Nicotinic Cholinergic Modulation as a Novel Treatment Strategy for Aggression Associated With Autism
Phase: PHASE1
Conditions: Autism Spectrum Disorder, Aggression, Irritability
Sponsor: Yale University

Official title of study: Nicotinic Cholinergic Modulation as a Novel Treatment Strategy 
for Aggression Associated With Autism  
 
NCT number: 02552147  
 
Document date: 8/29/2017 
 

Page 1 of 12 
 
RESEARCH PLAN 
 
1. Statement of Purpose:  
 
The scientific aims of this pilot study are to determine the feasibility, tolerability, and 
efficacy of transdermal nicotine to treat irritability and aggression comorbid with Autism 
Spectrum Disorder in adults without intellectual disability. 
 
2. Background:  
Autism spectrum disorder (ASD) is a heterogeneous disorder characterized by 
deficits in communication, social interactions, and behavior, presenting in the early 
developmental period that causes significant impairment in functioning[1]. The 
constellation of aggression and related behaviors, which include irritability, agitation, 
and aggression, are highly comorbid with ASD, with a large recent study reporting over 
two-thirds of children with ASD have demonstrated aggressive behaviors toward 
caregivers[2]. Aggression in ASD, both toward others or as self-injurious behavior, 
significantly increases the risk for acute hospitalization or residential placement[3], 
increases parent and/or caregiver stress[4], and places the patient at risk for abuse[5]. 
Current treatment options for aggression in ASD include behavioral approaches 
and pharmacotherapy[6-8]. Atypical antipsychotics, especially risperidone and 
aripiprazole, have emerged as the accepted first-line treatment of aggression in ASD 
and pervasive developmental disorders (PDD), and are complimented by other 
antipsychotics and mood stabilizers[7, 9]. Risperidone demonstrated a large effect on 
aggression in children with ASD [10]. Despite this, treatment-resistant cases are 
common, with one large recent study of individuals of all ages with ASD finding almost 
40% were resistant to pharmacotherapy over a 3-5 year period[11]. Complicating this 
problem are significant side effects of these medications, particularly metabolic 
derangements, which appear to be even more significant in an ASD population[12]. For 
instance, children prescribed risperidone gained on average 5.6 kg (16.7%) over 6 
months[10, 13], increasing the risk for obesity and diabetes. The reality of aggression 
and its consequent harms weighed against partially effective treatments with significant 
side effects is one faced every day for patients with ASD, their parents and caregivers, 
as well as healthcare providers. 
None of the pharmacotherapies used for aggression in ASD are specific for 
treating aggression in ASD, and only a limited understanding exists regarding the 
neurobiological mechanisms driving aggression and irritability in ASD. The study of 
aggression in rodent models has benefited substantially from novel technologies such 
as rodent fMRI[14] and optogenetic techniques[15, 16], yet further knowledge of 
neurobiological mechanisms of aggression in general, as well as in ASD specifically, is 
critically needed. We rationalized that a logical foundation on which to design novel 
treatments for aggression in ASD is to identify systems important for modulating 
aggressive behavior that are also consistently misregulated in ASD cohorts. Modulation 
of nicotinic cholinergic signaling through nAChRs represents a novel strategy for 
influencing aggression in ASD. Multiple lines of evidence have identified abnormal 
cholinergic signaling and changes in nAChRs in patients with ASD[17]. 
Neuropathological studies from adults with ASD demonstrate significant misregulation 

Page 2 of 12 
 
of β2-containing and α7 nAChRs in diverse brain regions, including the cortex[18], 
cerebellum[19, 20], and thalamus[21], while studies of infantile autism identified 
abnormalities in basal forebrain cholinergic neuron development[22]. Genetic and 
epigenetic studies have also demonstrated that changes in nAChR subunits might 
contribute to ASD in humans, with the preponderance of evidence pinpointing changes 
in CHRNA7, the gene coding for the α7 subunit[23-25]. The 15q13.3 microdeletion 
syndrome results from a copy number variation encompassing roughly 6 genes, one of 
which is CHRNA7, and affected subjects can present with ASD, aggression, epilepsy, 
and schizophrenia[26, 27], which is replicated by a 15q13.3 mouse model[28]. Deletion 
of CHRNA7 itself is likely responsible for at least some of the phenotypic findings of 
15q13.3 microdeletion syndrome[29], directly linking abnormal α7 expression and 
signaling with ASD and aggression. Commonly used mouse models of ASD and related 
neurodevelopmental disorders, including the BTBR mouse[30] and the Mecp2-null 
mouse[31] demonstrate reduced prefrontal cortical acetylcholine levels and altered 
nAChR currents, respectively. The BTBR mouse also shows increased impulsivity[30], 
which could predispose to aggression in ASD. Taken together, human 
neuropathological studies, genetic studies, and animal models implicate alterations of 
nAChRs and cholinergic signaling in ASD throughout the lifespan.  
Pharmacological strategies designed to normalize or augment the cholinergic 
misregulation in ASD might be beneficial for treating its symptoms. Donepezil, an 
acetylcholinesterase inhibitor, improves cognitive rigidity and social deficits in the BTBR 
mouse model of ASD[30, 32] and has demonstrated encouraging, though as yet 
inconclusive, results for certain core symptoms in humans with ASD[33-35]. Despite 
these promising results, research on the use of cholinergic agents in ASD will have 
greater impact if experiments focus on specific ASD-related behaviors that are most 
likely to be improved by cholinergic modulation. Substantial evidence demonstrates that 
nAChR agonists, especially nicotine, reduce aggression in animal models with height-
ened aggression, including cats, rats, and mice[36-41], thereby acting as a serenic 
agent[42]. The pharmacological or neural circuit mechanisms underlying such serenic 
effects are unknown. Given the cholinergic changes in ASD and the ability of 
nAChR agonists to act as serenic agents, we hypothesize that nicotinic 
pharmacotherapies will be specifically therapeutic for aggression in ASD. Other 
disorders with cholinergic dysfunction and persistent aggression have been successfully 
treated with transdermal nicotine, including dementia[43, 44] and schizophrenia[45]. 
Nicotinic AChR agonist therapy has not been systematically described to treat ASD, 
however use of varenicline, an α4β2 partial agonist with activity at α7, was reported in a 
9-year old boy with ASD, with good tolerability and efficacy for social, communication, 
and repetitive behavior measures[46].  
 
3. Research Plan:  
 
The study will take the form of a small, randomized-controlled trial with a cross-over 
design. The basic study design is as follows: 
 
1. Participants: Participants will be recruited through the Yale Child Study Center 
(YCSC) Autism Program, local support and advocacy groups for young adults 

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with Autism Spectrum Disorder (ASD), local therapeutic schools, and local 
mental health providers who work with individuals with ASD. We will invite these 
entities to inform their patients/students about our study, and invite them or their 
parents (if legal guardians) to contact the PI (Alan Lewis) if they are interested in 
learning more about the study, and potentially participating, or having their adult 
children participate. Pamphlet advertisements with contact information will be 
placed at the meeting sites of these groups, with permission. Clinicians or group 
leaders will also offer the participants the option of submitting their telephone 
numbers in order for us to contact them directly. 
 
2. Consent: After telephonic contact is made, participants or parents who express 
interest in participation will be invited for an initial screening visit. At this point it 
will be determined whether the participants are able to give informed consent in 
accordance with the inclusion criteria of the study.  Once this is established, 
informed written consent will be obtained. Participants will be invited to attend the 
screening visit with a primary caregiver. If participants are unable to provide 
consent, a parent, if still their legal guardian, will be allowed to provide informed 
consent on their behalf. In the case where the participant is unable to provide 
informed consent and it is provided by the participant’s parent, assent will be 
obtained from the participant and the participant will still sign the consent form to 
confirm willingness to participate. Once participants are enrolled, this contact will 
be considered Visit 1, and relevant behavioral baseline data will be obtained, as 
discussed under study design. 
 
3. Study design: This is a double blind study such that subjects and all 
investigators will be unaware of treatments during the data collection phase of 
the study. It will be placebo controlled using identical appearing transdermal 
patches that either contain or do not contain nicotine. After informed consent is 
obtained, participants will be scheduled for a total of three visits with a member of 
the study team at the YCSC or at Services for the Underserved (SUS), Bronx, 
NY, as noted above. The purpose of conducting the study at SUS is to enable a 
subject who would otherwise meet criteria for the study but clinical considerations 
make the logistics of transportation to YCSC unacceptably challenging.  
 
a. Visit 1: As described above, informed consent will be obtained from 
participants or their legal guardian parent on meeting inclusion criteria and 
without exclusion criteria. Assent will be obtained from the participant in 
cases where informed consent is provided by a parent. Baseline 
behavioral rating scales will be performed. Specifically, we will obtain the 
Aberrant Behavior Checklist (ABC), State/Trait Anxiety Inventory (STAI), 
and Social Responsiveness Scale-Adults (SRS-A). These will be obtained 
from patients and primary caregivers as appropriate. Vital signs will be 
obtained. All participants will be given three weeks of identical appearing 
transdermal patches. Participants are randomized into two groups, one of 
which receives nicotine patches first and the other receives placebo 
patches first. Randomization will be performed by block randomization 

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using blocks of 4 subjects, and allocation concealment performed via the 
use of sequentially numbered, sealed opaque envelopes, on which the 
participant’s identifying information is written prior to opening the 
envelope. In collaboration with the Investigational drug service at YNHH, 
we will link the allocation to a set of patches that are numbered in a simple 
fashion to help participants use the correct patch on the correct day. The 
first group will receive 7 days of nicotine patches, dose = 7 mg patches 
followed by 14 days of identically appearing placebo patches. The second 
group will receive 14 days of placebo patches, followed by 7 days of 7 mg 
nicotine patches. This design allows each participant to serve as his/her 
own control, with a seven-day washout period, which will eliminate 
carryover effects for those subjects receiving nicotine during the first 7 
days of the study. Subjects will be asked to place the nicotine patch on 
their skin in the morning and remove the patch prior to sleep at night prior 
to sleep. This counter-balanced design is aimed to control for expectation 
bias and maximize validity. Duration of visit: 90 minutes. 
 
b. Visit 2: Participants will return for a subsequent visit at day 8, at which 
point the above rating scale data will again be collected to measure both 
primary and secondary outcomes. Tolerability will be assessed as 
determined by vital signs, open-ended inquiry, and screening for common 
adverse effects of transdermal nicotine as observed in previous studies 
with non-smoking subjects. Open-ended inquiry for tolerability will be 
performed by asking subjects “What types of side effects or discomfort did 
you experience while wearing the patch during the preceding week?” and 
subject responses will be recorded by detailed notes. This approach may 
allow the investigators to gain a better understanding of the experience of 
the patch in individuals with ASD who might experience side effects 
differently than neurotypical individuals. Brief qualitative reports of the 
patient’s subjective experience will be recorded by asking “In what ways 
did you find the patch helpful or harmful during the past week, if at all?”. 
Detailed notes of responses will be written. Duration of visit: 60 minutes 
 
c. Visit 3: Participants will return for a third and final visit at day 22, at which 
point data will again be collected to measure primary and secondary 
outcomes, tolerability, and subjective experience (as detailed in Visit 2, 
above). Duration of visit: 60 minutes 
 
4. Data analysis: Study results will be tabulated and subjected to statistical 
analysis to determine the presence of significant differences in primary and 
secondary outcomes between control and intervention. Statistical methods are 
outlined under ‘statistical considerations’ and performed by a trained statistician 
the YCCI and Yale Center for Analytical Sciences. Data regarding tolerability will 
be compared between control and interventions arms. 
 

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5. Deviations from standard of care: Current pharmacological standard of care 
for treatment of agitation and irritability in the population from which we are 
recruiting include mood stabilizers and antipsychotics[7, 9]. Patients currently 
taking these agents will not be required to discontinue this treatment, and the 
intervention is not expected to interfere with this treatment. Therefore, during the 
placebo period patients will not experience treatment below the standard of care, 
and during the active period they will continue to receive the standard of care, 
plus the active agent with its potential positive and negative effects. 
 
 
4. Subject Population: Provide a detailed description of the types of human subjects who will 
be recruited into this study. 
 
Our study population will consist of adult subjects with ASD who have primary 
caregivers. Specifically, our population is individuals over the age of 18 participating, 
or with parents participating in local support and advocacy groups for ASD. 
Participants in this study will have prior, confirmed diagnoses of ASD in order to be 
considered for inclusion. In this pilot study we will include both higher functioning 
individuals with ASD (who will provide their own consent), as well as less cognitively 
able individuals (who will have consent provided by a guardian parent). All 
participants will be living with or closely engaged with a primary caregiver, and are 
expected to have at least moderate impairment related to social and communication 
deficits. Please see the study inclusion and exclusion criteria for a quantitative 
description of the study subjects. 
 
5. Inclusion/Exclusion Criteria: What are the criteria used to determine subject inclusion or    
exclusion? 
 
Inclusion criteria: 
• Age: 18-60 
• Gender: All 
• Language: Communicative in English 
• Participants with a prior diagnosis of DSM-5 ASD at some point in their lifetime OR a 
DSM-4 diagnosis of Autism OR a DSM-4 diagnosis of Asperger’s syndrome OR a 
DSM-4 diagnosis of Pervasive Developmental Disorder Not Otherwise Specified. 
• Symptoms of irritability, agitation or aggression as reported by parent and/or 
participant 
• Aberrant behavior checklist – Irritability Subscale (ABC-I) score of 16 or higher 
• No changes in psychotropic medications within the past 14 days. 
• Either lives with a primary caregiver or closely engaged with a primary caregiver who 
interacts with the patient daily 
• BMI > 17.5 and < 45 
 
Exclusion criteria: 
• Age < 18 or > 60 
 

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• BMI < 17.5 or > 45 
• Currently using tobacco or any nicotine products (transdermal, gum, e-cigarettes) 
• Changes in psychotropic medication management within the past 14 days 
• Previous allergy to transdermal patches 
• Patients with heart rate  > 100 or < 50 or known history of cardiac rhythm 
abnormalities 
• Systolic blood pressure > 150 or < 95; diastolic blood pressure > 90 or < 50 
• No symptoms of irritability, agitation, or aggression as reported by parent and/or 
participant 
• ABC-I score of less than 16 
• No primary caregiver, or primary caregiver unable to assist with rating scales 
 
6. How will eligibility be determined, and by whom?  
 
Eligibility will be determined by the investigators. Once potential participants or a parent 
makes contact telephonically, we will explain briefly some information about the study, 
and offer them the opportunity to present for a screening visit. We will explain that a 
diagnosis of ASD is required for participation in the study, and encourage participants or 
their parent to bring information about their prior diagnosis to the screening visit, but will 
not request any PHI over the phone. At the screening visit, we will again explain the 
study procedures, and determine whether participants meet inclusion and exclusion 
criteria. In order to confirm diagnosis, we will require potential participants to provide 
evidence of a prior diagnosis done by a psychiatrist or clinical psychologist. 
 
7. Risks: Describe the reasonably foreseeable risks, including risks to subject privacy, 
discomforts, or inconveniences associated with subjects participating in the research.  
 
The study has a number of risks: 
• 
 Medication related: Side effects of transdermal nicotine exposure have been well 
studied, and participants may experience nausea, insomnia, headache, skin irritation, 
palpitations, and dizziness. These side effects were found to be dose related in healthy, 
non-smoking subjects, with 15 mg nicotine patch significantly less likely to cause side 
effects than higher dose such as 30 mg[47]. Specifically, in this study 8 non-smoking 
patients were given a 15 mg nicotine patch. Five of 8 patients experienced mild nausea 
and lightheadedness after one hour, while 3 of 8 did not have any side effects. Seven of 
8 patients continued with the study. Studies of chronic transdermal nicotine in non-
smoking populations are very encouraging regarding tolerability. In a large study of 
transdermal nicotine for treating mild cognitive impairment in older adults, tolerability 
was excellent in the over 30 subjects randomized to nicotine treatment [48]. It is not 
known whether subjects with ASD will be more or less susceptible to the common side 
effects of transdermal nicotine, however, transdermal nicotine use has been reported in 
case series or trials of patients with advanced dementia and age, a population that is 
likely to be increasingly sensitive to nicotine than younger, healthier subjects[43, 44, 48-
50].  
 

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The question of development of physiological dependence or even nicotine use disorder 
secondary to patch exposure in non-smoking participants is an important one. We can 
base the likelihood on previous studies in non-smoking participants that were conducted 
for significantly longer periods of time than in the proposed study. Nicotine patch has 
been used for 4-weeks in patients with mild-moderate Alzheimer’s Disease for 16 hours 
per day, initially using 5 mg patches for 7 days, 10 mg patches for 14 days, and finally 5 
mg patches for 7 days [49]. In general, patches were well tolerated, with only one of 8 
subjects discontinuing. There were no observed nicotine withdrawal symptoms in these 
participants. Sleep interruption was not problematic as the patches were removed at 
bedtime. An almost identical study was performed with 15 subjects with age-associated 
memory impairment using the same nicotine patch dose[50]. Only one of 15 subjects 
withdrew from this study due to nicotine-related side effects, in this case, nausea. In this 
study, the most common side effects were local skin irritation, mild nausea and 
abdominal discomfort, and lightheadedness. Two subjects reported palpitations, but 
there was no change in body weight, blood pressure, or heart rate in subjects receiving 
nicotine compared to receiving placebo. No subjects appeared physiologically or 
psychologically dependent on the nicotine patches. In a 6-month trial of transdermal 
nicotine or placebo to treat mild cognitive impairment, no subjects (0 of 34 subjects) 
developed withdrawal or continued to use nicotine products following the study[48]. 
Data from studies of smokers, who might be predisposed to difficulty discontinuing 
nicotine-containing products, demonstrated that difficulty with discontinuation of 
transdermal nicotine after use for smoking cessation was rare (2%), with the percentage 
of smokers continuing to use nicotine replacement products proportional to the rate of 
nicotine delivery[51]. Taken together, these previous studies are reassuring regarding 
the safety of nicotine products from an addiction potential standpoint in non-smoking 
subjects. Whether addiction potential differs in ASD populations remains to be seen, but 
epidemiological data demonstrating reduced smoking in ASD subjects argues that this 
group will not be predisposed to development of nicotine use disorder[52].  
 
• 
Inconvenience: Participants with significant symptoms of irritability may be 
negatively affected by not being able to make other medication changes for the duration 
of the study unless they withdraw from the study. In this instance, subjects will be 
withdrawn from the study and instructed to seek further treatment from their medical 
providers. 
 
8. Minimizing Risks:  
 
• Dependance: Please see above (#8) for a detailed discussion regarding the risk of 
development of dependence. To further reduce the risk in our study we will 
administer the active agent for no longer than one week, which is substantially 
shorter than the trials cited above that demonstrated no risk of dependence with 
transdermal nicotine. We chose transdermal delivery of nicotine because its slow 
rate of delivery significantly reduces the risk of dependence development[51]. 
Finally, our protocol involves removal of nicotine patch prior to sleep, which 
significantly reduces the total exposure to nicotine within the study.   
 

Page 8 of 12 
 
• Discomfort: The potential for discomfort will be clearly explained to subjects and 
where appropriate and permissible, their caregivers. In order to minimize the risk of 
discomfort, participants will be provided a low dose of 7 mg patches to be used for 
16 hours a day while the subjects are awake. Similar to a previous study[53], study 
subjects who experience unpleasant side effects above and beyond those on which 
subjects were counseled during the first visit will be asked to contact study staff and 
report their symptoms. Subjects experiencing expected, non-dangerous side effects 
such as moderate nausea will be instructed to remove the patch for that day and 
resume using the patch the next morning. Individuals experiencing side effects 
considered medically concerning, including severe headache, chest pain, or severe 
palpitations, will be instructed to discontinue patch use altogether and will be 
withdrawn from the study. Placebo patches will be handled in exactly the same 
manner, with the same ability to remove as described above. These data will be 
important for not only understanding tolerability of nicotine in subjects with ASD, but 
also tolerability of skin patches in populations with ASD, who are known to have 
altered somatic sensation. 
 
It should be noted that nicotine patches have been used successfully in populations 
with cardiovascular disease [54]. Although subjects with known cardiovascular 
disease, including hypertension, history of myocardial infarction, heart failure, or 
arrhythmia will be excluded, these data are reassuring given the possibility of 
enrolling a subject with a previously undiagnosed cardiovascular condition.  
 
• Inconvenience: In order to be included in our study, participants should not be 
undergoing active changes in their psychotropic medications, reducing the likelihood 
that this may become a problem in the course of the study. We have limited the 
number of visits to three, which we believe to be the minimum number of visits 
yielding scientifically useful data.  
 
 
9. Statistical Considerations: Describe the statistical analyses that support the study design.  
 
a) We plan to enroll approximately 16 subjects in this pilot study.  
 
b) The major hypothesis of the study is that transdermal nicotine will reduce symptoms 
of aggression as quantified by the Aberrant Behavior Checklist (ABC) – irritability 
subscale. For comparison, risperidone in a large clinical trial (n=63) had a large effect 
size of d>1.0 as determined by an ABC-irritability subscale reduction from a mean 
baseline score of 26.6 to a score of 9.5 after 8 weeks of medication exposure[10]. To 
determine power estimates for our study, we used 26.6 as our mean estimate for the 
placebo group. Based on a literature review, we conservatively estimated a standard 
deviation of 10. We then calculated power for a range of effect sizes, estimating a 
correlation of 0.70 for repeated measures. Alpha was set at 0.05.The power estimates 
are displayed in Table 1.   
 
Table 1: Power estimates 

Page 9 of 12 
 
Placebo Mean 
(SD) 
Treatment 
Mean (SD) 
Effect size (d) 
Power 
26.6(10) 
22 (10) 
0.59 (small-
medium) 
0.57 
26.6(10) 
20 (10) 
0.85 (medium) 
0.86 
26.6(10) 
15 (10) 
1.27 (large) 
0.99 
 
These figures demonstrate our study is highly powered to detect medium and large-
sized differences between placebo and treatment groups.   
 
c) For the major hypothesis we will collect ABC rating scale data as well as at both 
baseline as well as at two other timepoints: after 7 days of transdermal nicotine 
treatment as well as after 7 days of placebo patch. Repeated measures analysis of 
variance (ANOVA) will be used with treatment (nicotine or placebo) as within-subjects 
factor and order of treatment (nicotine-placebo-placebo or placebo-placebo-nicotine) as 
a between-subjects factor. We will first test models with between and within subjects 
factors only. We will then test the interaction between treatment and order, in order to 
test whether treatment effects varied by administration order. McNemar’s test or similar 
will be used for categorical variables such as whether or not a common, pre-defined 
side effect is reported. We will collect and report subjective experiences from patients 
and their caregivers but these will not be subjected to statistical analysis.  
 
 
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