Single Dose Intranasal Oxytocin (IN-OT) Versus Placebo in Autism: Examining Cognitive Effects
Phase: PHASE2
Conditions: Autism Spectrum Disorders
Sponsor: University of Minnesota

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PROTOCOL 
 
Title:   Single Dose Intranasal Oxytocin (IN-OT) Versus Placebo in Autism: 
Examining Cognitive Effects 
 
Running Title:   IN-OT in ASD 
 
 
Protocol Number:    
 
Protocol Version:    
 
Date:    
 
Principal Investigator:   Suma Jacob, M.D., Ph.D. 
University Status:   Associate Professor 
Department:   Psychiatry 
College:   Medicine 
 

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Purpose 
 
There are more than 1 million people living with autism spectrum disorder (ASD) 
according to the most recent report from the US Centers for Disease Control and 
Prevention (1). Unfortunately, current treatments targeting the core ASD symptom 
domains are insufficient because there is a gap in the knowledge of understanding the 
underlying mechanisms. Aside from doing treatment studies, our goal is to study how a 
particular drug changes a core symptom. This will lead to developing better outcome 
measures and ways to test new drugs that influence the same neurochemical pathways. 
Single-dose oxytocin (OT) administration in ASD facilitates retention of social cognition 
increases emotion recognition measured by reading the mind from the eyes task (RMET) 
(2), and promotes social behavior in games (3). Only one study focused on repetitive 
behaviors, and showed less severity, frequency, and number of repetitive behaviors 
during OT infusion versus placebo in a small sample (4). No studies with intranasal OT 
(IN-OT) to date have focused on mechanisms of how OT may change restrictive/rigid 
behaviors in ASD, which is a unique goal of this preliminary study. 
 
 
Lay Summary 
 
Autism spectrum disorder (ASD) is a group of severe, life-long developmental disorders. 
Oxytocin (OT) is a neurohormone involved in both repetitive/rigid and social behaviors. 
This study is focusing on how a single dose of intranasal OT (IN-OT) affects cognitive 
rigidity and social perception tasks. Taking OT as a spray through the nose increases 
social and decreases repetitive behavior in some adults with ASD, and we are exploring if 
it helps children with ASD similarly. However, it is unclear whether every person with 
ASD has an abnormal OT level, and if OT affects restrictive or social behavior differently. 
Consequently, we aim to study whether OT treatment can be effective in treating 
subgroups with specific features of ASD. We will use approaches utilizing both 
behavioral and physiological responses to clarify the role of OT in ASD. We will develop a 
deeper understanding of the range of social and rigid behaviors and use that information 
to identify persons with ASD who would benefit from OT treatment. Potential subjects 
will be asked if they want to participate in two sessions in our clinical laboratory where 
they will get either single dose IN-OT or placebo. After receiving the substance, they will 
be asked to do a handful of tasks while we monitor heart rate, eye movements, and 
collect baseline and post intranasal blood, urine and saliva. The levels of hormones, 
metabolites and peptides related to or interacting with OT will be measures in the 
collected samples of blood plasma, urine and saliva. Additionally DNA will be extracted 
from the blood samples to study genes related to OT and ASD if the participant consents 
to blood collection for genetic analysis.. 
 
 
Background 
 
The goal of this study is to understand how oxytocin (OT) relates to restricted social 
interests and cognitive inflexibility in autism. Impairments in social functioning and 
cognitive inflexibility represent two of the most agreed upon impairments in autism that 
affect quality of life. There is an unmet need in understanding the neural mechanisms 
that underlie these social communication and cognitive flexibility deficits, as well as 
treatments to alleviate specific symptom domains in autism. 

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OT & behavior: OT is involved in modulating social and repetitive behavior. Intranasal 
OT (IN-OT) administration in healthy human subjects promotes prosocial, trust 
behavior (5-7), increases gaze toward the eye region of faces (8), improves facial memory 
(9), enhances salience of social cues (10), and improves performance on the reading the 
mind in the eyes (RMET) task (11). Animal studies knocking out OT receptor (OTR) or 
OT regulators report that these mice have decreased social memory or recognition (12). 
Recently, OTR knockout mice also showed decreased cognitive flexibility and a 
resistance to change a learned pattern of behavior, comparable to restricted/repetitive 
interests (13). Both social deficits and behavioral rigidity were ameliorated by OT 
administration (13). 
 
OT dysregulation & ASD: Children with autism spectrum disorder (ASD) have lower 
average levels of blood OT compared to typically developing children (14). Single-dose 
OT administration in ASD facilitates retention of social cognition (15) and promotes 
social behavior in games (3). It heightens skill recognizing and complex psychological 
states measured by the RMET (proposed as a primary outcome measure in this study (2). 
Only one study has focused on the effects of OT on repetitive behaviors, and it showed 
less severity, frequency, and number of repetitive behaviors during the OT infusion 
versus placebo in a small sample (4). No studies with IN-OT have focused on 
mechanisms of restrictive/repetitive behaviors in ASD. The question remains as to 
whether all persons with ASD have significant disruptions in the OT system or whether 
OT disruption affects rigid/repetitive behavior or social behavior differentially. Given 
increased prosocial behaviors with IN-OT in healthy, typical individuals, we expect that 
many children or adults with ASD will benefit from IN-OT, but will have variable 
responses within each symptom domain based on individual differences (e.g. genetic 
and/or plasma level). Another question concerns whether such relationships are 
observed in subclinical expression of ASD related traits among relatives, believed to 
represent genetic liability. Demonstration of such relationships would provide powerful 
evidence that disruption in the OT system pays a role in the etiology of ASD related 
features. 
 
Neural circuits, neuropeptides, social & restrictive/repetitive behaviors: One of the 
longer-term goals, to determine neural mechanisms by which IN-OT may improve social 
behaviors and behavioral flexibility, is in preclinical study. In a complementary manner, 
molecular studies of both OT and AVP in individuals with ASD may inform an 
understanding of the relationship among OT, neural processes and behavior. There is 
growing evidence that although social/communicative and restrictive/repetitive 
behaviors are core to the diagnosis of ASD, they may be independent groups of traits 
with distinct neural pathways (16). OTR and the receptors for vasopressin (AVP), a 
evolutionarily related nonapeptides, are found differentially in these functional pathways 
(17). Consequently, OT’s action on its own receptor and cross-talk with AVP receptors 
may influence both flexibility/rigidity and social learning, but through different 
pathways. In this study, we will determine whether a single treatment with OT is 
effective in alleviating behavioral inflexibility impairments for the first time while 
attempting to replicate reported social perception improvements in the same subjects. 
 
 
Significance 
 

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Autism is a developmental disorder characterized by qualitative impairments in social 
functioning, communication, and repetitive behaviors and restricted interests (APA, 
2000). The prevalence of autism is reported to be on the increase, with the most recent 
figures suggesting that 1 in 88 individuals in the US are affected by some form of autism 
(1). Autism is a disorder with high caregiver burden. Up to 50% of mothers and 21% of 
fathers of children with autism have elevated depression scores (18). It severely impacts 
both affected individuals and their families. The financial burden on these families is 
also quite substantial. The cost of disability related to autism in the US alone is estimated 
to be $30 billion a year (19) 
 
In 2003, the Interagency Autism Coordinating committee, IACC, convened an expert 
panel which created the IACC Autism Research Matrix; a road map for autism research. 
A major goal in the road map is to establish “Efficacious drug treatments that target core 
symptoms”. This was identified as a high risk long term goal (7-10 years), which 
positions this study within the guidelines of the matrix. Now, the IACC has released a 
blueprint for autism research (2009) (20). The document specifically states that: 
“Medications to improve some of the symptoms associated with autism have been 
studied. However, thus far, no medication has been shown in controlled trials to enhance 
social behavior or communication….biological and pharmacological treatments that have 
been investigated in small studies and may warrant fuller attention include …oxytocin”. 
Thus, this project could lead to a new treatment for alleviating core deficits in a major 
neurodevelopmental disorder. 
 
 
Project Plan 
 
We will collect pilot data and develop pilot research in order to develop into a federally 
funded proposal. A unique aim of this study is to test hypotheses about alterations in 
specific neurocognitive processes as potential contributors to cognitive rigidity which is 
the opposite of cognitive flexibility. Alterations in behavioral flexibility will be assessed 
using probabilistic reversal learning (PRL) paradigms. In PRL, a subject must learn to 
choose between two stimuli. The “correct” choice is reinforced on a majority of trials 
(80%), and the other choice reinforced on 20% of trials. Subsequently, the contingencies 
are reversed between choices. Our recent work found that individuals with autism are 
impaired on PRL. We also showed that reversal learning errors are related to clinical 
measures of repetitive behaviors. Furthermore, individuals with autism also exhibit 
social deficits. Thus, it is critical to expand on the center’s focus to build a more 
comprehensive understanding of autism, as well as to develop the most efficacious and 
rationale treatments. 
 
As an essential first step to achieving these longer-term goals, we have the following 
specific aims. Our goal is to recruit 60 individuals with autism who have already 
demonstrated an ability to perform the reversal learning paradigm. 
 
Aim 1: Examine whether acute IN-OT treatment vs. placebo improves social cognition 
and replicates other ASD studies to date. 
 
Aim 2: Determine whether acute IN-OT treatment vs. placebo improves cognitive 
flexibility as a novel drug target or future treatment outcome measure. 
 

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Preliminary human data with this Protocol: Preliminary PRL and genetic data has been 
collected at the University of Illinois at Chicago (UIC) on over 300 individuals. Also at 
UIC, the same protocol for the Single Dose Intranasal Oxytocin (IN-OT) Versus Placebo 
in Autism study was completed while Dr. Jacob, the PI, moved to the University of MN.  
Eighteen subjects safely completed this protocol between May and October of 2012 (UIC 
IRB Protocol #: 2011-1082; Title: Single Dose Intranasal Oxytocin (IN-OT) Versus 
Placebo in Autism: Examining Cognitive Effects). Subjects tolerated the drug and 
protocol well and all safety data was sent to the FDA, IND #114259 (UIC IND). 
 
 
Task Data: Rigidity was examined using computer paradigms that were designed to 
closely approximate 2-choice T-maze and 4-arm radial maze tasks, which are widely 
used in studies of reversal learning in 
rodents (13, 21). It was found that children 
with ASD demonstrate impairments in 
flexibly shifting from preferred behavioral 
patterns 
to 
new 
adaptive 
response 
strategies. The ASD group required more 
trials to successfully complete their reversal 
phase (t(79)=-2.08, p=0.04) and made 
significantly 
more 
regressive 
errors 
(p=0.019). In addition, the number of 
regressive errors were correlated with total 
score on the RBS-R (p=0.003) (Figure 1). 
 
Physiology Data:  Autonomic Nervous 
System (ANS) measures as physiological 
indicators of emotional state were also 
studied. Preliminary results of reactivity to 
stressful challenges show that children with ASD have a higher heart rate (HR; F(1, 
51)=5.53, p=0.023)) and lower respiratory sinus arrhythmia (RSA; F(1,51)=4.25, 
p=0.045) than controls, indicating a tendency for decreased flexibility during novel or 
stressful situations. Only one study has examined ANS with IN-OT (20 IU) in 40 adults 
and reported that it increased RSA but less in individuals who reported high levels of 
loneliness (22). Given that children with ASD may be less able to self-report emotional 
state information, ANS measures may also help us assess baseline anxiety or social 
aloofness and see how they correlate to response outcomes with intranasal challenge 
(IN-C). 
 
Eye Gaze Tracking:  During facial/video recognitional task the movement of the eyes will 
be tracked.  This measurement allows us to correlate the gaze area and time spent gazing 
with the subject’s ability to interpret emotions.  An earlier study showed that children 
with less severe ASD symptoms and ASD children with increased gaze to the eye region 
were able to more accurately recognize emotions (23). 
 
 
Methods 
 
We will contact families through the Autism Spectrum and Neurodevelopmental 
Disorders (AS/NDD) clinic. In addition, potential participants will be recruited from the 
Fairview Health System list after IRB approval has been obtained. Fairview will be asked 
Figure 1. Repetitive Behavior Scales-Revised (RBS-R).  
r2=0.45, p=0.003 

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to provide the names and addresses of the parent/caregiver of children with Autism ages 
5-17 and adults with Autism (or their legal guardians) ages 18-40. Study staff will send 
the standard letter from Fairview and a study specific recruitment flyer in the mail, and a 
consent form will be signed when the families come into the clinic face-to-face. They can 
voluntarily call if they are interested in participating. If they call and are interested, study 
staff will schedule an appointment for the first evaluation. 
 
We will also advertise at ASD Intervention Service Providers, Autism Matters and Fraser 
Child and Family Center. Fraser is a nationally renowned Minnesota nonprofit serving 
children and adults with special needs through comprehensive education, healthcare and 
housing services. Fraser is a leading provider of autism services. Autism Matters is a 
center-based applied behavior analysis (ABA) therapy center run by licensed speech-
language pathologists that has recently expanded to two locations. At the intervention 
centers, this study will be advertised with a flyer. A copy of the proposed flyer is enclosed 
with this application. If a parent/caregiver calls to say they want to participate, study 
staff will schedule an appointment for the first evaluation. 
 
We also will recruit potential participants using the Interactive Autism Network (IAN), 
an online environment that disseminates information on autism to families and 
facilitates research. Through IAN researchers can apply to receive help with subject 
recruitment. If our study qualifies, IAN queries its research database based on the 
study's recruitment criteria, identifies participants who qualify, and sends potential 
participants the study's IRB-approved recruitment flyer via email. Those interested in 
participating will contact the study for further information. 
 
Individuals will come to the lab to participate in a randomized, placebo-controlled, 
crossover single dose challenge study to examine the effects of IN-OT challenge on social 
as well as cognitive flexibility outcomes. After collecting baseline data, the IN-C will be 
given on two visits separated by approximately two weeks. Vital signs will be taken and 
subjects will be monitored for adverse events (AEs). Blood, urine and saliva will be 
collected before and after IN-C at both study visits. We will measure plasma, urine and 
saliva for levels of hormones, metabolites and peptides related to or interacting with OT. 
Additionally DNA will be extracted from the blood samples to study genes related to OT 
and ASD if the subject consents to genetic analysis. Subject assessments will be done by 
an evaluator who is blinded to content of IN-C. All ASD studies published to date report 
24-48 IU of OT to be well-tolerated and intranasal-placebo>intranasal-oxytocin side 
effects. Subjects will be monitored for side effects. Safety evaluations will be done by a 
blinded MD investigator. 
 
OT acquisition & dosing schedule: IN-OT is currently not manufactured in the U.S. but 
has been obtained from Victoria Apotheke (Zurich, Switzerland). They are also providing 
the drug for Dr. Jacob’s current 12-week intranasal trial in autism. We will use a weight-
adjusted dose based on the 40 IU dose, block design based on 0.6 IU/kg/dose. The 
randomized IN-OT versus IN-placebo session will be separated by approximately two 
weeks. Placebo will be identical to the OT formulation with the exception of the active 
compound. It is prepared under aseptic conditions with carrier ingredients identical to 
OT nasal spray. 
 
Aim 1: Social tasks: ASD subjects exhibit impairments in processing both social visual 
and auditory cues. The purpose of Aim 1 is to determine whether a single dose of IN-OT 
reduces deficits in processing social and auditory recognition tasks, especially, tasks that 

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require assessment of an emotional state to make a decision. The data collected will 
indicate the degree of impairment in the subject’s ability to recognize social visual and 
auditory cues. 
 
Autonomic Nervous System (ANS) and Emotional Regulation: Because ANS measures 
can serve as physiological indicators of emotional state, we hypothesize that autistic 
subjects will have altered ANS functioning during tests that require more emotional 
regulation. Also, given that children with ASD may be less able to report emotional state 
information, ANS measures may help us assess baseline anxiety or social withdrawal and 
see how they correlate to response outcomes with IN-OT vs. placebo. Recently, it has 
been shown that ASD children focus more on non-eye areas of the face than those in the 
control group when fear was presented. This effect remained even when age and IQ 
(verbal, non-verbal, and composite) were used as covariates (cite). HR and RSA, used to 
assess reactivity to stressful challenges, showed that during a two minute baseline 
period, children with ASD had a higher HR and lower RSA indicating a tendency for 
decreased flexibility in stress reactivity. Only one study has examined ANS with IN-OT 
(20 IU) in 40 adults and reported that it increased RSA but less in individuals who 
reported high levels of loneliness (22). 
 
Aim 2: Cognitive Flexibility task: The PRL task will assess individuals’ ability to 
learn associations between stimuli and subsequent reward or punishment, as well as 
adaptive control of behavior following changes in reinforcement contingencies. To 
examine cognitive flexibility, we have been using computer paradigms that were 
designed to closely approximate the 2-choice T-maze and 4-arm radial maze tasks widely 
used in studies of reversal learning with colleagues at UIC (24). We found that 
individuals with ASD demonstrate impairments in flexibly, shifting from preferred 
behavioral patterns to new adaptive response strategies. The participant’ cognitive 
flexibility, as well as, social abilities will be assessed by answering questions and 
performing tasks such as those in Table 1. 
 
Taken together, the integrative approach to systematically investigating the effects of IN-
OT on social functioning and cognitive flexibility in ASD subjects is a critical first step for 
determining whether OT may be an effective treatment for these deficits in ASD, as well 
as for the longer-term goal of investigating what may be the neural mechanisms by 
which OT affects social and cognitive functioning. 
 
 
Design 
 
The treatment intervention will be a double-blind placebo-controlled cross-over 
challenge study of IN-OT versus placebo separated by a drug-free period of 
approximately two weeks. Enrollment will be up to 30 subjects. The primary outcome 
measuring changes in cognitive flexibility will include regressive errors on our PRL task. 
In addition, to examine changes in social cognition RMET will be utilized. We will test 
cognitive flexibility along with social tasks via computer. One of the goals of this initial 
study is to choose the best outcome measures for future trials. Additionally, the 
standardized clinical measurements, Repetitive Behavior Scale-Revised (RBS-R), State-
Trait Anxiety Inventory for Children (STAI; or Adults based on age and developmental 
abilities) and ABC-CV stereotypy, will be included. 
 
Table 1:  Study Outcome Measures for Aim 1 and Aim2  

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Inclusion Criteria 
 
Participants will be between 5 and 40 years of age. All subjects will have a diagnosis of 
autistic disorder or ASD that was confirmed by administration of the Autism Diagnostic 
Observation Schedule-WPS (ADOS-WPS) (30). Eligible participants must be able to 
perform the cognitive learning tasks and must have an IQ score of ≥70.. 
 
Exclusion Criteria 
 
Although we acknowledge that concomitant medications, or other types of intervention, 
may potentially bias study results, participants will be allowed to stay on concomitant 
medications and non-pharmacologic treatments, provided that no changes are made 
within 3 months prior to baseline and that no changes are made during the study. 
Individuals that are on antipsychotic drugs will be excluded from participation. All 
subjects must lack a significant medical history. Subjects with any condition, including 
alcohol and drug abuse, which might interfere with the conduct of the study, confound 
interpretation of the study results, or endanger their own well-being will be excluded. 
This includes, but is not limited to impairment of renal function, evidence or history of 
malignancy or any significant hematological, endocrine, respiratory, hepatic, 
cardiovascular or gastrointestinal disease. All female subjects of childbearing capacity 
will have a urine pregnancy test (a positive test will exclude the subject from 
participation). A pregnancy test will be conducted at both visits prior to drug 
administration. Uterine contractions may occur in women and are more likely to occur in 
pregnant women, especially towards the end of pregnancy. As a result, we exclude 
pregnant female patients, sexually active female patients on hormonal birth control 
sexually active females who do not use two types of non-hormonal birth control. In 
Social Cognition Tasks 
Primary Social Outcome 
Reading the Mind in the Eyes Task (RMET) 
Extended Social Battery 
 
Includes computerized & photo tasks such as Diagnostic 
Analysis of Nonverbal Accuracy (DANVA2; has auditory 
emotion ID component) (25), an updated version of 
Lets Face IT! computer task (26) 
Cognitive Rigidity Tasks 
Primary Rigidity Outcome 
Probabilistic Reversal Learning (PRL) Task (27)  
Extended Rigidity Battery 
Includes Rapid Automatized Naming (RAN) (28), Stop 
Signal Task 
Brain-Body Physiology Measures 
Autonomic Nervous System 
(ANS) 
Data will be collected continuously during the session to 
examine if specific tasks require more emotion 
regulation. Blood pressure will be collected twice during 
the session, once pre challenge and once post challenge. 
Eye Tracking with Face 
Attribution Tasks 
Eye tracking paired with Dynamic Affect Recognition 
Evaluation (DARE) (23) and facial/video recognitional 
task images. 

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addition, we will exclude any nursing females. Sexually active male participants must 
agree to use an effective form of contraception for the duration of the study. 
 
All interested potential subjects will be contacted via phone. If they meet eligibility 
criteria, two sessions that are approximately two weeks apart and approximately the 
same time of day will be scheduled. At their first visit, we will review the study and 
undergo informed consent procedures. Overall study procedures per visit are estimated 
to take approximately 2-3 hours per session, and are detailed in Table 2. 
 
Table 2:  Schedule of Procedures 
Procedure 
Screening 
Visit 
Study Visit 
1 
Study Visit 
2 
Obtain consent 
X 
X 
 
Diagnostic 
Assessments 
X 
 
 
IQ Test 
X 
 
 
Medical history 
review and past 
medications 
X 
X 
 
Vitals (blood 
pressure, pulse, 
height, weight) 
X 
X 
X 
Review current 
medications 
X 
X 
X 
Blood draw 
 
X 
X 
Urine collection 
 
X 
X 
Pregnancy Test 
 
X 
X 
Saliva Collection 
 
X 
X 
Receive study 
drug or placebo 
 
X 
X 
ANS 
measures/EKG 
 
X 
X 
Behavioral 
outcomes 
 
X 
X 
Eye-tracking 
 
X 
X 
Monitor side 
effects 
 
X 
X 
Post follow up 
phone call 
 
X 
X 
 
 
Participants, caretakers, and investigators involved with clinical ratings and research 
assessments will be blinded to IN-OT versus placebo assignment throughout the study. 
 
ANS measures: We will use a system with ultra miniature recorders enabling us to record 
EKG waveforms in children and adults without the need for belt mounted recorder or 
lengthy wires. ANS data will be collected and analyzed with current software at baseline 

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and throughout the two IN-C sessions, but data analysis will focus on resting intervals 
between tasks similar to other ASD studies (cite). 
 
Eye-tracking: A computerized system will be utilized to collect and quantify the eye-gaze 
data. This system has previously been successful in our research group for collecting data 
from ASD subjects. It is composed of a camera, a video head movement unit, a control 
computer, and a stimuli presentation computer. The eye-tracking system uses algorithms 
to locate and track corneal reﬂection and bright-pupil location and collects the 
coordinates of the separation between these two using an optical camera. The system 
then transposes the coordinates to corresponding locations on the monitor showing the 
stimuli being viewed. Data will be collected during the DARE task and facial/video 
recognitional task. 
 
Blood work for biomarkers: During the two blinded, randomized IN-C sessions, blood 
will be drawn at baseline and 60 
minutes post IN-C 
administration. The timing is 
according to differences reported 
in plasma OT after IN-OT in ASD 
(3). The Jacob lab has extensive 
experience for optimizing OT 
collection methods. See plasma 
level time course from two 
control subjects previously 
obtained by Dr. Jacob and a 
collaborator (figure 2).  
 
Anticipated results & interpretation: This study will be essential in allowing us to 
examine mechanisms of OT modulation of social or rigid behaviors in ASD. Our primary 
analyses will focus on IN-OT versus placebo differences in RMET for social perception 
and the PRL task as a measure of rigidity. Additional social cognition and rigidity tasks 
will be explored to assess appropriate measures for future studies investigating 
mechanism of OT change or treatment outcomes. Our ultimate goal is to see if IN-OT 
can be used to augment social or behavioral learning therapies and compare the risk and 
benefits of paired IN-OT treatment versus chronic administration (currently being 
conducted at UMN) in targeting specific subdomains within ASD across development. 
 
 
Sample composition: Sample heterogeneity, for instance with respect to symptom 
severity, might affect treatment response. Restricting the sample to prescreened subjects 
who can perform cognitive tasks reduces heterogeneity. 
 
 
Concomitant medication and treatments: Although we acknowledge that 
concomitant medications, or other types of intervention, may potentially bias study 
results, participants will be allowed to stay on concomitant medications (except 
antipsychotic drugs) and non-pharmacologic treatments, provided that no changes are 
made within 3 months prior to baseline and that no changes are made during the study. 
Information from the treatment history form can be used in ad hoc analyses to highlight 
potential confounding factors. 
 
Figure 2. Plasma OT Time Course changes within Subjects 
with IN-OT vs. Placebo 

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Intervention attrition and missing data: An intent-to-treat analysis will be used 
including every randomized participant who has baseline data. We will make every effort 
to minimize dropouts in order to preserve the integrity of the study; however, we 
recognize that missing data in pre-post designs are inevitable. For missing data, multiple 
imputation methods will be considered. Logistic regression will be used to obtain 
adjusted estimates of treatment effects for the primary measures. We will document 
reasons for dropouts and we will compare the characteristics of the subjects who drop out 
to those who complete. 
 
Data management & Sources of Material 
The sources of research material obtained from each participant will be in the form of 
information gathered specifically for research purposes. This will include data from 
diagnostic testing, cognitive and neuropsychological data, clinician and evaluator ratings 
(e.g. safety measures), and blood, urine and saliva samples. Additionally, DNA will be 
extracted from the blood samples to study genes related to OT and ASD if the participant 
consents to the collection. Data and records will be rigorously protected. To protect 
participants’ confidentiality, only authorized persons will have the right to review 
research records. Confidentiality of those records will be protected to the extent 
permitted by law. For all data, the identity of study participants will be kept confidential, 
such that all participants will be assigned a unique identifying number under which all 
obtained information will be coded. Research records will de-identified or be kept in 
secure, encrypted, password-protected databases and will be accessible only by key study 
personnel and will not be released without the subject’s consent unless required by law 
(e.g. suspected abuse) or a court order. All biological samples will be labeled using 
unique numerical identifiers that can only be linked to subject identity by a key stored in 
the locked archive. When the results of this research are published or presented at 
scientific meetings, the identity of subjects will not be disclosed. 
 
Potential Risks & Safety Monitoring and Assessment 
This study may present greater than minimal risk to subjects. For the screening, 
behavioral testing and evaluations, and blood draw, the risks to the subjects are minimal, 
sometimes involving test anxiety, apprehension and fatigue, discomfort, and breach of 
confidentiality. For the administration of IN-OT, the risk to subjects may be greater than 
minimal. We will take numerous safety precautions to ensure the safety and well-being 
of participants. 
 
Intranasal oxytocin: For the OT component of the study, the primary risks are related to 
potential side effects. Side effects of IN-OT may include nasal irritation, runny nose, or 
tearing of the eyes. Additional rare side effects reported in single cases and of unknown 
relationship with the medication include allergic dermatitis, unusual bleeding, difficulty 
passing urine, sudden weight gain, convulsions, nausea, drowsiness, headache and rarely 
confusion, anxiety, sad mood, fast or irregular heartbeat (palpitations) and unusual 
swelling. Large doses of intravenous OT decrease both systolic and diastolic blood 
pressure through a transient relaxation of vascular smooth muscle. Any OT-induced 
decrease in blood pressure is followed by a mild but sustained increase. IN-OT, which 
will be used in the present study, has not been found to substantially affect blood 
pressure. 
 
Medication: Exogenous OT is metabolized by chymotrypsin in the GI tract and thus 
cannot be administered orally. It can be administered in either an intravenous or an 

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intranasal form. Currently, the only form of OT that is available in the US is the 
intravenous form. Although this formulation has been found to produce positive effects on 
social cognition in ASD (11), it is invasive to administer and the extent to which this 
formulation crosses the blood-brain barrier (BBB) is unknown. One alternative is IN-OT, 
which is absorbed through the highly permeable nasal mucosa and has been shown to 
pass the BBB (25). IN-OT remains on the market outside the US and Canada (e.g., 
Syntocinon®, NOVARTIS, Switzerland). The applicant currently holds INDs for studying 
oxytocin in ASD children and adults (IND#109833; #114259). The effect on smooth 
muscle after intranasal administration of 10-20 IU lasts approximately 20 min, which 
reflects its duration of action in the periphery and outside the BBB (26). Mean CSF 
concentrations of peptides like OT begin to rise within 10 minutes of intranasal 
administration and last for up to 90 minutes after intranasal administration (25). OT is 
distributed throughout the extracellular fluid and most is rapidly destroyed in the liver 
and kidneys. Only small amounts are excreted unchanged in the urine. IN-OT will be 
accessed in the same ways as our chronic OT treatment studies, and will be obtained the 
same way for this project. 
 
Safety: A recent literature review summarizing the safety data of 1,529 individuals 
exposed to IN-OT reported that it produces no reliable side effects and is not associated 
with adverse outcomes in doses of 18-48 IU in the short term (31). The majority of the 
reviewed studies were single dose studies; four multi-dose studies (7 days to 13 weeks) 
were also included. In our Toronto collaborator’s 6-week feasibility study using 24 IU 
IN-OT (morning & noon) in adults with ASD, IN-OT was well tolerated and no serious 
adverse effects were reported (32). Three participants discontinued early, 2 in the 
placebo group and one in the active group. Early terminations in the placebo group were 
due to 1) worsening tics, and 2) panic attack in response to the nasal spray use. In the 
active group, one participant with stable epilepsy disorder was noted to have starring 
spells by his wife. The events were not observed by a physician or at any visit; the 
participant terminated for safety reasons. There were no significant differences between 
IN-OT and placebo in any of the blood work and no abnormal EKGs. In addition, the 
same protocol was performed at UIC (UIC IRB Protocol #: 2011-1082) using a weight-
adjusted dose based on the 40IU dose, block design based on 0.6IU/kg/dose. In this 
pilot 18 subjects safely completed the study, 4 AEs were reported. Three of the AEs were 
mild and possibly related to the study intervention. The mild AEs reported were a bloody 
nose, fatigue reported by the mother, and pain in the roof of the mouth. The fourth AE 
was moderate in severity and also possibly related to the intervention of the study. This 
subject reported a headache. Finally, previous research that investigated the role of IN-
OT in enhancing lactation in women has reported no safety concerns (33, 34). 
 
Definition of Adverse Event (AE) 
An AE is defined as any untoward medical occurrence in a patient or clinical 
investigation subject administered a pharmaceutical product whether or not the event 
has a causal relationship with the treatment. An AE may be any unfavorable and 
unintended sign, symptom, or disease temporally associated with the use of a study 
medication, whether or not considered causally associated with the use of the study 
medication. 
 
The following guidelines will be used to characterize AEs. 
Attribution is classified as follows:  
• 
Definite: AE is clearly related to the study drug.  
• 
Probable: AE is likely to be related to the study drug. 

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• 
Possible: AE may be related to the study drug.  
• 
Unlikely: AE is doubtfully related to the study drug.  
• 
Unrelated: AE is clearly not related to the study drug.  
 
Severity of AEs is classified as follows: 
• 
Mild: Transient discomfort that does not interfere with the participant’s normal 
functioning. 
• 
Moderate: Produces limited impairment of function and can require therapeutic 
intervention, but unlikely to produce sequelae. 
• 
Severe: Results in marked impairment of function and can lead to temporary 
inability to resume usual life pattern. The AE may produce sequelae that require 
prolonged therapeutic intervention. 
 
Definition of a Serious Adverse Event (SAE) 
An SAE is defined as any event that meets the following criteria: 
• 
It results in death or is life threatening (i.e., presents an immediate risk of death 
from the event as it occurred). This criterion is not intended to include an AE 
that, had it occurred in a more severe form, might have caused death. 
• 
It results in persistent or substantial disability or incapacitation. (This criterion is 
not intended to include events of relatively minor medical significance, such as 
uncomplicated headache, diarrhea, or sprained ankle.) 
• 
It results in inpatient hospitalization. 
• 
It results in prolongation of an existing hospitalization. 
• 
It is a congenital anomaly or birth defect. 
• 
It requires medical or surgical intervention to prevent any of the above outcomes. 
 
Medical and scientific judgment should be exercised in determining whether an AE is 
serious when considering important medical events that may not be immediately life 
threatening or result in death or hospitalization, but may jeopardize the patient or may 
require intervention to prevent any of the other outcomes listed. Examples of such 
medical events that may also be considered serious include, but are not limited to, 
intensive treatment in an emergency room or at home for allergic bronchospasm, blood 
dyscrasias, or convulsions that do not result in hospitalization. 
Hospitalization for elective treatment of a pre-existing condition that did not worsen 
from baseline does not meet the definition of an SAE. 
Social or convenience admission to a hospital or prolongation of a hospitalization for 
social or convenience reasons not associated with the occurrence of an AE does not meet 
the definition of an SAE. 
 
Recording, Evaluating and Reporting Adverse Events and Serious Adverse Events 
The investigator will attempt to establish a diagnosis of the event on the basis of signs, 
symptoms, or other clinical information. In such cases, the diagnosis, not the individual 
signs or symptoms, should be documented as the AE or SAE. Any adverse events--
whether due to assessment, imaging, medication or other aspects of any project--will be 
reported to the principal investigator or the co-investigator of each study. Unanticipated 
(non-serious) events will be reported to the IRB within 30 days of the principal 
investigator becoming aware of the event, via submission of an Adverse Event Report. 
Adverse events will be documented and reviewed by the principal investigator within 24 
hours of the principal investigator becoming aware of the event. Serious events will be 
reported the IRB and FDA within 48 hours of the principal investigator becoming aware 

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of the event. Notification will be by phone, email, or fax. A completed Adverse Event 
Report will be submitted within 10 days of initial IRB notification. All deaths will be 
reported to the IRB and FDA within 48 hours of the principal investigator becoming 
aware of the event. All adverse events will be compiled, and reported in summary form to 
the IRB, on an annual basis and at the conclusion of the study. 
 
Dosage: The question of appropriate dosage remains. The majority of studies of IN-OT in 
ASD have used 24 IU per dose to document behavioral and biological (fMRI) effects (2). 
The range reported across neuropsychiatric disorders including ASD is 18-48 IU per dose 
(35). At this point, we plan to use a weight-adjusted dose based on the 40 IU dose, i.e. 0.6 
IU/kg/dose with the assistance of University of Minnesota Medical Center, Fairview, 
Investigational Drug Services Pharmacy. We have had to make such a weight adjustment 
for our current international multidose study in adolescents with ASD. In the case that 
regulatory agencies raise an issue with the chosen dosage, we will follow their guidelines. 
Participants will be asked to take the dose of IN-OT or placebo in the form of intranasal 
spray and then begin the tasks. 
 
Side effects observed during our current multidose IN-OT feasibility trial in Toronto, 
Canada and our intravenous OT pilot study at UIC in autism were mild in nature. 
Because very few studies have reported on the extended use of IN-OT, there may be 
other unknown side effects. The PIs and study physicians (or a covering clinician) will be 
available at all times to study participants in the event of a clinical emergency. Both 
availability and contact information in the case of an emergency will be clearly 
communicated orally and in writing to study participants and their families. 
 
Psychological stress and anxiety: Fatigue, anxiety, discomfort or stress are potential 
adverse effects associated with the content or length of clinical and medical evaluations, 
behavioral testing and administration of rating scales. The evaluations will be conducted 
by investigators and staff with extensive experience and expertise who are sensitive to 
the clinical state of participating individuals. Experienced clinicians will always be 
available to help in efforts to reduce any discomforts. These effects may also be 
encountered during other study procedures (e.g., blood draw) and we will attempt to 
minimize them by familiarizing participants with the personnel, setting and closely 
monitoring them during the study. Well-trained and supervised personnel administer 
tests, and participants are debriefed after each session. Parents will be present either in 
the same room or a nearby room. Research staff working on the project all have 
experience working with children and adolescents and will therefore do everything 
possible to accommodate the children and adolescents who come to our centers as part 
of this study. Participants will be allowed to take breaks and stop at any time to minimize 
fatigue and discomfort. Each visit will be organized to involve breaks for the family and 
to allow the child to rest between procedures. There may be discomfort associated with 
participation, but in our experience subjects who are well informed on the purpose of the 
study and who are accompanied throughout the procedures by a member of the research 
team tolerate this discomfort well and without complications. The research team is 
trained to provide appropriate responses, both clinically and legally, to any reports of 
abuse or harm. 
 
Blood draw: Risks to the study subjects might stem from blood drawing, which can result 
in temporary bruising or, very rarely, infection. There is the possibility of some 
discomfort from the withdrawal of blood. A topical anesthetic will be offered to the 
participant to minimize this discomfort, if necessary. The amount of blood removed is 

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minimal and generally will not cause significant hemodynamic problems. The 
venipuncture is performed by nurses and staff who have received special training and are 
skilled at drawing blood from children and adolescents. 
 
Breach of Confidentiality: Because this study involves clinical evaluation there is the risk 
that, if this information became public, both participants and their family members 
could have adverse consequences. We will make every effort to protect the individual’s 
confidentiality; confidentiality is carefully guarded to address issues of privacy and 
insurability. However, the greatest risk to participants is the rare possibility of an 
inadvertent breach of confidentiality that might adversely affect a participant’s future 
employability, insurability, or family and personal relations. This loss of confidentiality 
is unlikely because the investigators will not disclose information to anyone outside of 
the immediate research group working on this project. However, there is no absolute 
legal protection against discrimination based on such information. To minimize the risk, 
each subject is assigned a study number immediately upon recruitment, all research 
records will be kept confidential and all research information will be associated with 
codes and not personal identifiers. Although a list linking the study number to the 
subject’s name is maintained for future studies and contact, the name does not appear on 
any other document or laboratory specimen. The families are informed of this link and 
are assured that only relevant study staff has access to that list. The purpose of the list 
linking study ID number to the name is to record who we have already recruited so that 
we do not accidentally re-approach any family. 

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