was <7%, and although a higher dropout rate is expected with a longer, multi-site 
trial, the excellent safety profile and incentive of the open-label extension at the end of the trial 
should maximize retention. The addition of a delayed recall score should increase sensitivity for 
aMCI participants. For MRI endpoints, sample sizes needed to detect a 25% slowing in mean 
rate of decline in the entorhinal cortex over a 12-month period ranged from 70 for AD to 280 for 
aMCI (Holland, Brewer et al. 2009). Given that our n=240-300, and the sample will be 
composed of both aMCI and AD, we should have adequate power. 
 


 
 
  
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Table 5 | Range of estimates needed to detect absolute change score differences 
between groups ranging from 1.6-2.6 for dropout rates between 0.15-0.25 
 
 
 
13.0 
POTENTIAL RISKS 
 
13.1 
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 IU 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 IU once a day for three weeks in 20 
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. 
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there are fewer safety data for INI use by patients with AD and it is possible that long-term 
administration may induce CNS hyperinsulinemia or insulin resistance that may have 
deleterious effects. Such effects would presumably manifest as accelerated cognitive or 
functional decline. Thus an intensive safety-monitoring plan will be in place for this study that 
should detect such patterns and a conservative duration of treatment that balances safety and 
scientific considerations. 
 
13.2 
Risks associated with use of the POD device  
 
There are risks that may occur from use of the investigational intranasal drug administration 
device, although these are not expected to be serious. It is important that participants carefully 
follow the instructions they are given about how to administer the study drug and how to 
maintain the intranasal drug administration device and its components. If they don’t carefully 
follow the instructions it is possible that they may not receive the correct dose. They could also 
experience some discomfort to their eyes or face if they do not hold the device to their nose as 
directed. In addition, the contents of the POD device’s canister are under pressure and should not 
be punctured or stored near heat or open flame.  Finally, the POD tip on POD device is small and 
could be a choking hazard for children. The participant and their Study Partner will be trained on 
proper device operation and asked to maintain the device and its components properly.   
 
13.3 
Optional 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%.   
 
13.4 
MRI 
 
The risks of MRI primarily arise from the possible introduction of ferromagnetic objects into a 
high magnetic field, which can create a dangerous projectile or lead to dysfunction or heating of 
an implanted medical device.  All participants will be rigorously screened by MRI personnel to 
be certain that they do not have any medical contraindications for MRI, which include metallic 
foreign bodies in the brain or eye or cardiac pacemaker. This safety screening is part of routine 
clinical practice at MRI centers and is performed before any participant is permitted to enter the 
scanning room.  There is a slight risk of anxiety due to claustrophobia. Any participant who 
experiences anxiety when placed into the MR scanner will be removed from the scanner, 
offered reassurance by the MR tech doing the scan, and offered the option of continuing or 
terminating the scan. If the participant decides that the anxiety associated with MRI is 
uncomfortable for them and they wish to terminate the scan, then the examination will be ended 
at that time. There will be no attempt to coerce participants to complete exams that they are 
uncomfortable with.  Participants uncomfortable with MRI scans should preferably not be 
included in this study. Use of anxiolytic agents for completion of MRI scans is discouraged, but 
allowed at the discretion of the site clinician.  
 


 
 
  
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13.5 
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 150 mls of blood will be 
drawn for routine and biomarker laboratory assessments over the course of this trial.   
 
14.0 
PERSONNEL REQUIREMENTS 
 
The following staff-member roles will be required to conduct the protocol at each site.  Details 
will be provided in the procedures manual. 
 
! 
Site Protocol Principal Investigator: The Site Protocol Principal Investigator (PI) is 
responsible for the overall conduct of the study at the site. 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. The PI may also perform certain ratings him or herself. 
! 
Study Clinician: This person 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 protocol.  The study clinician will also perform the optional lumbar 
punctures.  The study clinician may perform the CDR-SB. The study clinician may be a 
physician, or if consistent with local practice and regulations, a nurse practitioner or 
physician’s assistant. The site Principal Investigator may also serve as the study clinician. 
! 
Study Coordinator: This person 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, 
maintaining a log of treatment adherence, serving as liaison with the clinical monitor, 
coordinating clinic visits, and administering cognitive assessments. 
! 
Interviewer/Psychometrician:  This person will be responsible for administering the 
cognitive and functional assessments.  The same individual should not administer both the 
ADAScog and the CDR-SB. 
! 
CDR Rater: This person will render the CDR-SB rating based on clinical assessment of 
participant and study participant. Since the CDR-SB is a secondary outcome measure, it is 
important for the CDR rater to remain blinded to ADAS-cog data. 
! 
ADAS-Cog12 Rater: This person should be blinded to the CDR-SB data. 
! 
Regulatory Affairs: This person will be responsible for managing all regulatory related 
documents for the duration of the trial, including submitting all required regulatory and 
essential documents to the Coordinating Center Regulatory Affairs. 
! 
Billing Remittance and Statement: This person will be responsible for reviewing and 
verifying payments from the Coordinating Center are in alignment with procedures 
completed, along with accepting and processing payments from the Coordinating Center. 
! 
MRI Contact: This person will be responsible for conducting phantom and human volunteer 
scans using the appropriate scanning sequence, for site qualification purposes and as 
needed to assess for drift. As well as conducting participant MRI scans per protocol. 
  
 


 
 
  
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15.0 
STUDY DRUG 
 
All participants will take two daily doses of INI (20 IU bid for a total of 40 IU daily) or placebo, 
administered approximately 30-60 minutes after breakfast and dinner.  Participants should be 
instructed to skip the morning dose of study drug on study visit days to avoid the acute effects of 
insulin. 
 
15.1 
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 
Placebo (Sterile Diluent) 
 
The matching placebo/sterile diluent (NDC: 0002-0800-01, Eli Lilly & Company) will be 
comprised of: Glycerin, 16 mg; Metacresol, 1.6 mg; Phenol, 0.65 mg; Sodium Phosphate 
Dibasic, 3.8 mg dissolved in 1 ml distilled water. 
 
 
15.3 
Randomization 
 
We will use a semi-crossover design (Jarjoura 2003). Initially, eligible participants will be 
randomized on a 1:1 schedule to receive placebo or 20 IU bid of INI using the following 
randomization strategy: ApoE-e4 carrier status (yes or no), gender, MMSE score (high, low). 
Study site and age will be weighted in a covariate-adaptive randomization strategy to achieve 
optimal balance between treatment arms on important factors that may influence treatment 
outcome. After 12 months of double-blinded treatment with placebo or 20 IU bid of insulin, all 
participants will receive 20 IU INI bid in a 6-month, open-label extension. 
 
15.4 
Blinding 
 
For the initial 12-month period, neither participants, study partners, site personnel, most 
Coordinating Center staff, nor PI will know whether the participant is receiving active study drug 
or placebo. Exceptions may be personnel involved in preparing the DSMB reports, members of 
the DSMB and personnel involved in managing certain aspects of the study drug supply chain. 
 
 
15.5 
Study Drug Administration 
Participants will use a needleless syringe and a vial adapter to load study drug into the POD tip.   
Package labels and study diary will instruct participants and their study partners to administer 
each dose 30-60 minutes after breakfast and dinner. If a dose is missed, it should not be 
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15.6 
Intranasal Administration 
 
The POD device specifically targets olfactory delivery to maximize drug transport to the CNS by 
releasing a measured insulin dose (0.1 mL) into a POD tip that rests inside the participant’s 
nostril.  The insulin or placebo is delivered by actuating a propellant, delivering 10 IU to the 
upper nasal cavity each time the device is activated for a total daily dose of 40 IU.  A pilot study 
(Core Human Factors, Inc., 2012) evaluating the usability and tolerability of the POD device 
demonstrated that participants preferred the POD device over a conventional nasal pump.   
 
Participation of the study partner in the administration of the study drug is recommended for all 
participants regardless of which device is used. If site clinicians determine that assistance is 
needed, study partners will participate in placing the study drug into the device and 
administering the insulin or placebo twice daily (after breakfast and dinner). Participants and 
study partners will be trained in use of the delivery device. If the study partner is not available to 
assist with the administration of the study drug twice a day, another person (such as a family 
member or friend or other) may be appropriate to assist the participant. The person (or persons) 
who assists with the study drug administration must be properly trained prior to assisting.  
 
15.7 
Compliance  
 
Compliance will be assessed by on-site vial counts preferably during each study visit. 
Participants will be instructed to return their study diary and all used and unused study drug 
vials at each visit. Site personnel will count returned vials and evaluate compliance preferably 
while the participant and study partner are still on-site. Participants found to be more than 20% 
noncompliant will receive specific instruction from site personnel, including re-instruction on 
study procedures and additional telephone contact between visits. 
 
15.8 
Dose Adjustments 
 
Dose adjustments by the Investigator are generally not permitted, however if a participant 
experiences an AE that is clinically significant, the Investigator has the option of interrupting 
therapy. The Investigator should contact the Project Director to obtain approval and instructions 
for a re-challenge. 
 
15.9 
Breaking the Blind   
 
In the case of an adverse event requiring treatment, sites will be directed to treat each 
participant as if he/she were receiving active study drug.  Decisions regarding breaking the blind 
must be made in conjunction with the Project Director or Medical Director.  If the blind has been 
broken in any instance, the PI must document the following information on the participant’s case 
report form: the date, the site personnel exposed to the treatment assignment, and the reason 
the blind was broken.   
 
15.10 Storage   
 
Participant and study partners will be instructed to keep the study drug at a controlled 
temperature in the refrigerator. 
 
 


 
 
  
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15.11 Drug Accountability   
 
Participants must return all used, unused and partially used study drug vials to the investigator.  
All returned study drug vials will be accounted for by the clinical monitor during monitor visits 
and disposed of according to local site procedures.  
 
16.0 
ADVERSE EVENTS 
 
16.1 
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. 
 
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. 
 
16.2 
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. All pertinent information will be entered into the e-
CRF. All adverse events will be reported to the DSMB. 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.  
 
17.0 
SERIOUS ADVERSE EVENTS (SAE) 
 
17.1 
Definition  
 
A serious adverse event is defined as per the Code of Federal Regulation Title 21 Part 312  
 
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17.2 
Reporting SAEs 
 
Any serious and adverse event due to any cause, which occurs during the course of the 
investigation (i.e. anytime after informed consent, regardless of study drug exposure) or within 
30 days of receiving the last dose or last study visit, whichever is longer, must be reported to the 
Project Director and the Coordinating Center within 24 hours of learning of the event. All serious 
adverse events will be reported to the DSMB. 
 
18.0 
ETHICS & REGULATORY CONSIDERATIONS 
 
18.1 
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.   
 
18.2 
Institutional Review Board (IRB) 
 
Each participating institution must provide for the review and approval of this protocol and the 
associated informed consent documents and recruitment material by an appropriate IRB 
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. In the 
United States, only institutions holding a current US Federalwide Assurance issued by OHRP 
may participate.  
 
The investigator must obtain approval from the IRB for all subsequent protocol amendments 
and, when warranted, changes to the informed consent document. Protocol and informed 
consent form amendments can be made only with the prior approval of the Coordinating Center.  
The investigator may not implement any protocol deviation without prior notification to the 
Coordinating Center and prior review and documented approval of the IRB, except where 
necessary to eliminate an immediate hazard to study participants, or when change(s) involve 
only logistical or administrative aspects of the trial (ICH 4.5.4). The investigator shall notify the 
IRB of deviations from the protocol or serious adverse events occurring at the site, in 
accordance with local procedures.  
 
 
 


 
 
  
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18.3 
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, biological sample storage and imaging scans as well. The consent for 
storage will include consent to access stored data, biological samples, and imaging data 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. MRI scan findings of clinical significance, 
determined by the site radiologist, can be shared with participants per site clinician discretion.  
 
Consent forms will be developed by the Coordinating Center in collaboration with the Project 
Director (PD). The sample consent form includes all of the required elements of informed 
consent required by the FDA and GCP. Once developed and approved by the PD, the sample is 
sent to sites participating in the protocol where it is tailored to include site-specific information as 
well as to meet local IRB consent form regulations. Each site’s PI, under the guidance of his/her 
IRB, is responsible for ensuring that all applicable state laws are met with regards to judgment 
of competency and the consent form process. Each study site must submit its letter of IRB-
approval and the approved consent form to Regulatory Affairs at the Coordinating Center along 
with other required regulatory records and essential documents in order to be approved to 
receive study drug/supplies and enroll participants into this study. During the first monitoring 
visit following participant enrollment, the clinical monitors will review the consent forms and 
verify that the proper signatures have been obtained.  
 
18.4 
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 of the sponsoring institution. Authorized 
representatives of the Coordinating Center may inspect all documents and records required to 
be maintained by the Investigator, including but not limited to, medical records (office, clinic or 
hospital) and pharmacy records for the participants in this study.  The clinical study site will 
permit access to such records. 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 


 
 
  
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the subject, except as necessary for monitoring by the Coordinating Center, their 
representatives,, 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. 
 
19.0 
GENETIC RESEARCH & STORAGE OF GENETIC MATERIAL  
 
The DNA is banked in locked freezers in the ADCS Biomarker Core at UCSD.  Sample tubes 
are bar-coded and linked to participant ID number only and banked without personal identifiers. 
The presence of the sample is recorded into a computerized inventory database that is 
encrypted and password-protected. 
 
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 qualified investigators to probe candidate 
genetic polymorphisms as predictors of outcome in future studies.  The samples will be stored 
by the ADCS as long as funding is available from the NIH.  If funding should lapse completely, 
the UCSD ADRC will provide responsible custodianship of the ADCS biospecimen bank. 
  
An online request form is available through the ADCS website for investigators wishing to 
access DNA samples stored by the ADCS.  Specific procedures for requesting samples for 
future genetic studies have been prepared by the ADCS DNA subcommittee in accordance with 
the recommendations of the NBAC Human Biological Materials Report.  These DNA guidelines 
were developed in accordance with the American Society for Human Genetics’ position paper 
on the NBAC report and the Ad Hoc Committee on Stored Tissue of the College of American 
Pathologists.  
 
19.1 
Storage of Biospecimen Samples  
 
All biospecimens being banked for future AD biomarker research will be shipped to and stored 
by the ADCS Biomarker Core at UCSD.  
 
20.0 
RISKS AND BENEFITS ASSOCIATED WITH THIS STUDY 
 
20.1 
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 


 
 
  
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Page 47 of 55 
memory and function in these patients. The trial is designed to answer the important question of 
whether intranasal insulin can safely provide clinically meaningful benefit to adults with AD and 
aMCI.  
 
There are significant potential scientific and clinical benefits for the participant population. 
Currently, little is known about the relationship between insulin abnormalities, Aβ regulation, and 
cognitive impairment in AD. As impaired insulin metabolism may contribute to the symptoms 
and pathophysiology of AD, this study has the potential to identify significant mechanisms 
related to cognitive decline and suggest novel therapeutic strategies for patients with AD. In 
contrast, the relatively minor risks posed by the intranasal administration, cognitive testing, MRI 
and LP are outweighed by the value of the scientific investigations outlined in this study. 
 
20.2 
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 and Retention group overseen by the 
Coordinating Center. Each site will be encouraged to employ specific efforts (e.g. media and 
community outreach activities) to attract appropriate minority participants to the trial. Spanish 
translation is available for a variety of instruments deployed in this trial. The success of minority 
recruitment is based on the specific efforts of each site (e.g. media and community outreach 
efforts) as well as the criteria for entry. This trial has been designed to maximize minority 
participation in both ways. No participant will be excluded due to his or her sex, race, or ethnic 
group.  
 
No studies have been completed that examine racial or ethnic differences in response to 
treatment with intranasal insulin. One study has been published examining gender differences in 
which 32 healthy participants were administered 160 IU regular human insulin or placebo 
intranasally; acute effects on cognitive performance were examined as outcomes. Their findings 
indicated that women showed the most cognitive benefit on hippocampal-dependent measures, 
and no sex differences on hippocampal-independent tasks (Benedict, Kern et al. 2008). 
Although this one study provides only modest support for sex differences, the current study will 
conduct secondary analyses designed to detect significant differences in intervention effect 
between sexes. 
 
20.3 
Inclusion of Children as Participants in Research Involving Human Subjects 
 
Children will not be included. 
 
21.0 
STUDY MONITORING & DATA COLLECTION 
 
Clinical monitors are responsible for contacting and visiting the investigator for the purpose of 
inspecting the facilities and, upon request, inspecting various records of the trial (e.g., case 
report forms and other pertinent data).   
 
Clinical monitors are responsible for inspecting the online case report forms at regular intervals 
throughout the study to verify adherence to the protocol, completeness and accuracy of the 


 
 
  
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Page 48 of 55 
data, and adherence to local regulations on the conduct of clinical research.  The investigator 
agrees to cooperate with the monitor to ensure that any problems detected in the course of 
these monitoring visits are resolved. 
 
In accordance with ICH GCP, representatives of the Coordinating Center may select this study 
for audit.  Inspection of site facilities (e.g., pharmacy, laboratories) and review of study-related 
records will occur to evaluate the trial conduct and compliance with the protocol, GCP, and 
applicable regulatory requirements.  
 
Results of locally administered tests such as ECG tracings and local clinical laboratory reports 
must be retained as source documents.  
 
No study document shall be destroyed without prior written agreement between the 
Coordinating Center and the investigator.  Should the investigator wish to assign study records 
to another party or move them to another location, he/she may do so only with the prior 
notification and written consent of the ATRI/RA.  
 
21.1 
Communication Plan & Site Monitoring  
 
Communication between the PI, the Coordinating Center, and the sites will occur through 
several mechanisms. Sites will communicate performance data to the clinical operations team, 
Regulatory Affairs and the Clinical Monitoring groups on an ongoing basis. These statistics will 
be reported during bi-weekly conference calls attended by the PD and the study team. Sites will 
also receive formal monitoring visits approximately twice yearly in which all ongoing trial 
operations and source documents are reviewed. Visit finding information is provided to the PI, 
following each monitoring visit. 
 
22.0 
DATA AND SAFETY MONITORING PLAN AND BOARD 
 
The Medical and Safety Core will be responsible for medical management of the trial including 
standardized coding of AEs and central medical monitoring. Safety reports will be created for 
the DSMB on a quarterly basis. Operations Core personnel will generate any aggregate 
summary tables, as needed, to the PI and the clinical sites to fulfill their regulatory/reporting 
obligations to the FDA, their respective site IRBs, as well as to the NIA, and other oversight and 
regulatory agencies. The Data Safety and Monitoring Board will monitor this trial. No 
investigator involved in the trial is a member of the DSMB. The initial task of the DSMB will be to 
review the protocol to identify any necessary modifications. If modifications are necessary, 
revisions will be reviewed by the DSMB prior to its recommendation on initiation of the project. 
The DSMB, based on its review of the protocol, will identify the data parameters and format of 
the information to be regularly reported. The DSMB will be informed of the occurrence of any 
serious adverse events and immediately notified of fatal or life threatening events. The DSMB 
may at any time request additional information from the study PI. The DSMB will initially be 
provided with data blinded to treatment status, but they may request unblinded data if there is a 
safety concern. The DSMB and NIA representative will meet in person or by conference call on 
a quarterly basis. Based on the review of safety data, the DSMB will make recommendations 
regarding the conduct of the study. These may include amending safety monitoring procedures, 
modifying the protocol or consent, terminating the study or continuing the study as designed. 
The Board will also be informed in a real-time basis of all immediately reportable AE (FDA 
defined serious AE). All reports are stripped of identifying information. These processes ensure 
substantial oversight and case review to alert the investigators, in a timely manner, to any safety 


 
 
  
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issues that may arise.  
 
23.0 
PUBLICATION POLICY 
 
The results of this study will be published.  To coordinate dissemination of data from this study, 
a publication committee will be formed.  The committee will consist of the Protocol Committee, 
interested Principal Investigators and appropriate Coordinating Center personnel.  The 
committee will solicit input and assistance from other Investigators as appropriate and adhere to 
all Coordinating Center Publications Policies. 
 
24.0 
CLINICAL TRIAL REGISTRATION & SHARING OF FINAL RESEARCH DATA 
 
This trial has been registered on www.ClinicalTrials.gov (NCT01767909).  
 
Data from this research will be shared with other researchers pursuant to the 02/26/2003 “NIH 
Final Statement on Sharing Research Data”.  The research grant contains a data sharing policy 
consistent with the goals of the NIH but which also respects the rights of commercial partners.  
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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Page 50 of 55 
25.0 
TABLE 4: SCHEDULE OF PROCEDURES AND ASSESSMENTS 
 
Visit # 
1 
2 
3 
4 
5 
6 
7 
8 
Visit Name/Month 
Screen 
Baseline 
3 
6 
9 
12 
15* 
18* 
 
 
 
 
 
 
 
 
 
Informed Consent 
X 
 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
 
Medical History 
X 
 
 
 
 
 
 
 
Concomitant Meds 
X 
X 
X 
X 
X 
X 
X 
X 
Nasal Exam 
X 
X 
X 
X 
X 
X 
X 
X 
Physical and Neurological Exam 
X 
 
 
X 
 
X 
 
X 
Vital Signs 
X 
X 
X 
X 
X 
X 
X 
X 
Height 
X 
 
 
 
 
 
 
 
Weight 
X 
X 
X 
X 
X 
X 
X 
X 
ECG 
X 
 
 
 
 
 
 
 
Modified Hachinski 
X 
 
 
 
 
 
 
 
Logical Memory II Subscale 
(Wechsler Memory Scale) 
X 
 
 
 
 
 
 
 
ADAS-cog12 
 
X 
X 
X 
X 
X 
X 
X 
MMSE 
X 
 
 
X 
 
X 
 
X 
Memory Composite (Story Recall, 
FCSRT) 
 
X 
 
X 
 
X 
 
X 
Trail-making Test (Part A & B) 
 
X 
 
X 
 
X 
 
X 
ADCS-ADL-MCI 
 
X 
 
X 
 
X 
 
X 
NPI 
 
X 
 
X 
 
X 
 
X 
CDR 
X 
 
 
X 
 
X 
 
X 
Treatment Blinding Questionnaire  
 
 
 
 
 
X 
 
 
Research Satisfaction Survey 
 
X 
X 
X 
 
X 
 
X 
Adverse Events 
X 
X 
X 
X 
X 
X 
X 
X 
Urinalysis 
X 
 
 
 
 
 
 
 
Blood Draw 
X 
X 
 
X 
 
X 
 
X 
- Clinical Labs 
X 
 
 
X 
 
X 
 
X 
- ApoE Genotyping | DNA 
Banking1 
X 
 
 
 
 
 
 
 
- Biomarkers | Serum | PBMCs  
| Sample Banking 
 
X 
 
X 
 
X 
 
X 
Optional LP 
 
X 
 
 
 
X 
 
 
- CSF Biomarkers | Banking2 
 
X 
 
 
 
X 
 
 
- post-procedure safety 
telephone check 
 
X 
 
 
 
X 
 
 
MRI 
X3 
 
 
 
 
X 
 
 
INI Device Training 
 
X 
 
 
 
 
 
 
Distribute replacement POD device 
components 
 
 
X 
X 
X 
X 
X 
 
Study Diary Return 
 
 
X 
X 
X 
X 
X 
X 
Drug Compliance 
 
 
X 
X 
X 
X 
X 
X 
Return of devices and components 
 
 
 
 
 
 
 
X 
*Open Label visits 
1DNA banking is optional  
2CSF banking is optional 
3Screening MRI to be conducted after confirmation from clinician that the participant has met inclusion/exclusion criteria 


 
 
  
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Page 51 of 55 
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SAP_001.pdf: 
CONFIDENTIAL DOCUMENT
Statistical Analysis Plan (SAP)
Therapeutic effects of intranasally-administered insulin (INI)
in adults with amnestic mild cognitive impairment (aMCI) or
probable mild Alzheimer’s disease (AD)
June 22, 2018
Author:
Chung-Kai Sun, MS
From the ATRI Biostatistics Team


CONTENTS
2
Contents
1
Introduction
3
2
Abbreviations
3
3
Study Design/Summary
4
3.1
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4
3.2
Study Aims . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4
3.2.1
Primary Aim . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4
3.2.2
Secondary Aims
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4
3.3
Study Population . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4
3.4
Power and Sample Size Determination
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4
3.5
Treatment Allocation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5
4
Study Outcome Variables
6
4.1
Primary Outcome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6
4.2
Secondary Outcomes
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6
4.3
Safety Outcomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6
4.4
Biomarker and Imaging Outcomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6
5
Sequence of Planned Analysis
7
5.1
Interim Analysis
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7
5.2
Final Analyses and Reporting
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7
6
Statistical Methods
8
6.1
Analysis Populations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8
6.2
Multiple-testing Strategy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8
6.3
Incomplete Follow-up/ Missing Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8
6.4
Covariates
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8
6.5
Data Management and Analysis Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9
7
Statistical Analysis
10
7.1
Study Flow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10
7.2
Visit Windows
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10
7.3
Patient characteristics and baseline variables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11
7.4
Analysis of Primary outcome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12
7.5
Analysis of Secondary Outcomes
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12
7.6
Analysis of Safety Outcomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12
7.7
Analysis of Biomarker and Imaging Outcomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12
7.8
Exploratory Subgroup Analyses
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13
8
Approvals
15
9
Revision History
15
CONFIDENTIALITY STATEMENT: This report from the USC Alzheimer’s Therapeutic Research Institute (ATRI) is for the sole use of the intended recipient(s)
and contains conﬁdential and privileged information. Any unauthorized review, use, disclosure, copying or distribution is strictly prohibited.


1 Introduction
3
1
Introduction
This Statistical Analysis Plan (SAP) describes the planned analyses and reporting for the study Therapeutic effects of intranasally-
administered insulin (INI) in adults with amnestic mild cognitive impairment (aMCI) or probable mild Alzheimer’s disease (AD).
The planned analyses identiﬁed in this SAP, will be included in future manuscripts. Exploratory analyses not necessarily
identiﬁed in this SAP may be performed to support planned analyses. Any post-hoc or unplanned analyses not speciﬁed in
this SAP will be clearly identiﬁed as such in the manuscripts for publication.
The following documents were reviewed when preparing this SAP:
• INI Clinical Research Protocol (INI_Protocol_v5.0_20161205.pdf)
• Case report forms (CRFs) for INI
• ATRI Statistical Analysis Plan SOP (ATRI SOP BI-002 Statistical Analysis Plan v1.0 20150908.pdf)
Readers of this SAP should read the Clinical Research Protocol for details on the conduct of this study and the operational
aspects of clinical assessments and timing for completing a patient in this study.
2
Abbreviations
ABBREVIATION
DEFINITION
ADAS-Cog
Alzheimer’s Disease Assessment Scale-Cognitive
ADCS-ADL MCI
Alzheimer’s Disease Cooperative Study-Activities of Daily Living - MCI
AE
Adverse Event
AMCI
Amnestic Mild Cognitive Impairment
ANCOVA
Analysis of Covariance
APOE/APOE4
Apolipoprotein (APOE) Epsilon 4 (APOE4)
ATRI
Alzheimer’s Therapeutic Research Institute
CDR-SB
Clinical Dementia Rating – Sum Of Boxes
CMRgl
Cerebral Metabolic Rate for glucose
CRF
Case Report Form
CSF
Cerebrospinal Fluid
DSMB
Data and Safety Monitoring Board
FDG
Fluoro Deoxy Glucose
FCSRT
Free And Cued Selective Reminding Test
mITT
modiﬁed Intention-To-Treat
MMSE
Mini- Mental State Examination
MRI
Magnetic Resonance Imaging
NPI
Neuropsychiatric Inventory
PBMC
Peripheral Blood Mononuclear Cell
PET
Positron-Emission Tomography
p-tau 181
phospho-tau 181
PK
Pharmacokinetic
SOP
Standard Operating Procedure
SAE
Serious Adverse Event
SAP
Statistical Analysis Plan
SPM
Statistical Parametric Mapping
sROI
statistical region of interest
CONFIDENTIALITY STATEMENT: This report from the USC Alzheimer’s Therapeutic Research Institute (ATRI) is for the sole use of the intended recipient(s)
and contains conﬁdential and privileged information. Any unauthorized review, use, disclosure, copying or distribution is strictly prohibited.


3 Study Design/Summary
4
3
Study Design/Summary
This section is from the Clinical Research Protocol, INI_Protocol_v5.0_20161205.pdf
3.1
Overview
The study will consist of a multisite, randomized, double-blind trial comparing the effects of INI (20 IU bid for total daily dose of
40 IU) and placebo for 12 months, followed by a 6-month open-label period in which all participants will receive INI. Participants
with aMCI or probable mild AD (n=240-300) will be enrolled. The primary outcome measure will consist of the ADAS-Cog12.
Secondary measures will include a memory composite, an executive function test, CDR Sum of Boxes (CDR-SB), and the
ADCS-ADL MCI. The ADAS-Cog12 will be administered at 3 month intervals to optimize imputation of missing data. Other
cognitive and functional measures will be administered at 6 month intervals. MRI measures of entorhinal cortex and hippocam-
pal atrophy will be obtained at screening and 12 months. CSF and plasma biomarkers, as well as PBMCs and APOE-ϵ4 allele
carriage will also be assessed.
3.2
Study Aims
3.2.1
Primary Aim
To test the hypothesis that 12 months of treatment with INI (compared to placebo) in adults with aMCI and probable mild AD
will improve performance on a global measure of cognition (ADAS- Cog12).
3.2.2
Secondary Aims
1. To test the hypothesis that 12 months of treatment with INI (compared to placebo) in adults with aMCI or probable mild
AD will improve performance on a memory composite (Story Recall and FCSRT) and on daily function in adults with
aMCI and probable mild AD.
2. To test the hypothesis that INI treatment reduces the rate of hippocampal and entorhinal atrophy as measured by MRI,
and conduct exploratory analyses of other brain regions.
3. To test the hypothesis that INI will favorably alter CSF Aβ and the CSF Aβ/tau ratio, and will modulate inﬂammatory
markers.
4. To examine whether baseline AD biomarker proﬁle, APOE4 allele carriage and gender predict treatment response.
5. To determine whether further improvement occurs after 18 months of treatment.
3.3
Study Population
A total of 240-300 adults diagnosed with aMCI or probable mild AD will be enrolled in this trial. We expect to enroll no more
than 50% of participants with probable mild AD and no more than 60% participants with aMCI diagnosis. To determine eligi-
bility, all participants will undergo cognitive assessment, physical and neurological examination, ECG, clinical/safety laboratory
assessment, and interviews of the participant and study partner.
3.4
Power and Sample Size Determination
Power calculations to estimate the sample sizes required to detect a difference between the rate of change in two groups
were based on the two-sample t-test using the formula in Statistical Methods in Medical Research (Armitage and Berry 1994).
Sample sizes per arm were estimated using ADNI 12-month change scores for the ADAS-Cog12 for the combined MCI and
AD population, targeting power=.80, Type 1 error level=0.05, SD=5.72. Table 1 shows the range of estimates needed to
detect absolute change score differences between groups ranging from 1.6-2.6 for dropout rates between 0.15-0.25. These
estimates were derived with 6 month intervals between testing, and thus more frequent administration of the ADAS-Cog12 in
CONFIDENTIALITY STATEMENT: This report from the USC Alzheimer’s Therapeutic Research Institute (ATRI) is for the sole use of the intended recipient(s)
and contains conﬁdential and privileged information. Any unauthorized review, use, disclosure, copying or distribution is strictly prohibited.


3.5
Treatment Allocation
5
this trial should provide even greater power. Similarly, a 4-month pilot trial (Craft, Baker et al. 2012) dropout rate was <7%,
and although a higher dropout rate is expected with a longer, multi-site trial, the excellent safety proﬁle and incentive of the
open-label extension at the end of the trial should maximize retention. The addition of a delayed recall score should increase
sensitivity for aMCI participants. For MRI endpoints, sample sizes needed to detect a 25% slowing in mean rate of decline in
the entorhinal cortex over a 12-month period ranged from 70 for AD to 280 for aMCI (Holland, Brewer et al. 2009). Given that
our n=240, and the sample will be composed of both aMCI and AD, we should have adequate power.
Table 1: Sample Size Estimate
1.7
1.8
1.9
2.0
2.1
2.2
2.3
2.4
2.5
2.6
2.7
Dropout rate .15
431
382
341
306
276
250
228
209
192
177
163
Dropout rate .175
436
387
345
309
279
253
231
211
194
179
165
Dropout rate .20
441
391
349
313
282
256
233
214
196
181
167
Dropout rate .225
446
395
353
317
286
259
236
216
198
183
169
Dropout rate .25
451
400
357
320
289
262
239
218
201
185
171
3.5
Treatment Allocation
The two interventions are as follows:
• Group 1: active, 20 IU Humulin RU-100 bid for a total daily dose of 40 IU
• Group 2: placebo, matching placebo (sterile diluent)
Initially, eligible participants will be randomized on a 1:1 schedule to receive placebo or 20 IU bid of INI using the following
randomization strategy:
• MMSE range (<=25 vs. >25)
• Site
• ApoE-e4 carrier status (yes or no)
• Gender (weight: 2)
• Age (<=70 vs. >70)
will be weighted in a covariate-adaptive randomization strategy to achieve optimal balance between treatment arms on impor-
tant factors that may inﬂuence treatment outcome. After 12 months of double-blinded treatment with placebo or 20 IU bid of
insulin, all participants will receive 20 IU INI bid in a 6-month, open-label extension.
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and contains conﬁdential and privileged information. Any unauthorized review, use, disclosure, copying or distribution is strictly prohibited.


4 Study Outcome Variables
6
4
Study Outcome Variables
4.1
Primary Outcome
Alzheimer’s Disease Assessment Scale-Cognition 12 (ADAS-Cog12)
4.2
Secondary Outcomes
• CDR-SB
• memory composite (Immediate Paragraph Recall, Delayed Paragraph Recall and FCSRT)
• ADCS-ADL MCI
4.3
Safety Outcomes
• Adverse Events (AEs)
• Serious Adverse Events (SAE)
• Deaths
• Hospitalizations
• Vital Signs
• Standard Laboratory Tests
4.4
Biomarker and Imaging Outcomes
• MRI volumes (hippocampus, entorhinal cortex)
• Aβ42, CSF total tau, p-tau 181, Aβ42/tau, p-tau 181/Aβ42, Aβ42/Aβ40 ratio.
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and contains conﬁdential and privileged information. Any unauthorized review, use, disclosure, copying or distribution is strictly prohibited.


5 Sequence of Planned Analysis
7
5
Sequence of Planned Analysis
5.1
Interim Analysis
AEs and SAEs are being monitored on a regular basis by a Data and Safety Monitoring Board (DSMB). There are no planned
interim analysis being conducted for this study, but the DSMB may modify this during ongoing safety monitoring.
5.2
Final Analyses and Reporting
All ﬁnal planned analyses identiﬁed in the research plan and in this SAP will be performed in two phases:
• blinded phase:
After the last participant has completed all assessments scheduled for blinded phase (12 months) of the study and the
database for this phase has been cleaned and locked. This analysis will only include visits up to and including 12 months.
• follow-up phase:
After the last participant has completed assessments scheduled for the study (18 months) and the complete database
has been cleaned and locked. This analysis will only include visits up to and including 18 months.
Any post-hoc exploratory analyses performed to provide support for planned analyses but not identiﬁed in this SAP will be
documented and clearly identiﬁed as unplanned analysis in the manuscript.
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and contains conﬁdential and privileged information. Any unauthorized review, use, disclosure, copying or distribution is strictly prohibited.


6 Statistical Methods
8
6
Statistical Methods
6.1
Analysis Populations
• Intent-to-treat (ITT) population: All eligible individuals who are randomized.
• Modiﬁed ITT (mITT) population: Subjects who are randomized and have at least one post-baseline assessment.
• Compliance population: Subjects complete the study on treatment with ≥80% treatment compliance based on study
diary.
6.2
Multiple-testing Strategy
We propose a multiple-testing strategy using a basic serial gatekeeping procedure to maintain over-all experiment-wise Type I
error at α = 0.05 (two-sided). The four hypotheses regarding (i) ADAS-Cog12, (ii) CDR-SB, (iii) memory composite (Immediate
Paragraph Recall, Delayed Paragraph Recall and FCSRT), (iv) ADCS-ADL MCI will be tested in order using a basic serial
testing strategy. If, for example, pADAS ≥0.05, no subsequent hypotheses will be declared statistically signiﬁcant. Otherwise
if pADAS < 0.05, the CDR-SB hypothesis is tested by comparing pCDR to 0.05. This strategy continues until a p-value is
≥0.05, or the ADCS-ADL MCI hypothesis is declared statistically signiﬁcant with pADL < 0.05.
Regardless of the outcome of the multiple testing strategy, p-values and 95% conﬁdence intervals will be reported for all
planned analyses.
6.3
Incomplete Follow-up/ Missing Data
• If any of the individual items for the clinical outcomes are missing, every effort will be made to obtain the score for the
missing item or items.
• ADAS-Cog12 and CDRSB Scoring: Total scores for assessments with missing item scores will be imputed using a
proration strategy. If the maximum score with the non-missing items represents ≥70% of maximum possible total score,
the total score will be imputed. The score observed with the non-missing items will be prorated to the score with all
items, as follows:
Imputed score = Maximum score with all items × Score observed with non-missing items
Maximum score with non-missing items
The imputed score will be rounded to the nearest integer. If the maximum score with the non-missing items represents
< 70% of the maximum score with all items, the total score for the assessment at that visit will not be imputed and will
be considered missing.
• ADCS-ADL MCI Scoring: The response of "Don’t Know" for any individual question will be scored as 0 (the same score
as response of "no").
• FCSRT scoring: Sum of total free and total cued recall score. The score is missing if cued recall is missing.
• Memory Composite (Immediate Paragraph Recall, Delayed Paragraph Recall and FCSRT): Each of the three component
scores is divided by the baseline sample standard deviation of that component to form standardized Z scores. Three Z
scores are summed to form the memory composite. If any of Immediate Paragraph Recall, Delayed Paragraph Recall
and FCSRT is missing, the memory composite is missing.
6.4
Covariates
Covariates to be included are speciﬁed for each analysis, when applicable.
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6.5
Data Management and Analysis Software
9
6.5
Data Management and Analysis Software
Most data manipulation, tables, ﬁgures, listings and analyses will be created using R programs and performed using R statistical
software (http://www.r-project.org). R version used will be speciﬁed in the analysis reports.
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and contains conﬁdential and privileged information. Any unauthorized review, use, disclosure, copying or distribution is strictly prohibited.


7 Statistical Analysis
10
7
Statistical Analysis
7.1
Study Flow
A CONSORT style ﬂow diagram will illustrate patient progression through the trial from initial screening for eligibility to com-
pletion of the ﬁnal primary outcome assessment. Number (percentage) will be included for subjects screened, screen failed
(with reasons), randomized, study discontinued (with reasons), treatment discontinued (with reasons), analyzed, excluded from
analysis (with reasons). The diagram will be separated by treatment groups starting from randomization.
7.2
Visit Windows
As subjects do not always adhere to the protocol visit schedule, the following rules are applied to assign actual visits to analysis
visits. Listed below are the visit windows and the target days for each visit. The reference day is Study Day 1. If a subject
has 2 or more actual visits in one visit window, the visit closest to the targetday will be used as the protocol visit for that visit
window. Listed below (Table 2) are the visit windows and the target daysfor each visit deﬁned in the protocol.
Table 2: Visit Windows for Analysis
Visit Code
Time Interval
(days)
Target
(days)
bl
(-30, 1)
1
m03
(2, 136)
91
m06
(137, 225)
181
m09
(226, 315)
271
m12
(316, 379)
366
m15
(380, 502)
458
m18
(503, 554)
541
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7.3
Patient characteristics and baseline variables
11
7.3
Patient characteristics and baseline variables
Baseline demographics and clinical characteristics will be summarized by frequencies and percentages for categorical vari-
ables, continuous variables will be summarized by mean and standard deviation as well as quartiles. Group comparisons will
be performed using Student t-tests or Wilcoxon Rank-Sum tests, as appropriate, for continuous variables. Fisher’s Exact test
will be used for categorical data. The following baseline demographic and clinical characteristics will be presented:
• Demographics
– Age
– Gender
– Race
– Ethnicity
– Language
– Marital status
– Retirement
– Living situation
– Education
– ApoE-e4 carrier status (yes or no)
• Clinical
– ADAS-Cog12
– Memory Composite (Story Recall, FCSRT)
– MMSE
– NPI
– ADCS-ADL MCI
– CDR-SB
– Modiﬁed Hachinski
– Logical Memory II Subscale (Wechsler Memory Scale)
– Trail-making Test
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7.4
Analysis of Primary outcome
12
7.4
Analysis of Primary outcome
Individuals in the active arm with Impel device will demonstrate slower rate of decline on ADAS-Cog12 in comparison to the
placebo arm. Mixed Model repeated measures analysis of variance (MMRM) will be used to assess the difference between
treatment groups in change from baseline for performance on the ADAS-Cog12. Participants will be included in the model if
they have both a baseline and at least one post-baseline ADAS-Cog12. Change from baseline at each post-baseline visit for
the ADAS-Cog12 will be the dependent variable. We will use a heterogeneous autoregressive AR(1) covariance structure. If
the heterogeneous AR(1) covariance structure results in a lack of convergence, the following covariance structures will be used
in sequence: heterogeneous compound symmetry covariance, homogeneous autoregressive AR(1) covariance, homogeneous
compound symmetry covariance. Fixed effects in the model will include the following terms: (i) baseline ADAS-Cog12 score, (ii)
treatment, (iii) visit, (iv) treatment-by-visit interaction, and (v) APOE-ϵ4 genotype (Y/N) (vi) gender (vii) baseline MMSE score
(viii) baseline age. Visits (baseline, month 3, month 6, month 9 and month 12) will be treated as a categorical variable. The
null hypothesis is that the treatment difference between active and placebo with Impel device for the ADAS-Cog12 at month 12
is equal to zero.
7.5
Analysis of Secondary Outcomes
Secondary Aims I
Individuals in the active arm with Impel device will demonstrate slower rate of decline on secondary outcomes including CDR-
SB, memory composite (Story Recall and FCSRT), and ADCS-ADL MCI in comparison to the placebo arm. Mixed Model
repeated measures (MMRM) models similar to the primary analysis will be used.
Secondary Aims V
To determine whether further improvement occurs after 18 months of treatment.
• The analysis will be the same as the primary analysis with data up to the month 18. The null hypothesis is that the
treatment difference between active and placebo with Impel device for the ADAS-Cog12 at month 18 is equal to zero.
• Delayed-start analysis is to compare ∆2, the treatment differences between active and placebo at the month 18 (delayed-
start period) with ∆1 that at the end of the placebo-controlled period with noninferiority margin as 50% of ∆1 [1, 2].
7.6
Analysis of Safety Outcomes
To assess the safety and tolerability of treatment with INI over a 12 month compared to placebo . Tolerability and safety will
be determined by serious adverse events, adverse events and treatment discontinuation rate of INI/placebo as determined by
the participant and the physician. AEs and SAEs will be recorded and categorized by body system, event type, attribution,
frequency, severity, and course. Differences for rates of AEs, SAEs or other abnormalities between groups will be assessed
using Fisher’s Exact tests. Changes in vital signs, and laboratory tests between groups will be assessed using a MMRM
approach analogous to the primary analysis.
7.7
Analysis of Biomarker and Imaging Outcomes
Secondary Aims II and III
Individuals in the INI arm will demonstrate slower rate of decline on MRI volumes and CSF biomarkers in comparison to the
placebo arm.
Analysis Plan: The rate of change in MRI volumes and CSF biomarkers will be compared between the two treatment groups
using a linear mixed-effect model with MRI volumes and CSF biomarkers at each time point (including baseline) as the outcome
variable. The model will include ﬁxed effects for (i) time (as a continuous variable), (ii) time-by-treatment interaction, (iii) APOE-
4 genotype (Y/N), (iv) gender (v) baseline MMSE score (vi) baseline age; and participant-speciﬁc random intercepts. The
model will constrain the two treatment groups to have the same mean at baseline. The hypothesis will be tested using the
p-value corresponding to the mean group difference in rate of change in MRI volumes and CSF biomarkers.
CONFIDENTIALITY STATEMENT: This report from the USC Alzheimer’s Therapeutic Research Institute (ATRI) is for the sole use of the intended recipient(s)
and contains conﬁdential and privileged information. Any unauthorized review, use, disclosure, copying or distribution is strictly prohibited.


7.8
Exploratory Subgroup Analyses
13
7.8
Exploratory Subgroup Analyses
Secondary Aims IV
All analyses described above will be repeated on the following subgroups to explore the differential treatment effects:
• Compliance population
• Amyloid Positive (< 600pg/ml) vs Negative (>= 600 pg/ml) based on Aβ42 MSD platform.
• Number of ApoE-e4 alleles (0, 1, 2).
• ApoE-e4 carrier status (Positive vs Negative).
• Gender Male vs Female.
• Screening MMSE median split.
• Clinical Diagnosis MCI vs AD.
Supporting analysis
Two sets of analyses will be conducted as the part of the primary, secondary and subgroup analysis.
• individuals randomized to Kurve device alone
• all individuals in the study with both Impel and Kurve device. Device and device by treatment interaction are included as
ﬁxed effects in the model. If the device by treatment interaction is not signiﬁcant (p > 0.05), only device is included as a
ﬁxed effect.
CONFIDENTIALITY STATEMENT: This report from the USC Alzheimer’s Therapeutic Research Institute (ATRI) is for the sole use of the intended recipient(s)
and contains conﬁdential and privileged information. Any unauthorized review, use, disclosure, copying or distribution is strictly prohibited.


REFERENCES
14
References
[1] Liu-Seifert, Hong, Scott W. Andersen, Ilya Lipkovich, Karen C. Holdridge, and Eric Siemers. 2015. "A Novel Approach
to Delayed-Start Analyses for Demonstrating Disease-Modifying Effects in Alzheimer’s Disease." Edited by Rochelle E.
Tractenberg. PLOS ONE 10 (3): e0119632. doi:10.1371/journal.pone.0119632.
[2] Liu-Seifert, Hong, Eric Siemers, Karen C. Holdridge, Scott W. Andersen, Ilya Lipkovich, Christopher Carlson, Gopalan
Sethuraman, Sharon Hoog, Roza Hayduk, Rachelle Doody, Paul Aisen. 2015. "Delayed-Start Analysis: Mild Alzheimer’s
Disease Patients in Solanezumab Trials, 3.5 Years." Alzheimer’s Dementia: Translational Research Clinical Interventions
1 (2): 111–21. doi:10.1016/j.trci.2015.06.006.
CONFIDENTIALITY STATEMENT: This report from the USC Alzheimer’s Therapeutic Research Institute (ATRI) is for the sole use of the intended recipient(s)
and contains conﬁdential and privileged information. Any unauthorized review, use, disclosure, copying or distribution is strictly prohibited.


8 Approvals
15
8
Approvals
Approved by
Title
Signature
Date
Suzanne Craft, PhD
Principle Investigator
Paul Aisen, MD
Director, ATRI
Rema Raman, PhD
Lead Biostatistician
9
Revision History
Version
Number
Date
Revision Summary
v1.0
20180205
Original
v1.1
20180607
Add a subgroup and correct typos
CONFIDENTIALITY STATEMENT: This report from the USC Alzheimer’s Therapeutic Research Institute (ATRI) is for the sole use of the intended recipient(s)
and contains conﬁdential and privileged information. Any unauthorized review, use, disclosure, copying or distribution is strictly prohibited.