Intranasal Insulin and Post-stroke Cognition: A Pilot Study
Phase: PHASE2
Conditions: Stroke
Sponsor: Wake Forest University Health Sciences

Title:  Intranasal Insulin and Post-stroke Cognition:  A Pilot Study  
Protocol ID:  IRB00029022 
NCT02810392 
Date Approved:  3.15.18 
 
   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
 
 
 

1. SPECIFIC AIMS  
Nearly 800,000 people in the U.S. have strokes each year,1 and about two-thirds of stroke 
survivors have cognitive impairment, manifested as memory, language, and judgement 
problems. Cognitive impairment, even after a mild stroke, impairs survivors’ return to gainful 
employment2 and social function.3 It also reduces life satisfaction and reintegration to normal 
living.4  Importantly, the degree of a stroke survivor’s cognitive deficits is a primary predictor of 
caregiver burden.5  Post-stroke cognitive impairment at 2 weeks is a significant predictor of 
long-term functional outcome,6 and cognitive impairments impede functional outcome and 
ability to participate in rehabilitation.7,8  Inflammation is a likely underlying contributor to 
cognitive impairment.9,10 Our preliminary data suggest that the inflammatory biomarker, VCAM-
1, is linked to post-stroke cognitive dysfunction, specifically memory. 
Currently, no treatments exist  that boost cognitive recovery during the early post-acute stroke 
period.11 Thus, the National Institute of Neurological Disorders and Stroke (NINDS) has listed 
the study and treatment of vascular cognitive impairment as one of their highest priorities in 
stroke research. Intranasally-administered insulin (INI) is a promising new therapy for 
enhancing memory in patients with mild cognitive impairment (MCI) and Alzheimer’s disease 
(AD).  Multiple randomized controlled studies performed by our study team have shown that 
INI at doses of 20 IU BID for a minimum of 3 weeks improved verbal memory and caregiver 
rating of functional status in patients with MCI and mild to moderate AD.12,13  The likely 
mechanisms of benefit are intranasal insulin’s ability to restore normal cerebral insulin 
signaling.14  Cerebral insulin resistance15 is associated with inflammation and oxidative 
stress,16 and occurs in both AD and post-stroke cognitive impairment.15  We hypothesize that 
intranasal insulin administered in the post-acute period will improve cognition in patients with 
ischemic stroke or intracerebral hemorrhages (ICH) and reduce caregiver burden. Here we 
have designed an innovative proof-of-concept, feasibility trial to provide pilot data on the effect 
of intranasal insulin on: 1) stroke survivor cognitive impairment and caregiver burden; and 2) 
inflammatory biomarkers, including VCAM-1.  Thus, our Specific Aims are as follows:   
Aim 1.  To determine whether patients with ischemic stroke or ICH randomized to INI 20 IU 
BID for 3 weeks have improved cognition, compared to patients who receive intranasal saline.  
The primary outcome is the composite of (a) memory and executive function z scores, based 
on a cognitive battery recommended by the Vascular Cognitive Impairment (VCI) 
Harmonization;17 and (b) verbal memory, using story recall.12,13  
Aim 2.  To assess the impact of INI vs saline on inflammatory biomarker levels (VCAM-1, 
TNF-α, TNFRI and II). (DNA) 
Aim 3.  To measure differences in burden among caregivers of participants in the INI vs 
intranasal saline groups. 
Exploratory Aim.  To develop a repository of DNA on participants to assess any genetic 
differences in responses for those receiving insulin vs saline.  
When insulin is delivered intranasally, it binds to receptors in the hippocampus and frontal 
cortex within 60 minutes without any systemic absorption,14 does not cause systemic 
hypoglycemia, and is safe in elderly patients with MCI or AD.12,13  In our trial, we will 
prospectively enroll 20 subjects (10 per group) and randomize to intranasal insulin or saline 
treatment in blocks of 4.  All participants will undergo baseline cognitive testing 6 weeks to 6 
months post stroke, then receive the assigned treatment for 3 weeks, followed by a 3-week 

washout period, with cognitive testing performed after the treatment and washout periods.As 
recommended by FDA, the first block of 4 randomized patients (2 patients receive insulin) will 
undergo treatment, and the safety results from this group will be reviewed prior to proceeding 
with the study.      
 
2. RATIONALE 
 
Post-stroke cognitive deficits are a public health problem.  The prevalence of stroke in the U.S. 
is estimated at 6.7 million, or 2.8% of the population.1  About 30% to 40% of patients with 
stroke will have cognitive impairment 3 to 6 months post-stroke,18,19 which increases after 12 
and 24 months.20  Overall, almost two-thirds of stroke survivors have cognitive impairment – 
manifested as memory, language, and judgment problems – and almost one quarter have 
deficits of higher cortical function without any obvious motor, sensory, or visual deficits.21  
Therefore, at least 4 million people suffer from cognitive impairment due to stroke alone.  
Stroke-related deficits can be an important cause of early-onset cognitive impairment.22   
Cognitive impairment post-stroke impairs survivors’ return to gainful employment2 and social 
function,3 and reduces life satisfaction and reintegration to normal living, even after a mild 
stroke.4  In stroke patients, both basic and instrumental activities of daily living (IADLs) are 
directly influenced by cognitive impairment.7,8 As a result, it is also a major strain on caregivers 
because the stroke survivor’s cognitive deficits are a primary predictor of caregiver burden.5   
 
Inflammation and the association with post-stroke cognitive impairment.  Inflammation is a 
likely candidate mechanism for cognitive impairment and for decline over time after stroke, and 
a variety of inflammatory cytokines have been implicated in baseline and subsequent cognitive 
decline.10,23  One study showed that systemic inflammation was associated with worse 
memory scores in stroke patients, and independently associated with smaller hippocampal 
volumes on MRI, suggesting that inflammation may lead to cognitive decline via hippocampal 
pathways.9   
Effective interventions to treat post-stroke cognitive impairment are lacking.  Treatments that 
could boost cognitive recovery during the early post-acute stroke period are not established.11 
Thus, the NINDS has listed vascular cognitive impairment as one of their highest priorities in 
stroke research.  In an open-label randomized study, citicholine showed a significant benefit in 
attention-executive function at 6 and 12 months vs. usual treatment, but no difference in 
memory, language, spatial perception, or motor speed.24  Three trials of intensive lifestyle and 
risk factor management to prevent VCI and dementia are ongoing in Europe.25   
 
Treatment for post-stroke cognition: overlap with mild cognitive impairment (MCI) and 
Alzheimer’s disease (AD). We have designed an innovative proof-of-concept study to test 
whether post-stroke cognitive impairment is improved with intranasal insulin. An overlapping 
mechanism between ischemic stroke and AD is cerebral insulin resistance. An area of the 
brain important in both stroke and AD is the hippocampus. Thus, this study is designed to test 
mechanisms at work in a defined area of the brain, in both disorders. 
Shared pathology involving insulin resistance and tumor necrosis factor (TNF):  
Cerebral insulin resistance may occur as a result of decreased response in the setting of 
normal levels of insulin, or the necessity for more insulin to achieve a normal response.15 
Because insulin receptors are densely localized in the hippocampus, and insulin beneficially 
modulates toxic Aβ amyloid species, reduced levels of insulin and of insulin activity may 
contribute to the pathology of AD. Insulin modulates the levels of soluble amyloid-β deposits 

(endoproteolytic products of amyloid precursor protein) in the hippocampus, which are 
synaptotoxic.26  Cerebral insulin resistance is potentiated by excess TNF in the brain. TNF 
appears to be regulated by gonadotropins, an imbalance of which leads to excess brain TNF 
levels.15  This situation, and the subsequent cytokine cascade, may be viewed as the pre-
illness step in the development of AD.15  TNF also has multiple pleiotropic effects in stroke.27  It 
initiates the ischemic cascade, and promotes progression of stroke through inflammation, 
apoptosis, and necrosis. Increased levels of TNF and the subsequent inflammatory responses 
during and after ischemic stroke may therefore may be a common pathway for the 
development of cerebral insulin resistance,15 and cognitive impairment via hippocampal 
damage.9 Similarly, this association fits with the vascular contribution toward the development 
of AD-related cognitive impairment.28  Insulin resistance is also associated with inflammation 
and oxidative stress, both important in acute stroke.  In a study of stroke patients, elevated IL-6 
and reduced IL-10 levels, oxidative stress, and higher NIH Stroke Scale (NIHSS) scores were 
all associated with insulin resistance.16   
Shared pathology involving the hippocampus as a target: Cerebrovascular pathology 
may contribute to the pathology of AD and is thus often referred to as “co-pathology”.28 In 
addition, the cerebral circulation disturbances related to small vessel disease and hypoxia 
caused by systemic vascular disease likely lead to hippocampal and brain atrophy noted in 
stroke patients.29 In patients who died after large ischemic stroke in the middle cerebral artery 
(vs age-matched controls), cerebral ischemia led to up-regulation of cyclin-dependent kinase 5 
(Cdk5),30 a pathogenic process also important in AD because of its role in tau phosphorylation 
and amyloid plaque formation.31 Another study showed that elevated systemic inflammation in 
patients with first-ever mild to moderate ischemic stroke was associated with hippocampal 
atrophy and worse cognitive performance, particularly memory.9  These shared pathologies 
between ischemic stroke and AD suggest the possibility of designing of treatments with 
benefits to both disorders.   
 
A novel treatment for post-stroke cognitive impairment.  Intranasal administration of insulin is 
delivered directly to the brain and binds to receptors in the hippocampus and frontal cortex 
within 60 minutes without systemic absorption.14 In multiple randomized controlled studies, our 
team showed that intranasal insulin (20 IU BID to 40 IU BID) for a minimum of 3 weeks 
improved verbal memory and caregiver rating of functional status in patients with MCI and mild 
to moderate AD.12,13 The likely mechanisms of benefit are intranasal insulin’s ability to restore 
normal cerebral insulin signaling, and thus synaptic survival in the hippocampus.14 Given the 
beneficial actions of insulin in the brain in both AD and ischemic stroke, we hypothesize 
that intranasal insulin administered in the post-acute period will lead to improved 
cognition in patients with ischemic stroke.   
 
Post-stroke cognitive problems determine, in large part, both the patient’s ability to return to 
previous activities and the degree of caregiver burden.  The mechanisms behind post-stroke 
cognitive impairment and/or decline are not well-established, but there is an overlap with 
mechanisms in Alzheimer’s disease-related cognitive dysfunction.  Gaps this project fills and 
relevance to the AHA mission:  Our project builds on what is known about post-stroke 
cognition and the role of inflammation, and applies a promising and novel intervention for 
treatment of post-stroke cognitive dysfunction.  Multiple overlapping pathologies and pathways, 
which culminate in abnormal insulin activity in the brain, make the testing of intranasal insulin 
administration in stroke patients a logical next step. If successful, this study will provide the first 
evidence that intranasal delivery of insulin could improve cognition if administered soon after a 
stroke.  This is an extremely important and relevant goal matching the AHA mission to improve 
the lives of stroke patients.   

 
Neurologic deficits of ischemic and hemorrhagic stroke are identical. Patients with hemorrhagic 
stroke commonly have cognitive deficits post-stroke and are therefore being included in this 
study. 
 
3. METHODS  
 
Our hypothesis is that INI could improve post-stroke cognitive impairment if administered in the 
early post-acute stroke phase.  The same cohort of participants in this randomized controlled 
trial will be analyzed for all 3 aims (shown in Figure 4). 
  
Methods/procedures common to all aims: 
Design:  Block randomized, double-blind placebo-controlled trial of 20 patients, 10 per group.  
Study population:  Ischemic or ICH stroke patients with complaints of cognitive impairment 
post-stroke and a Montreal Cognitive Assessment  (MoCA) score above the 5th but at or below 
the 50th percentile of norms based on age, race, and education (Table 1) OR a MOCA score of 
2 out of 5 in the delayed recall portion and between the ages of 21 and 89 [Dr. Kaycee Sink, 
personal communication based on >9,000 diverse participant scores]. Patients will be asked 
permission for the study team to administer the 5-minute MoCA over the phone if they do not 
have a MoCA documented in their chart, or if they do not have an upcoming appointment 
where a full MoCA can be performed to assess eligibility. Patients who score ≤21 or ≤2 out of 5 
on the delayed recall on the 5-minute MocA over the phone will be eligible. 
Inclusion criteria:  Patients as above 
with clinical ischemic stroke or ICH 
and measurable deficit who are able to 
sign informed consent, have a 
caregiver (for patients with deficits that 
would require them to have assistance  
with carrying out study tasks), and live 
within a reasonable driving distance 
from WFBMC.  
Exclusion criteria:  Patients under age 
21 or 90 years or older, those 
transferred to skilled nursing facility, or 
with severe stroke deficits that prohibit 
participation in cognitive testing (global 
or receptive aphasia, or severe 
expressive aphasia,32 or those with 
diabetes taking insulin, unstable or 
severe psychiatric disorders that 
would impair the ability to undergo 
cognitive testing; severe head trauma, 
alcoholism, neurologic disorders other 
than stroke, end stage kidney disease 
that requires dialysis, acute renal 
failure that is causing delirium or other kidney disease that would impair the ability to undergo 
cognitive testing, end stage liver disease that is associated with hyperammonemia or 
metabolic encephalopathy, chronic obstructive pulmonary disease severe enough that the 
patient has known hypoxia or hypercarbia, is on chronic oxygen or their lung function is severe 
enough to impair the ability to undergo cognitive testing; unstable angina or poorly controlled 
Table 1.  Distribution of MoCA scores for eligibility based 
on race, age, and education. 
 
<70 years 
70-79 
80+ 
Whites <12 years education 
5th to 50th 
17-23 
16-22 
14-20 
Blacks < 12 years education 
5th to 50th  
14-20 
13-19 
11-17 
Whites 12 years education 
5th to 50th 
18-24 
17-23 
15-21 
Blacks 12 years education 
5th to 50th  
15-21 
14-20 
12-18 
Whites >12 years education 
5th to 50th  
21-26 
19-25 
17-23 
Blacks >12 years education 
5th to 50th  
18-23 
16-22 
14-20 

heart failure that would limit the patient’s 
ability to undergo cognitive testing,12  and 
those with prior deficits in ADLs.  We 
have not pre-specified a maximum NIHSS 
at onset for exclusion, since waiting for at 
least 6 weeks allows patients with severe 
deficits to recover naturally, and still 
potentially be able to participate.  It also 
allows most patients to complete inpatient 
rehabilitation.  
 
Recruitment:  WFBMC admits 
approximately 460 ischemic stroke 
patients and approximately 400 ICH 
patients per year.  We anticipate 40% 
(n=270) of these patients will be eligible 
after excluding for aphasia, severe stroke, 
and insulin-dependent diabetes; of those, 
a pool of 180 patients per year could be 
enrolled.  Patients will be identified while 
hospitalized on the WFBMC stroke 
service. Recruitment strategies have 
been developed to identify and approach 
potential participants. The study 
coordinator will screen the stroke service 
census and communicate with the study 
team if a potential patient is identified. 
Patients that are deemed 1.) not eligible 
at the time of initial screening and 2.) do 
not meet exclusion criteria, will be 
followed and approached when/if they 
become eligible. An invitation letter and/or 
recruitment phone call script will be used 
if patients were not able to be approached 
during their inpatient stay or follow up 
clinic visit. Patients may also be 
approached by phone and/or during other WFBMC clinic visits (i.e. rehab). Any MoCA 
performed as part of inpatient or outpatient assessments prior to enrollment will be used for 
screening eligible patients. If there is no recent MoCA assessment, the screening MoCA will be 
performed by the study team either during the phone screen or during a visit day and time that 
is convenient for the patient. If the patient scores within the range of eligibility OR a score of ≤2 
out of 5 in the delayed recall portion of the MoCA, the coordinator will obtain consent from the 
patient or a legal representative, and the initial cognitive testing/blood draw will be scheduled. 
We will aim to enroll patients that are between 6 weeks and 6 months post-stroke onset.  We 
will also advertise the study on the WFBMC web site, in local newspapers, and possibly TV 
and radio. Patients may also be referred to the study from local Neurology practices. The 
recruitment letter and recruitment phone call script and phone screen MoCA have been written 
to allow patients outside of WFBH to contact the study team. 
 
Patients with ischemic stroke or ICH meeting eligibility 
criteria (n=20) 
                                  Consent  
Screening MoCA, 2 wks-6 mths post-stroke, 
if eligible score then randomize 
Baseline cognitive battery 
Intranasal 
insulin x 3 
weeks 
(n=10) 
Intranasal 
saline x 3 
weeks 
(n=10) 
Cognitive testing, outcomes, biomarkers  
Treatment period end  
Washout x 3 weeks 
Cognitive testing, post-washout / End of 
Study 
Figure 1.  Flow diagram of the study procedures 

Randomization and Blinding: The participant will be randomized and undergo the full baseline 
cognitive battery.  Caregivers will also sign a separate consent to complete the Caregiver 
Strain Index.  Subjects will be randomized to intranasal insulin (20 IU BID) vs saline BID 
according to a stratified permuted block randomization of 4 as per our statistician, stratified by 
age 40 to 69 years vs > 70 years) and presence of language deficit (based on NIHSS) at the 
time of randomization. Insulin and saline will be packaged in identical single-dose ampules that 
will be opened and inserted into the chamber of the VianaseTM device. Ampoules will be 
dispensed in 3 week supplies at each study visit. Package labels will instruct participants to 
administer each dose 30-60 minutes after breakfast and dinner. If a dose is missed, it will not 
be replaced. The insulin and saline will be identical in packaging except for randomization 
code. Patients/caregivers, investigators, and outcome assessors will be masked to the group 
assignment. All subjects will receive their first dose in the clinic and wait for 30 minutes to 
determine any adverse effects of the inhaled dose, while remaining blinded to the treatment.  
The glucose level will be measured with the glucometer to monitor for hypoglycemia and 
documented.  All subjects will additionally check peak dose blood sugar levels with a 
glucometer, 3 times per week during insulin or saline treatment.  The patients and caregivers 
will receive a handbook which describes the method for loading the device, inhalation, 
methods for fingersticks, use of the glucometer and test strips, a blood sugar log, and the signs 
and symptoms of hypoglycemia.  They will also document any other adverse effects on the 
weekly log.   
 
Timing for the procedures (Figure 1).  Cognitive impairment measured early after stroke 
predicts functional outcomes in stroke patients at 13 months.6  Furthermore, waiting until 6 
weeks after the stroke to conduct baseline cognitive testing and treatment allows enough 
recovery and completion of intensive rehabilitation for those patients who may not be able to 
complete cognitive testing immediately after stroke, but takes advantage of the potential 
benefits of insulin in the early phase, shown in animal studies.33  In addition, we potentially 
reduce the variability of the biomarkers by avoiding the acute phase of stroke.  The frequency 
of cognitive testing is based on trials of intranasal insulin for MCI and AD.12,13 
Patients with a history of psychiatric medication use for the management of mental and 
emotional disorders prior to their stroke will not be excluded from the study. However, as these 
medications may have an effect on cognitive testing, initiation of psychiatric medications 
should be avoided from the time of study enrollment through the post wash out period unless it 
is clearly indicated to be in the patient’s best interest. Patients will be informed of the prohibited 
medications during consent and will be asked to immediately inform the study team of any new 
medications or changes in existing medications. 
 
Baseline Data collection.  We will collect the baseline data described below as in the VCI 
Harmonization standards:17   
Demographics:  Birth date, sex, race/ethnicity, years in current country of residence, number of 
years of education, occupation, literacy, living situation and level of independence, type of 
residence, marital status, and name and contact information for a family member or caregiver  
will be collected.   
Proxy/informant information collected:  Birth date, sex, race/ethnicity, relationship and length of 
time of relationship with the patient, education, and living status with respect to the subject.  
Family history information collected:  First-degree relatives with stroke, myocardial infarction, 
and dementia, approximate age at onset, and age at death. 
Stroke hospital data collected: Severity (NIHSS) at onset, location of stroke, vascular territory, 
modified Rankin score at discharge, short physical performance battery score, length of stay, 

stroke complications, stroke prevention medications at discharge, stroke risk factors (diabetes, 
hypertension, hyperlipidemia, smoking, alcohol/substance abuse, atrial fibrillation, carotid 
stenosis, hypercoagulable state), and stroke subtype (NINDS classification). 
Intranasal insulin administration:  We will use an innovative investigational device developed 
by Kurve Technology (Fig 5A). Typical spray bottle administration results in large droplets that 
penetrate only within the first 20% of the lower nasal cavity, and due to gravity and insufficient 
airflow, ~90% of the droplets wind up in the stomach (Fig.5B). The ViaNaseTM device delivers 
a substance throughout the nasal cavity, to the olfactory region and paranasal sinuses, thereby 
maximizing access to nose-to-brain channels (Fig.5C). This distribution occurs because 
droplet size is adjusted according to the weight of the substance, through an individually 
optimized droplet generator resulting in maximal vortical distribution (Fig.5D). We have used 
the ViaNaseTM device with excellent results in two pilot trials (described above).12,13  A total 
volume of 20IU of insulin or placebo (saline) will be administered each time. Caregivers will 
supervise participants in administering intranasal treatment 2 times per day, after breakfast 
and dinner. Participants and caregivers will be trained in use of the delivery device. In previous 
studies, participants with aMCI/AD have found the device to be easy and pleasant to use, with 
compliance rates of 95 to 97%.  
Cognitive battery:   
Aim 1 focuses on two primary outcomes: (a) cognitive tests, including a summary memory 
score and a summary executive function score, and (b) the story recall test, which was 
sensitive to intranasal insulin effects in MCI/AD patients within 3 weeks of treatment.12 Since 
participant fatigue can be a major factor with these cognitive tests in stroke participants, we 
have designed a testing administration procedure that can be completed in fewer than 40 
minutes. Our battery and time for administration include:   
Montreal Cognitive Assessment (MoCA):34  The MoCA is a cognitive screening instrument 
which provides information about overall mental status.  It is useful for identifying overall 
cognitive impairment, and also provides information about core cognitive domains, such as 
visuospatial abilities, attention, and memory.  
Memory Tasks.  
Hopkins Verbal Learning Test-Revised (HVLT-R)35 is a measure of rote verbal learning and 
memory.  After a 20-minute delay, participants are 
asked to name all items in a 12-item word list they 
recall.  There are 6 lists available to facilitate repeat 
administration in future testing (10 minutes with 
delay and recognition).   
Brief Visual Memory Test-Revised (BVMT-R)36 is a 
measure of nonverbal learning and memory 
captured with immediate and delayed free recall 
trials, and a recognition memory task (10 minutes 
with delay and recognition).   
Executive Function Tasks 
Trail Making Test-A & B (public domain):  Part A is a 
measure of visuomotor speed and sequencing, 
where the subject is asked to connect numerical 
dots (1-25) in ascending order as quickly as 
possible. Part B, administered immediately 
afterwards, is a measure of executive functioning 
(working memory, mental flexibility) whereby the 
subject must connect dots to sequence consecutive 

numbers and letters (1, A, 2, B, etc.→12, L, 13).  Each task is scored in terms of time to 
completion, and number of errors (4 to 7 minutes). 
WAIS-III Digit Span subtest37 is comprised of two portions. Digits Forward is a measure of 
simple attention that requires the participant to repeat increasingly longer strings of digits in 
order, starting with 2 digits and progressing to 9.  The task is discontinued when both strings of 
a specific length are incorrect.  Digits Reverse is considered a measure of working memory, 
and requires repetition of increasingly longer strings of digits in the reverse order, starting with 
2 and progressing to 8 (1 to 5 minutes). 
WAIS-III Digit-Symbol Coding.37 A sensitive measure of visuomotor processing speed, this 
task requires involves rapidly coding geometric symbols given a number, by using a legend of 
number-symbol pairs at the top of the page (2.5 minutes). 
Verbal fluency:  Animal naming and FAS (public domain):   These tasks of verbal fluency 
provide the subject one minute to say as many words as possible. For Animal Naming, any 
living creature is counted (1.25 minutes). For FAS, words beginning with a given letter (F, A, 
and then S) are counted excluding proper nouns, numbers, and variations of the same word 
(3.5 minutes). 
Story Memory recall.  A narrative of 44 informational bits is read and the recalled information is 
recorded immediately and after 20 minutes. Information retained over the delay will be 
calculated as delayed recall/ immediate recall. The participant is asked to recite it immediately, 
and then following a 20-minute delay. This measure of verbal memory is less affected by 
executive difficulties experienced in those with history of stroke or vascular disease.  Multiple 
passages exist to allow for repeat testing.12  Total time for administration is 20 minutes with 
delayed recall.  
Other outcome scales: 
Functional outcome Instrumental Activities of Daily Living (IADL) scale:  We will use the Stroke 
Impact Scale ADL/IADL scale,38 and the SIS-16,39 both of which has been extensively used 
and validated in the stroke population. This scale is based on self-reported information about 
functional skills necessary to live in the community, is easy to administer, can be administered 
by proxy, and takes about 10-15 minutes.40   
Modified Caregiver Strain Index:  This survey includes 13 questions related to caregiver strain 
(sleep, finances, time, inconvenience, etc.); a score of 7 or greater is considered significant 
caregiver strain.41  Multiple studies of stroke and the elderly have measured caregiver burden 
with this scale.42,43  Coordinators will administer the scale to caregivers separately from the 
stroke patient. 
Table 2.  Measures and the timing of assessments 
Blood collection for cytokines 
and DNA: Following consent, 
blood will be collected from 
fasting subjects on the day of 
baseline cognitive testing.  One 
tube will be used to measure 
basal levels of cytokines; this 
blood will be processed 
immediately and stored at -
80°C.  One additional tube will 
be used for DNA analysis; the 
blood will be placed in a 
refrigerator (4°C) until 
personnel from the Center for Genomics & Personalized Medicine Research can transport the 
Measure/Task 
Study start 
Treatment period 
end  
3 wks post-
washout End 
of Study 
Randomization 
X 
 
 
Fasting glucose 
and insulin 
X 
X 
 
Cognitive testing 
X 
X 
X 
Depression (PHQ-
9) 
X 
X 
X 
Biomarkers 
X 
X 
 
DNA collection 
X 
X 
 
Caregiver strain 
index 
X 
X 
X 
IADL and SIS-16 
X 
X 
 
Modified Rankin 
score (mRS) 
X 
X 
 

sample to the laboratory. At the end of the study, all frozen blood samples collected pre- and 
post- insulin treatment will be shipped on dry ice to Assaygate (Ijamsvsille, MD) for cytokine 
quantification of VCAM-1, TNFα, sTNFRI, sTNFRII. 
Cytokine quantification: All samples in the study will be stored and sent for analysis in one 
batch. Plasma cytokine concentrations will be quantified by custom bead-based suspension 
array for human cytokines by Assaygate as previously described.44  Assaygate provides 
multiplex protein analysis services for a wide range of human and mouse inflammatory 
proteins. 
DNA analysis: Genomic DNA will be isolated from whole blood (yellow-top, ACD tubes). DNA 
will be isolated using the AutoPure LS (Qiagen, Inc.), and then bisulfite-converted using the EZ 
DNA Methylation Gold kit (Zymo, Irvine, CA). To determine the proportion of DNA methylation 
at each of over 485,000 CpG sites, we will use the HumanMethylation450 BeadChip (Illumina, 
Inc.) along with the iScan Reader (Illumina, Inc.).  
Aim 1 Analysis  
Cognitive Analysis:  The analysis will be based on intention to treat.  The primary outcomes 
are the executive function (Trails B, Digit Symbol, Digit Span) and Memory (HVLT and Brief 
Visual Memory Test-Revised) composite z scores at 3 weeks of treatment in both groups.    
Secondary outcomes:  Story Recall and MoCA score (out of 30), and IADLs.  Pre-specified 
confounders include age, sex, education, presence of language deficit, and depression, 
measured with the Patient Health Questionnaire-9 (PHQ-9). The PHQ-9 has been validated as 
a measure of depression severity (PHQ-9)45 including stroke,46 and these measures will be 
obtained from the stroke subject (not proxy). 
IADL analysis:  The summary scores of the SIS IADL scale and SIS-16 will be compared from 
baseline to 3 weeks post treatment in both groups.   
Functional covariates:  Disability and physical function measures will be collected to 
understand the trajectory of motor recovery and will be mapped to the cognitive trajectory from 
baseline (2 weeks to 20 weeks post-stroke).  Modified Rankin Scale (mRS):  A 
disability/dependence scale designed as an outcome measure for stroke,47,48 and is now the 
most commonly used endpoint in therapeutic clinical trials of stroke interventions, such as 
thrombolytic therapy.49 The score ranges from 0 (no symptoms at all) to 6 (death).  Exploratory 
analyses will be performed to determine whether INI leads to an improvement in IADLs, SIS-
16, and mRS. 
 
Aim 1 Statistical Analysis: The 3 week intervention effect of INI on the cognitive outcomes 
will be assessed using analysis of covariance (ANCOVA) adjusting for age, language deficit, 
and baseline cognitive score. We will assume a Type I error rate = 0.05 for all analyses. 
Adjusting for baseline characteristics will help attenuate the potential effect of differential 
dropout,50 but we will conduct a sensitivity analysis using multiple imputation to ensure dropout 
does not unduly affect our results.51 The observed estimates, standard deviations, and dropout 
rates will be used for the design of future studies of INI treatment for stroke patients. 
Exploratory analyses will examine response by subgroup for evidence of an enhanced 
treatment effect, including subgroups defined by participant age (<70 vs. >70 years), baseline 
cognitive impairment and the presence or absence of any degree of language deficit (aphasia). 
Covariates will be utilized so that they do not overlap with clinical subgroups. Because of the 
relatively small sample size, if the prespecified subgroups based on continuous characteristics 
are not sufficiently large, new clinically meaningful subgroups may be formed for exploratory 
analyses.  
Power Analysis: The primary purpose of the pilot study is to demonstrate feasibility and safety. 
The proposed study will collect data to estimate power in future trials, identify instruments most 

sensitive to the proposed cognitive effects, and anticipate recruitment and retention rates in the 
target population. However, the proposed study will have 83% power to detect a 1 standard 
deviation difference in cognitive scores between groups at 3 weeks (i.e. effect size = 1.0), 
assuming normally distributed outcomes, = 0.05, and a 10% dropout rate.  
 
Potential Problems and Alternative Strategies 
1)  Recruitment is slower than anticipated.  Recruitment will be monitored very carefully to 
ensure we are on target with the study timeline.  However, if distance from the medical center 
is the only reason that some patients are excluded, we will amend the protocol to include in-
home cognitive testing.  We will also extend recruitment to Moses Cone, advertise in the 
Greensboro newspaper, and the local radio stations. 
2)  Study compliance may be lower than anticipated.  We will consider additional phone 
monitoring and problem-solving with participants to overcome compliance barriers.  
Compliance was 95% to 97% in all prior studies. 
 
Aim 2 Rationale   
Aim 2 will test the hypothesis that INI’s effects on post-stroke cognition will suppress 
proinflammatory cytokines and enhance immune responsiveness. In addition to improving 
cognition, INI down-regulates systemic inflammation associated with obesity and diabetes by 
altering hypothalamic insulin growth factor signaling mechanisms. We hypothesize that INI will 
improve post-stroke systemic inflammatory responses by similar mechanisms.52–54 Our primary 
goal in Aim 2 is to assess differences in inflammatory biomarkers between the two groups.  
The secondary goal is to assess the relationship between these biomarkers and the primary 
cognitive outcomes (memory and executive function z scores, and story recall).  We will also 
assess whether the relationship between biomarkers and cognition is mediated by the 
treatment group.  
Biomarker panel:  VCAM-1, which is exclusively expressed in endothelial cells, is induced by 
TNF and IL-1.55  VCAM-1 levels likely reflect advanced atherosclerosis and endothelial 
dysfunction.56  TNFα is the putative cause of cerebral insulin resistance,15 one of the presumed 
mechanisms through which INI may improve memory, and was associated with cognitive 
impairment in the Framingham cohort.57 The soluble TNF receptor I (sTNFRI) was associated 
with stroke mortality in the Northern Manhattan study;58 therefore, we will measure sTNFR I 
and II as a reflection of TNF pathway activity.   
Approach and outcomes: Biomarkers and DNA will be measured 4weeks- 3 months 
after stroke onset (at baseline cognitive testing) and at 6-9 months post stroke.  
 
Aim 2 Statistical Analysis.  All biomarker assay results will be based on mean values of 
duplicate assay measurements.  The distribution of each biomarker will be assessed, and log-
transformations will be performed as appropriate.  Non-parametric tests will be used to 
compare medians (Wilcoxon rank sum or Spearman rank correlation) and parametric tests 
(Student t test, Pearson correlation) to compare means. We will assess the randomization 
effect using the same ANCOVA model described for Aim 1, adjusting for randomization 
allocation factors and baseline scores. When log-transformed biomarkers are used, the 
treatment effect will be assessed on the log-scale values. We will test for the association 
between cognition and serum biomarkers, both using the composite memory and executive 
function z scores and each of the component parts, as well as story recall and MoCA scores. 
We will use a linear regression approach adjusting for age, education, and baseline cognitive 
scores. Secondarily, we will perform a mediation analysis to determine the potential role of 
intranasal insulin in the relationship between inflammatory biomarkers and cognition 
relationship. The long-term cognitive randomization effect will be explored using a mixed 

model analysis controlling for within-subject variability including both the post-intervention and 
long-term follow-up time points, further adjusting for randomization factors and baseline 
cognitive score. Treatment effects at 20 weeks will be assessed using contrast statements. 
The results of these analyses will provide a new understanding of the relationships between 
inflammatory biomarkers and cognitive outcomes in stroke patients. These results can then be 
used to study a broader population of cognitive impairment after stroke and/or VCI.  
 
Potential problems and alternative strategies: 
1)  Multiple comparisons of biomarkers, outcomes, and covariates could be an issue, but the 
pilot nature of this small study does not require adjustment of the significance. 
2) Technical issues:  Dr. Vidula Vachharajani has extensive experience in sample preparation, 
shipment, data analysis and interpretation.  Assaygate’s methods for cytokine detection are 
highly sensitive and published performance characteristics show a low coefficient of variation. 
Thus, we do not anticipate any problems executing these studies.  
 
Aim 3 Rationale 
For caregivers of stroke patients, more than 60% of caregivers have significant strain at 28 
days after discharge, although this decreased over the 1 year after stroke.59 The goal of Aim 3 
is to assess whether intranasal insulin treatment improves cognitive impairment after stroke, 
and also improves caregiver burden.  This is an important outcome because a similar 
caregiver rating of functional status in the pilot studies of intranasal insulin for MCI/AD was 
significantly improved with this treatment.12,13   
 
Aim 3 Analysis   
For each outcome, we will analyze mean difference from baseline to post-treatment and other 
time points; therefore, two-sample t-tests will likely be used to compare mean differences in 
the 2 treatment groups.  Alternatively, the outcomes could be categorized as positive, 
negative, or no change using a nonparametric approach.  Specific analyses will include the 
following comparisons in the 2 groups:  CSI post-treatment , and atend of study, before and 
after adjusting for baseline CSI, NIHSS, and cognitive scores. We will perform multivariate 
linear regression analyses of each outcome adjusting for treatment group, age, sex, race, 
stroke severity, and co-morbidities (e.g. diabetes, hypertension, atrial fibrillation, CAD, CHF) at 
baseline. 
 
Potential Problems and Alternative Strategies 
Retention of caregivers will be a potential issue, and the goal is to identify a primary caregiver 
at the start of the trial and continue measures with the same caregiver throughout the trial.  
However, caregivers may change during the trial due to unforeseen circumstances.  We will 
therefore identify a secondary caregiver at enrollment for each stroke participant.   
 
Exploratory Aim Analysis 
The DNA will be processed and stored until additional funding is obtained to cover the costs of 
the analyses.  Participants will be given the opportunity to opt out of this portion of the blood 
collection on the consent form.  When funding has been obtained, we will submit an 
amendment to the protocol. 
 
Table 3. Timeline for the pilot study and procedures 
 
Y1 
Y2 
Start-up  
 
 
 
 
 
Enrollment  
 
 
 
 
 
Data collection  
 
 
 
 

 
Ethical aspects of the application Safety monitoring 
phone calls will occur at weeks 1 and 2 to ensure 
proper administration techniques, monitor blood 
glucose changes, and assess for any early adverse 
events. In-person visits will occur at each cognitive testing time frame as shown in Table 1. 
Potential risks with intranasal insulin: There are no known serious risks associated with INI 
without absorption enhancers as will be administered in our study. In our prior studies of INI, 
there were no episodes of severe hypoglycemia, and no adverse effects or functional 
disturbances following intranasal insulin administration. No other serious adverse effects of 
treatment were observed.12,13 It is possible, albeit unlikely, that increased glucose levels may 
occur. If participants show fasting glucose levels above 126 mg/dl or random glucose levels 
above 200 mg/dl on two occasions, they will be referred to their primary care physician for 
evaluation.  We will use the standing institutional Data Safety Monitoring Committee (DSMC).  
The investigators and the DSMC will develop a plan for interim data analyses to monitor 
adverse events and preliminary analyses. 
 
Summary and future research 
Our translational pilot study will provide important and highly relevant preliminary data on a 
novel therapy, INI, for cognitive improvement after ischemic stroke or ICH, while measuring 
physical function simultaneously. We will accumulate early efficacy data, in addition to 
feasibility and safety in stroke patients.  At the same time, we will determine the impact of the 
intervention on systemic inflammation using markers that we and others have shown to 
influence post-stroke cognition (VCAM-1) and cerebral insulin resistance (TNF). This project 
will be the first step to addressing 1) a significant need in the quality of life of ischemic stroke 
patients, ICH patients and their caregivers, and 2) a research priority for AHA and NINDS, and 
will therefore form the basis for larger therapeutic trial applications.  Our multidisciplinary team 
includes a vascular neurologist focused on stroke outcomes, a neuroscientist with expertise in 
neuro-inflammation, a cognitive neuroscientist with expertise in INI trials, and a statistician with 
experience in stroke and aging clinical trials.  We therefore have the expertise to conduct and 
complete this research and make a contribution to the lives of stroke survivors and their 
caregivers.  
 
4. PROTECTIONS FOR HUMAN SUBJECTS 
4.1 Risks to Human Subjects 
There are 3 potential risks of participation in this trial.  1) Hypoglycemia:  Insulin given 
intranasally with the nose-to-brain device used in these studies has not been associated with 
low blood sugar in previous studies.  In diabetic patients on oral hypoglycemic, we do not 
expect any additional risks. In a recent study testing intranasal insulin in this population, 
Intranasal insulin administration appeared safe and did not affect systemic glucose control. 60 
Nevertheless, the study staff will train participants and caregivers to recognize signs of low 
blood sugar such as shaking, fast heartbeat, sweating, dizziness, anxiousness, hunger, 
impaired vision, weakness, headache, and irritability.  If the participant develops these 
symptoms, they will be instructed to eat a snack, call the study staff at (336) 716-4101 and call 
his or her physician.  2)  Nasal irritation:  A few participants in previous studies complained of 
nasal irritation after being given the study drug, but these symptoms did not persist beyond the 
initial use of the study drug.  As with any drug, there may be unexpected side effects to nasal 
insulin.  Although insulin has been safely administered intravenously, the potential toxicity of 
Biomarker 
analyses 
 
 
 
 
 
Analysis, 
manuscript 
prep 
 
 
 
 
 

the proposed clinical formulation to the nasal cavity and other locally exposed tissues has not 
been evaluated in animal studies.  3)  Blood Draw:  There is no additional risk associated with 
the blood draw for this study compared with routine clinical care.  All blood draws will be 
performed by study personnel in a monitored setting (Neurology clinic or the CRU). 
There are no risks to performing the cognitive testing, although participants may become 
fatigued. If this occurs, the participant can stop testing at any time.  Participants may 
experience some distress if they are aware of the results of the caregiver strain index (CSI), 
completed by their caregiver.  In addition, caregivers may not feel comfortable providing 
truthful answers to the questions in the presence of the participant.  Therefore, the CSI will be 
completed on paper by the caregiver while the stroke participant is completing the cognitive 
testing.  
5. RISK PROTECTION  
We have minimized the risk of adverse effects from the inhaled study drug by monitoring 
participants in the clinic or CRU for at least 30 minutes after the first dose.  Also, we will review 
the blood sugar logs of the first block of 4 participants (2 insulin and 2 saline) prior to enrolling 
the next block (as per FDA recommendations).  In addition, the participants will monitor peak 
dose blood sugars 2 to 3 hours after the study drug and record blood sugars at least 3 times 
per week for the first week of the study treatment.        
To minimize the number of sticks for blood draws, blood collection will occur at the same time 
as a scheduled blood draw as often as possible.  To minimize the possible psychological 
stress of the participant’s caregiver in completing the CSI, this will not be shared with the 
stroke survivor unless the caregiver allows sharing of the index with the participant. 
Potential benefits to the subjects and others 
There are two potential benefits to participation in this study.  If there is indeed an 
improvement in cognition with intranasal insulin, the subjects in this group will reap the benefits 
of improved memory, cognitive function, and presumably, better quality of life and functional 
status. All participants will likely benefit from contact with study personnel who will be able to 
monitor for any changes in their functional status or depression after stroke and provide 
referrals, as needed.   
5.1. Data Safety Monitoring Board 
All adverse and serious adverse events will be monitored by the study coordinator and 
reported to the WFUHS IRB and the Data Safety Officer, William Applegate, MD.  Each event 
will be reviewed to determine whether it may be related to the intranasal insulin, to the natural 
history of an individual’s stroke, or to other existing conditions. The safety officer will have 
scheduled assessments of the data from the trial, after the first block of 4 randomized subjects, 
after 20 subjects, and then at the end of enrollment of 40 subjects (about 18 months total).  In 
compliance with the FDA IND, we will submit all adverse events to FDA and a yearly progress 
report within 60 days of the “may proceed” date of October 17, 2014. 
6. INFORMED CONSENT & HIPAA AUTHORIZATION FORM 
All study personnel who screen medical records will have completed Human Subjects 
Protection Education, have secure access to records, and have completed the WFBMC 
security and data protection requirements for accessing patient records.  All admissions to the 

WFBMC stroke service will be screened by the study coordinator and/or PI for eligibility.  
Eligible participants must be at least 6 weeks post-stroke. After a potential participant is 
identified, the study coordinator will contact the potential participant, explain the study and 
provide him or her with a copy of the consent and other study documents.  If the patient 
chooses to participate, written consent will be obtained prior to randomization.  All participants 
(and their caregivers) will sign the consent before they are enrolled in the study.  The consent 
form will be approved by the WFUHS Institutional Review Board prior to study start. 
6.1. Confidentiality and Privacy  
Personal information and such information that directly identifies the participants will be 
collected during this study. This includes names, addresses, telephone numbers and dates of 
birth.  Personal health information includes all health information created as related to the 
study and information that is maintained in the medical records at this institution.  Examples of 
this personal health information include:  health history, family health history, how the 
participant responds to study questions, and information obtained during study visits and 
phone calls.     
All study information will be secured under lock and key by the program manager and the 
primary investigator at WFUSM will be responsible for maintaining this secure file of the study 
information.  We will emphasize to subjects that our interest is in average responses as 
opposed to individual data. No individual data will be released without prior consent of the 
individual(s) in question. All data collected will be coded with all HIPAA identifiers removed and 
transferred electronically using encrypted data transfer techniques. We will abide by recently 
published HIPPA regulations, which are part of the review process for Human Subjects 
Research at our institution. The signed consents and information that directly identifies the 
participants (names, addresses, phone numbers and other contact information required for the 
follow-up telephone calls and for mailing additional information to participants) will be 
maintained separately from the Case Report Forms and the institutional IRB approval 
documents.   
The Case Report Forms will identify participants by a unique study ID and the list that links the 
study ID to the personal information will be maintained in a separate file from the Case Report 
Forms.  Paper copies of the data forms will be stored in a secure, locked location and all data 
collected as part of the study will be entered into a database to which only specific study 
personnel will be allowed access.  This system is encrypted and password protected.  Data will 
be uploaded into a web-based database housed within Public Health Sciences and maintained 
by the statistician (Dr. Beavers).  Data entry is completed through a secure, encrypted login, by 
the study coordinator.  Data elements are stored in a secure database with access limited to 
user access.  Data will be stored for at least six years following completion of the study.  No 
personal information that directly identifies the participant (name, address, phone number) will 
be entered into the study database.  Participants will not be directly identified in any publication 
or presentation that may result from this study.      
The subject, study coordinator, PI, and study personnel will be blinded to the intervention 
assignment until the end of the study.  The biostatistician will be the only unblinded study team 
member, and will be in charge of generating the reports for the DSMB review.  The study 
coordinator will maintain frequent contact with each participant throughout the treatment 
period.     

If necessary, the personal health information and/or information that identifies the participants 
will be shared with the WFSM IRB, sponsor representatives at the American Heart 
Association, FDA and/or representatives from government agencies such as the Department 
of Health and Human Services (DHHS) for the purposes of:  determining the results of the 
study, making sure that the study is being conducted correctly or providing the required 
reports. 
Importance of the knowledge to be gained  
From this trial we will determine whether intranasal insulin leads to cognitive improvement vs. 
placebo, with memory and executive function composite scores as the primary outcomes. This 
study is important because there are currently no other therapies available to improve post-
stroke cognition.  The data obtained in this trial will be used to design a larger clinical trial that 
will provide more definitive evidence of benefit and perhaps FDA approval for the indication of 
cognitive recovery after stroke. 
7. INCLUSION OF WOMEN & MINORITIES 
Based on the average distribution of gender in our stroke population at WFBMC, we anticipate 
about half of the participants will be women, and 25% will be African American.  Less than 1% 
of our stroke population is of Hispanic ethnicity, so we do not expect to enroll more than 1 in 
this trial.  Children will not be enrolled because the age requirement is over age 18 years.  
8. PARTICIPATION OF ADULT WOMEN WHO ARE PREGNANT OR POSSIBLY 
PREGNANT 
We will not recruit any women who are pregnant or who believe they may possibly be pregnant 
into the proposed trial due to insufficient research on the safety profile of intranasal insulin on 
pregnant women.  Pregnancy tests are administered as standard of care at hospital admission 
to women of childbearing age who suspect that they may be pregnant or could have become 
pregnant. Our rationale for omitting children is that they would require an entirely different 
intervention. 
 
 
 
 
 
 
 
 
 
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