Sulforaphane Treatment of Children With Autism Spectrum Disorder (ASD)
Phase: PHASE1
Conditions: Autism Spectrum Disorder
Sponsor: University of Massachusetts, Worcester

 
 
 
Study Protocol and Statistical Analysis Plan 
 
Title: Sulforaphane Treatment of Children with Autism Spectrum Disorder (ASD). 
NCT: NCT02561481 
Document Date: 11/28/2016, most recently approved by IRB 07/03/2019 
 

Sulforaphane Treatment of Children with Autism Spectrum Disorder (ASD).  
Investigator Study Plan v4 (11/28/2016) 
 
 
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1) 
Title 
Sulforaphane Treatment of Children with Autism Spectrum Disorder (ASD). 
2) 
IRB Review History* 
NA 
3) 
Objectives* 
 
We propose to conduct a phase I/II, randomized, double blind, placebo-controlled, one-
arm crossover clinical trial to test the safety and efficacy of orally administered 
sulforaphane in young boys and girls with autism.  
 
Our long term research goal is to discover and develop pharmaceuticals of low toxicity 
that treat core cellular mechanisms underlying behavioral features of ASD. Our objective 
in this application is to define the clinical effects of sulforaphane in children with ASD, 
along with the biomarkers and cellular mechanisms that may explain these effects.  
 
Our specific aims are: 
1. To determine if there are measurable effects on social responsiveness and 
problem behaviors during treatment with orally administered Sulforaphane-rich 
Broccoli Seed Powder (referred to as sulforaphane hereafter) in 3-12 year old boys 
and girls with ASD. Our working hypothesis, based on strong preliminary data from our 
recent trial of sulforaphane in young adults with ASD, is that sulforaphane will have 
similar and measurably greater clinical effects in children with ASD;  
 
2. To determine if treatment with sulforaphane is safe and well tolerated. Our 
working hypothesis, based on our previous work and other studies, is that there will be 
few or no signs of toxicity;   
 
3. To elucidate cellular biomarkers that support the hypothesized mechanism of 
action of sulforaphane in ASD. Our working hypothesis is that sulforaphane will 
increase levels of Nuclear factor-erythroid factor 2 (Nrf2), improve antioxidant capacity, 
upregulate expression of heat shock proteins, and modify mTOR signaling pathways and 
cytokine expression. 
 
Our overarching hypothesis is that ASD results from underexpressed or abnormal genes 
that converge on common metabolic pathways which affect synaptic development and 
function. Our long term goal is to discover agents, such as sulforaphane, that are safe and 
effective in treating ASD, based on their known effects on these pathways, and to define 
these effects insofar as it is reasonably possible, in clinical trials. Sulforaphane 
exemplifies this goal, in that it is a nontoxic natural substance that has the potential to 
improve both underlying cellular abnormalities, as reflected in redox imbalance1, Nrf22, 
heat shock proteins3 and mTOR expression,4 and their behavioral and cognitive effects in 
ASD. Furthermore, the beneficial effects of sulforaphane are likely to be more marked in 
children compared to adults with ASD, since they have greater potential for clinical 

Sulforaphane Treatment of Children with Autism Spectrum Disorder (ASD).  
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improvement due to the compounding effects of treatment during dynamic brain 
development in childhood.  
 
 
4) 
Background* 
 
Despite progress in genetic research in ASD, direct treatment of underlying physiologic 
mechanisms is limited. Several findings in ASD suggest that different types of cellular 
dysfunction, including neuroinflammation5, oxidative stress1, mitochondrial 
abnormalities6, and abnormal synaptic plasticity/connectivity7 may involve a number of 
related, interacting metabolic pathways.   
 
Fever effect: The widespread anecdotal reports that febrile illness dramatically but 
temporarily ameliorates the disordered behavior of a substantial fraction of autistic 
patients have been corroborated and may provide important clinical clues to cellular 
mechanisms in ASD8. The degree of improvement in these patients (mostly in reduced 
stereotypic behavior and speech) was unrelated to the severity of fever or of ASD. The 
mechanisms for this “fever effect” in ASD are unknown, but may include direct thermal 
effects, as well as indirect effects, such as increased cytokines, changes in the 
hypothalamic-pituitary-adrenal axis, locus coeruleus and central noradrenergic function9, 
and increased expression of heat shock proteins (HSP) and cellular stress responses3,10. 
Fever stimulates the HSP and cellular stress responses that may ultimately lead to 
changes in synaptic function and increased long-range connectivity,11 and thereby leads 
to behavioral improvements. The expression of gene transcription by NFE2L2 (Nrf2), 
which is reduced in ASD2, may also be increased during fever, thereby increasing gene 
transcription. Sulforphane, by activating Nrf2, enhances antioxidant expression in 
astrocytes and protects against neurotoxicity in experimental herpes encephalitis12. 
 
Sulforaphane, an isothiocyanate (1-isothiocyanato-4R-(methylsulfinyl)butane), is 
obtained from lyophilized extracts of 3-day-old broccoli sprouts or as its glucosinolate 
precursor glucoraphanin, from broccoli seeds. Both broccoli sprouts and seeds contain 
the glucosinolate precursor glucoraphanin; neither contains any sulforaphane, which is 
produced from glucoraphanin by the action of myrosinase in the gut. Sulforaphane 
induces HSP and Nrf2 with positive effects on redox regulation, DNA damage sensing 
and repair, molecular chaperones, fatty acid and lipid metabolism and energy 
metabolism13,14. Sulforaphane has been shown to suppress lipopolysaccharide induced 
inflammation in rat microglia15 and may regulate neuroinflammation in degenerative 
disorders of the CNS through its induction of Nrf216. Sulforaphane and related 
compounds are hormetic drugs that induce a general “cell-protective” response, as 
demonstrated in vitro in sickle cell disease as well as X-linked adrenoleukodystrophy17,18, 
fragile X syndrome and spinal muscular atrophy19. Sulforaphane crosses the blood brain 
barrier and is bioavailable orally20,21. Recent evidence shows that dysregulation in the 
mTOR/AKT signaling and cell survival pathway is present in animal models of ASD and 
tuberous sclerosis with ASD4. The lack of suppression of mTOR leads to excessive 
synapse formation due to failure of autophagy and pruning of synapses during brain 
development, which may be improved by administration of rapamycin22. Sulforaphane 

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has been shown to induce cell cycle arrest and apoptosis in primary lymphoblasts from 
children with acute lymphoblastic anemia (ALL), and does so, in part, through its 
inhibition of the AKT and mTOR survival pathways23. Sulforaphane, therefore, has at 
least several possible modes of action that may benefit ASD through common cellular 
mechanisms that underlie its heterogeneous phenotypes.  
 
Preliminary data: We completed a randomized, double blind, placebo-controlled pilot 
clinical trial at the Lurie Center/MassGeneral Hospital for Children from 01/2011 to 
12/2013 (clinical trials identifier NCT01474993)24 to test the efficacy of sulforaphane in 
44 male adolescents and adults (13-30 years) with ASD. Fifteen participants were 
randomized to placebo and 29 to sulforaphane. Forty participants (14 on placebo and 26 
on sulforaphane) completed the trial; of those 32 (80%) had a history of positive 
behavioral responses to fever. All participants were treated for 18 weeks with study visits 
at baseline, 4, 10 and 18 weeks, followed by a final visit at 22 weeks, 4 weeks after 
treatment ended. Parent/caregivers completed the Aberrant Behavior Checklist (ABC)25 
and Social Responsiveness Scale (SRS)26 and study physicians completed the Ohio 
Autism Clinical Impressions Severity and Improvement Scales (OACIS-S and OACIS-
I)27,28 to document severity of autistic behavior at each visit. OACIS-S and -I evaluate 
severity and improvement of ASD symptoms in the following subdomains: general level 
of autism, social interaction, aberrant/abnormal behavior, stereotypical behavior, verbal 
communication, non-verbal communication, hyperactivity and inattention, anxiety/fears, 
sensory sensitivities and restricted interests. Participants were monitored with regular 
physical examinations and clinical laboratory studies. 
 
Results from clinical trial in young adult males with ASD: Sulforaphane was well 
tolerated and there were no significant toxic effects. SRS scores decreased by an average 
of -15 units in the sulforaphane group compared to -3 units in participants on placebo  
(p=0.02); considering the 4 drop-outs as non-responders, 31% (9/29) sulforaphane 
participants vs 0% placebo participants had a 25% decrease in total SRS score from 
baseline (p=0.018). The average change in total ABC score was -18 units in the 
sulforaphane group as compared to -1 units in the placebo group (p=0.0001), and after 
considering the 4 drop-outs as non-responders, 52% (15/29) sulforaphane participants vs. 
13% (2/15) placebo participants had a 25% decrease in their total ABC score from the 
baseline (p=0.02). On the OACIS-I, participants on sulforaphane were much improved or 
very much improved on the following subdomains: social interaction, 41% (12/29) 
sulforaphane participants vs. 0% on placebo (p=0.003); aberrant/abnormal behavior, 48% 
(14/29) on sulforaphane vs. 6.7% (1/15) on placebo (p=0.007); repetitive/stereotypical 
behavior, 21% (6/29) on sulforaphane vs. 0% placebo (p=0.08); and verbal 
communication, 38% (11/29) on sulforaphane vs. 0% placebo (p=0.008). Seventeen of 
the 26 participants on sulforaphane who completed the trial were much or very much 
improved on at least one subdomain of the OACIS-I. Ninety-four percent (16) of those 17 
on sulforaphane had a history of positive behavioral effects during febrile illness. In 
comparison, of the remaining 9 participants who were taking sulforaphane but were not 
much/very much improved on OACIS-I, only 55% (5/9) had a history of positive effects 
of febrile illness (p=0.03).  

Sulforaphane Treatment of Children with Autism Spectrum Disorder (ASD).  
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5) 
Inclusion and Exclusion Criteria* 
 
 Inclusion criteria 
o Autism (ASD) diagnosis. Quantitative autism traits and severity for diagnosis 
of autism will be assessed by clinical judgment corroborated by the ADOS-G 
and DSM-V checklist of symptoms. ASD symptoms moderate to severe, 
based on ADOS criteria. 
o Age from 3 through 12 years. 
 
 Exclusion criteria for subjects will include:  
o Absence of a parent or legal guardian and consent 
o Inability to speak/understand English language 
o Seizure within 1 year of screening:  This exclusion is based on the theoretical 
concern that cellular activation by sulforaphane might exacerbate seizures in 
patients with known seizure disorders. As previously noted in our previous 
trial of sulforaphane in young adult males, a seizure occurred in each of 2 
participants: one during treatment (in a participant with a previously 
undisclosed seizure), the other 3 weeks after discontinuing sulforaphane. 
o Impaired renal function (serum creatinine > 1.2 mg/dl), impaired hepatic 
function (AST/ALT > 2x upper limit of normal), impaired thyroid function 
(TSH outside normal limits):  This exclusion is based on a theoretical 
possibility of activation of underlying cellular metabolic abnormalities by 
sulforaphane. Current infection or treatment with antibiotics:  this exclusion is 
to avoid complications of inter-current illness that may occur due to the 
clinical trial or obscure possible effects of sulforaphane. 
o Medications that may modify the course or testing of ASD parameters (e.g., 
prednisone):  This exclusion is necessary in order not to interfere with or 
complicate effects of sulforaphane.  
o Chronic medical disorder (e.g., cardiovascular disease, stroke or diabetes) or 
major surgery within 3 months prior to enrollment:  Serious medical illness in 
the child may be complicated by the clinical trial and make it difficult to 
discern a change in ASD associated with treatment.  
o Less than 3 years or more than 13 years of age: this age range was selected to 
cover the ages from usual diagnosis of ASD up to adolescence. 
o A diagnosis of autism spectrum disorder other than autism, for example, 
Asperger disorder, PDD-NOS, not consistent with moderate to severe autistic 
disorder, according to ADOS criteria. 
o Prisoners 
o Pregnant women 
 
6) 
Study-Wide Number of Subjects* 
NA 

Sulforaphane Treatment of Children with Autism Spectrum Disorder (ASD).  
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7) 
Study-Wide Recruitment Methods* 
NA 
8) 
Study Timelines* 
 
Duration of intervention: 15 weeks in first (double blind, Phase 1), and 15 weeks in 
open-label (Phase2).  
 
Prior to the main clinical trial, we will carry out a pilot study in 10 children with ASD, 6-
12 years of age, who will receive sulforaphane, 2.2 micromoles/kg daily for 14 days 
(details below in “10: Procedures Involved (Study design)”). 
 
The entire clinical trial is estimated to last 3 years. The following tables below depict our 
estimated timelines for study procedures and subject recruitment: 
 
SPECIFIC AIMS/TASKS 
TIMELINE 
(UMASS) 
TIMELINE 
(JH) 
SPECIFIC AIMS 1 AND 2:  Clinical trial: To determine if there 
are measurable effects on social responsiveness and problem 
behaviors during treatment with orally administered 
Sulforaphane-rich Broccoli Seed Powder (referred to as 
sulforaphane hereafter) in 3-12 year old boys and girls with ASD; 
To determine if treatment with sulforaphane is safe and well 
tolerated.  
 
 
  Task 1: Obtain regulatory approvals for clinical trial (submit 3+ 
mo. before start date)   
 
 
 
Subtask 1.1: USAMRMC Human Research Protection   
                      Office 
(-) 3-4 mo. 
 
Subtask 1.2: UMass and JH IRB application 
(-3) mo. 
(-) 3 mo.
Subtask 1.3: Apply to FDA for IND 
(-) 3 mo. 
 
Task 2: Organize study staff: recruit Primary Care MD and 
Clinical Research Assistant, Training sessions to assure ratings 
reliability on CGI and review consent/assent procedures and 
patient privacy protections. Review clinical data collection 
methods and safeguards 
0-1 mo. 
 
Task 3: Recruit for Pilot Study (10 subjects with ASD), 
administer SF (broccoli seed powder) for 2 weeks; collect 
blood and urine samples before and after BSP; process and ship 
samples to JH . 
1-3 mo. 
 
Task 4: Recruit for rolling enrollment in main SF Clinical Trial 
(50 subjects) 
 
 
Subtask 4.1: Screen potential participants by phone, email 
or in person. Obtain informed consent and assent when 
possible at screening/enrollment visit. Explain the study in 
detail and answer all questions. Availability 24/7 with 
pager/phone. Perform ADOS, physical exam, phlebotomy 
for screening clinical labs and baseline CGI-S, ABC and 
SRS on each participant. Obtain SF-BSP/placebo from 
2-24 mo. 
 

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Pharmacy at dose specific for child, dispense but hold 
before starting until review of clinical lab studies, then call 
to start medication.  
7-week visit: Review progress; Perform CGI-I; ABC and 
SRS by parents; physical exam; phlebotomy/urine for 
clinical and research lab studies. Dispense BSP. 
15-week visit: Review progress; Perform CGI-I; ABC and 
SRS by parents; physical exam; phlebotomy/urine for 
clinical and research lab studies. Dispense BSP. 
22-week visit: Review progress; Perform CGI-I; ABC and 
SRS by parents; physical exam; phlebotomy/urine for 
clinical and research lab studies. Dispense BSP. 
30-week visit: Review progress; Perform CGI-I; ABC and 
SRS by parents; physical exam; phlebotomy/urine for 
clinical and research lab studies. Discontinue medication 
36-week visit: Review progress; Perform CGI-I; ABC and 
SRS by parents; physical exam; phlebotomy/urine for 
research lab studies. Record exit comments on study. 
 
Task 5: Data storage and analysis
0-36 mo. 
 
SPECIFIC AIM 3: To elucidate cellular biomarkers that support 
the hypothesized mechanism of action of sulforaphane in ASD. 
Blood and urine samples will be collected at UMass in Pilot and 
Main clinical trial, processed, stored and shipped on dry ice to JH 
 
 
Task 1: Labwork at JH for Pilot Study of biomarkers: Nrf2, 
oxidative stress markers, heat shock proteins, mitochondrial 
functions, mTOR, cytokine 
3-6 mo. 
3-6 mo. 
Task 2: Labwork at JH for Main Clinical Trial: biomarkers and 
mechanisms 
6-36 mo. 
6-36 mo. 
 
 
ANTICIPATED TARGET ENROLLMENT (QUARTERLY): 
 
  
Year 1 
Year 2 
Year 3 
  
  
Q1 
Q2 
Q3 
Q4 
Q1 
Q2 
Q3 
Q4 
Q1 
Q2 
Q3 
Q4 
TOTAL
Target Enrollment Pilot 
Study  
10 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
10 
Target Enrollment Main 
Study (per quarter) (does 
not include the pilot 
study participants, who 
will not take part in the 
main study because they 
will no longer be 
sulforaphane naïve) 
- 
7 
10 
8 
8 
5 
3 
3 
2 
2 
2 
0 
50 
 
9) 
Study Endpoints* 
a) Primary study endpoint:  

Sulforaphane Treatment of Children with Autism Spectrum Disorder (ASD).  
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i) 
Ohio Autism Clinical Impressions Scale – Improvement (OACIS-I) 
Average Score. 
b) Secondary study endpoints: 
i) 
OACIS-I response rate (much or very much improved) on aberrant 
behaviors 
ii) 
OACIS-I response rate (much or very much improved) on social 
communication 
iii) 
Change in total ABC score (as well as subscales of Irritability, lethargy and 
social withdrawal, stereotypic behavior, hyperactivity and inappropriate 
language) from baseline 
iv) 
Change in total SRS score (as well as subscales of social awareness, social 
cognition, social communication, social motivation and autistic 
mannerisms) from baseline 
v) 
Determination of Nrf2 levels 
vi) 
Determination of oxidative stress biomarkers 
vii) 
Determination of Heat Shock Response biomarkers 
viii) Determination of mitochondrial function biomarkers 
ix) 
Determination of mTOR signaling pathway markers 
x) 
Determination of immune function and inflammatory biomarkers 
c) Safety endpoints 
i) 
Liver function tests (ALT/AST) 
ii) 
Renal function tests (serum creatinine) 
iii) 
Thyroid function (TSH levels) 
iv) 
Complete blood counts (WBC count, RBC count, platelet counts) 
v) 
Urinalysis 
Description of surveys used as the primary and secondary study endpoints: 
1. Ohio Autism Clinical Impressions Scale - Severity (OACIS-S)27,28 is a 7 point, 
10 domain scale that requires the clinician to rate the severity of the patient's ASD 
at the time of assessment, relative to the clinician's past experience with patients 
who have the same diagnosis. The questions are rated on a scale from 1 to 7 in the 
increasing order of severity, where “1” is normal (symptoms indistinguishable 
from typically developing children) and “7” is the most severe symptoms causing 
significant problems with individual functioning on a daily basis. The 10 items on 
the OACIS-S scale cover different domains of patients’ behavior, including global 
autism severity, social interaction, aberrant behavior, repetitive or ritualistic 
behaviors, verbal communication, non-verbal communication, 
hyperactivity/inattention, anxiety, sensory sensitivities and restricted/narrow 
interests. These are rated in a similar way to the NIMH CGI Severity scale, but it 

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is focused on autism spectrum symptoms. This scale will be used to acquire 
clinician-assessed severity of autistic symptomatology in the study. 
2. Ohio Autism Clinical Impressions Scale – Improvement (OACIS-I)27,28 is a 7 
point, 10 domain scale that requires the clinician to assess how much the patient's 
ASD has improved or worsened relative to a baseline state at the beginning of the 
intervention and rated as: “1”, very much improved; “2”, much improved; “3”, 
minimally improved; “4”, no change; “5”, minimally worse; “6”, much worse; or 
“7”, very much worse. The 10 domains in which improvement is assessed are the 
same as those used in the OACIS-S scale. For this study, the proportion of 
patients who were rated as improved or very much improved on each of 10 
domains of OACIS-I were compared between sulforaphane and placebo groups. 
This scale will be used to gauge the clinician assessment of improvement in 
autistic symptomatology at all follow up visits as compared to the previous study 
visits.  
3. Social Responsiveness Scale (SRS)26: SRS is a parent- and/or teacher-reported 
65 point scale covering 5 treatment subscales focusing on the social 
communication domain of ASD as observed in natural (non-clinical) settings. 
Each item on the scale inquires about an observed aspect of reciprocal social 
behavior that is rated on the questionnaire on a scale from “1” (never true), “2” 
(sometimes true), “3” (often true), and “4” (almost always true). Its quasi-interval, 
ordinal design facilitates assessing degrees of change and/or severity of symptoms 
in response to an intervention. The SRS scale scores is subdivided into 5 
subscales (social awareness, social cognition, social communication, social 
motivation and autistic mannerisms). This scale will be used at all study visits and 
the scores (as well as change thereof from the baseline visit) will be used as one 
of the clinical outcome measures assessing social interaction skills in the study. 
4. Aberrant Behavior Checklist (ABC)25: ABC is a 58-item rating scale was 
developed for persons with developmental disabilities living in the community, 
and to assess medication effects. Its 58 questions are rated by parents or teachers 
on a scale of 0 to 3, where a score of “0” for particular behavior is not a problem 
at all, “1” indicates that the behavior is a problem but slight in degree, “2” 
indicates that the problem is moderately serious, and “3” indicates that the 
problem is severe in degree. The ABC score is sub-divided into 5 subscales 
(Irritability, lethargy and social withdrawal, stereotypic behavior, hyperactivity 
and inappropriate language). This scale will be used at all study visits and the 
scores (as well as change thereof from the baseline visit) will be used as one of 
the clinical outcome measures assessing severity of aberrant/abnormal behaviors 
in the study. 
5. Autism Diagnostic Observation Schedule (ADOS)29: ADOS is an instrument 
for diagnosing and assessing autism. The protocol consists of a series of 
structured and semi-structured tasks that involve social interaction between the 
examiner and the subject. The examiner observes and identifies segments of the 
subject's behavior and assigns these to predetermined observational categories. 
Categorized observations are subsequently combined to produce quantitative 
scores for analysis. Research-determined cut-offs identify the potential diagnosis 
of autism or related autism spectrum disorders, allowing a standardized 

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assessment of autistic symptoms. ADOS as performed by a research reliable 
psychologist will be used as one of the primary measures to ensure eligibility 
criteria for diagnosis of ASD in the study. 
6. Vineland Adaptive Behavior Scale (VABS)30: VABS is a valid and reliable 
nonverbal test to measure a person's adaptive level of functioning. VABS aids in 
diagnosing and classifying cognitive impairment in ASD and developmental 
delays. The focus of VABS is the measurement of the adaptive behaviors, 
including the ability to cope with environmental changes, to learn new everyday 
skills and to demonstrate independence. VABS will be administered at all study 
visits to help assess changes in adaptive skills during the study. 
7. Leiter-3 scale31: Leiter scale is used as a measure of nonverbal IQ. It will be used 
at the screening visit in order to classify study participants’ nonverbal IQ.  
 
10) Procedures Involved* (Study design): 
 
Pilot Study to Determine Biomarkers: Prior to the main clinical trial, we will carry out 
a pilot study in 10 children with ASD, 6-12 years of age, who will receive sulforaphane, 
2.2 micromoles/kg daily for 14 days. We will collect blood and urine samples before and 
at the end of treatment, in order to measure several parameters that are likely to 
demonstrate expected effects of sulforaphane, to standardize the assays and procedures, 
and to determine the most effective measures. Further details of the pilot study are 
discussed below.*    
 
Study Design (Specific Aim 1): Following the pilot study for biomarkers, we will 
conduct a randomized, double-blind, placebo-controlled phase-2 clinical trial of 
sulforaphane in a single-arm crossover design in 3 phases, in 50 boys and girls (ages 3-12 
years) with moderate to severe ASD (see Figure 1 below). In Phase 1 we shall treat 
participants for 15 weeks (50% with sulforaphane ((2.2 micromoles/kg body weight)), 
50% with placebo of identical appearance). In Phase 2, all children will receive 
sulforaphane (2.2 micromoles/kg b.w.) on open label for an additional 15 weeks. In Phase 
3 (6 weeks), participants will receive no sulforaphane, then return for followup at the end 
of the study. We project that treatment with sulforaphane will be associated with 
statistically significant improvements in physicians’ global impressions, aberrant 
behaviors, social responsiveness and adaptive behaviors (see descriptions of outcome 
measures and statistical plan below). 
 
Study variables will include clinician and parent assessments, and both clinical and 
scientific laboratory test measurements to evaluate for signs of toxicity and potential 
biomarkers of sulforaphane’s effects. Our primary outcome measure will be the Ohio 
Autism Clinical Global Impression Scale (OACIS)27,28. Secondary outcome measures 
will include the Aberrant Behavior Checklist (ABC)25, Social Responsiveness Scale 
(SRS)26, and Vineland Adaptive Behavior Scale (VABS)30. The OACIS will be 
completed at each of 5 visits by clinicians; the ABC, SRS and VABS will be completed 
by the parents. All participants will have moderate to severe ASD, according to ADOS 
criteria as part of the screening process. Control subjects will be those children who 

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receive placebo for 15 weeks during Phase 1. All children will receive active 
sulforaphane during Phase 2.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 1: Flowchart of progress of study participants through the 3 phases of the clinical 
trial (flowchart not to scale) 
 
 
At an initial screening visit, following informed consent by the parents and assent by the 
child if he or she is able, a complete pre- and postnatal, pediatric and neurodevelopmental 
history will be obtained. We will also review the child’s history of behavioral responses 
to fever and illness, and rate the parents’ recall of subjective impressions of changes in 
ASD according to the OACIS. Children will be tested using the Autism Diagnostic 
Observation Schedule (ADOS)29 by a trained clinician. If the diagnosis of ASD is 
confirmed, nonverbal intelligence will be measured using the Leiter-331.  
 
Monitoring for safety (Specific Aim 2):  A complete physical exam will be performed 
at each of the 6 study visits. Phlebotomies will be performed 6 times, along with urine 
collection:  1) After screening and prior to randomization; 2) at 7 weeks after starting 
treatment in Phase 1; 3) at 15 weeks, at the end of Phase 1; 4) at 22 weeks; 5) at 30 
weeks, at the end of Phase 2; and 6) at 36 weeks, at the end of the study. Blood samples 
will be sent for routine laboratory studies at screening, 7, 15, 22 and 30 weeks: complete 
blood count with differential white blood count; comprehensive metabolic profile, 
including AST and ALT; thyroid stimulating hormone, in order to monitor for possible 
adverse effects of sulforaphane in children. Portions of the blood and urine samples will 
also be processed and stored for research studies as outlined below at screening, 7, 15, 
22, 30 and 36 weeks.   
Following screening, participating children will be assigned to receive either 
sulforaphane (2.2 micromoles/kg b.w.) or placebo in Phase 1 by block randomization 
SCREENING 
VISIT 
50% 
RANDOMIZED 
SULFORAPHANE 
15 WEEK VISIT - 
SINGLE ARM 
CROSSOVER: ALL 
ALLOCATED 
SULFORAPHANE
50% 
RANDOMIZED 
PLACEBO 
30 WEEK 
VISIT (Rx 
STOPS) 
36 WEEK 
VISIT (FINAL 
FOLLOW-UP) 
PHASE 1 (15 
WEEKS): DOUBLE 
BLIND PHASE 
PHASE 2 (15 WEEKS): 
OPEN LABEL PHASE 
PHASE 3 (6 
WEEKS): 
WASHOUT PHASE

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using a table of random numbers, by the Research Pharmacy at the UMass Memorial 
Medical Center. Medication will be supplied to the Research Pharmacy at UMass by the 
Nutramax Laboratories.  
 
The Research Monitor, Christy Stine, MD, is responsible to oversee the safety of the 
research and report observations/findings to the study DSMB and IRB of Record or a 
designated official.  The Research Monitor will review all unanticipated problems 
involving risks to subjects or others associated with the protocol and provide an 
independent report of the event to the IRB of Record. The Research Monitor may discuss 
the research protocol with the investigators and the DSMB, shall have authority to stop 
the research protocol in progress, remove individual human subjects from the study, and 
take whatever steps are necessary to protect the safety and well-being of human subjects 
until the IRB can assess the monitor's report; and shall have the responsibility to 
promptly report their observations and findings to the IRB or other designated official 
and the HRPO. 
 
We shall obtain an IND from the FDA, as well as approvals from the USAMRMC 
Human Research Protection Office, UMass and Johns Hopkins IRB for this study, and it 
will be listed on Clinicaltrials.gov. Study clinicians will remain blinded at all times to 
subject assignment until the last subject has completed the study. Blinding may be broken 
for any subject who develops adverse effects or illness, and every such event will be 
reviewed by the P.I. and DSMB. Stoppage will be determined by the P.I. in consultation 
with the DSMB, and appropriate medical care will be provided in consultation with the 
participant’s primary care physician. Subjects will be entered into the study on a rolling 
basis, according to the time of their screening and entry into the study. All families will 
be followed on a regular basis and encouraged to call with questions or concerns 
regarding possible side effects of sulforaphane. A visit will take place at 7 weeks into 
Phase 1, as well as at 15 weeks, for clinical testing and phlebotomy. At 22 weeks, 
progress will be reviewed and clinical testing and phlebotomy performed. At the end of 
Phase 2 (30 weeks), all treatment with sulforaphane will end and a fifth phlebotomy will 
take place, for clinical and research lab studies. Testing of OACIS, ABC, SRS and VABS 
will be obtained at each visit. At the end of Phase 3 (36 weeks), an exit interview will be 
carried out, along with repeat OACIS, ABC, SRS and VABS, and the sixth phlebotomy 
for research studies.  Subjects will receive a gift card for $15/visit for the 6 visits.  
 
Monitoring of biochemical and molecular biomarkers of ASD (Specific Aim 3):   
Over the last two decades many studies have shown that the ASD phenotypes (defined on 
the basis of behavioral manifestations) are associated with robust physiological 
abnormalities in a wide variety of systemic and central nervous system processes. These 
abnormalities include oxidative stress and reduced antioxidant capacity, depressed 
synthesis of reduced glutathione, dysfunction in mitochondrial energy capture, increase 
lipid peroxidation, immune dysfunction, and (neuro)inflammation5,32-34.  Although it is 
unclear whether these ASD-associated anomalies are of etiological significance or 
secondary manifestations of ASD, it is clear that their correction often improves 
behavioral abnormalities substantially35. Quantifying these biomarkers before, during, 
and after sulforaphane intervention can therefore provide biomarkers for monitoring the 

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effects of therapeutic interventions and for correlating the effects with behavioral 
changes. They are also likely to provide important information as to the mechanisms of 
action of sulforaphane in ASD.  
 
We propose to monitor selected examples of various types of biomarkers in peripheral 
blood mononuclear cells (PBMC), in plasma, and in urine of the patients undergoing 
sulforaphane treatment and compare them with matched placebo controls.  
Determinations will be made at the time of enrollment, at 7, 15, 30 weeks (end of 
treatment), and at 36 weeks, after discontinuation of the intervention.  
 
*Pilot study: Before running assays on the samples we collect from participants in the 
main study, we will first carry out a pilot study using administration of sulforaphane (1 
micromole/lb or 2.2 micromoles/kg) to 10 children (6-12 years old) with ASD who 
donate samples of blood (PBMCs) and urine, before and after 14 days of daily 
treatment. By doing so, we will be able to identify the precise biomarker endpoints and 
the reproducibility of individual biomarker values and magnitudes of changes resulting 
from (short term) sulforaphane intervention. We will standardize the assays below and 
in the process will also determine the most effective and parsimonious group of assays 
to perform on samples from the children in our main study. Peripheral blood 
mononuclear cells will be obtained by differential centrifugation in Ficoll gradients 
using Vacutainer Cell Preparation Tubes (Becton Dickinson). The following will be 
studied in the Cullman Chemoprotection Laboratory at Johns Hopkins University 
School of Medicine: 
 
1. Determination of Nrf2 Levels. The transcription factor Nrf2 (Nuclear factor-
erythroid factor 2) regulates a significant fraction of the genome (perhaps 4-5%) 
that codes mostly for cytoprotective gene products14,36,37. It orchestrates protective 
responses to a diversity of endogenous and exogenous stresses. Nrf2 levels are 
substantially depressed in ASD to 45% of typically developed children2.  
Determination of PBMC Nrf2 levels and the effects of sulforaphane treatment will 
be one of the primary biomarkers to be examined. Effects of sulforaphane treatment 
on the expression and activities of Nrf2-dependent enzymes such as nicotinamide 
nucleotide quinone oxidoreductase 1 (NQO1) and heme oxygenase 1 (HO-1) will 
be also examined in PBMC of autistic children.    
 
2. Oxidative Stress Biomarkers.  Most of the methods for these analyses are described 
in Rose (2014)38. These biomarker endpoints will be examined in PBMC, plasma, 
and urine, as appropriate. Biomarkers we will measure include: (a) levels of 
oxidized and reduced glutathione, and their ratios, (b) reactive oxygen species and 
their generation as determined by fluorescent probes, (c) plasma and urine F2 
isoprostanes which are considered the most sensitive indicators of redox 
dysfunction39, (d) plasma levels of 3-chlorotyrosine (measure of reactive nitrogen 
species and myeloperoxidase activity) and of 3-nitrotyrosine (measures of chronic 
immune activation and oxidative protein damage)40, and (e)  urinary levels of 8-
hydroxydeoxyguanosine.  
 

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3. Heat Shock Response Biomarkers. The heat shock response is complex and 
evolutionarily conserved. Much evidence points to the neuroprotective role of heat 
shock proteins (HSPs), and the enhanced susceptibility of cells to damage when 
HSPs are depressed. HSPs are involved in sensing and repairing DNA damage, and 
function as chaperones for a number of misfolded proteins41.  Since fever can 
dramatically but temporarily ameliorate abnormal symptoms in a substantial 
fraction of autistic patients8, and fever is associated with activation of heat shock 
proteins42, we will examine expression of heat shock proteins, focusing on heat 
shock factor 1 (HSF-1), HSP70 and HSP90, which are also upregulated by 
sulforaphane3,43. 
 
4. Mitochondrial Function Biomarkers. In the light of persuasive evidence for 
extensive mitochondrial dysfunction in autism2,44, we will survey mitochondrial 
oxygen consumption and evidence for increased glycolysis (pyruvate and lactate 
levels and ratios in plasma). Determinations will also include measurements of 
mitochondrial NADH oxidase and pyruvate dehydrogenase activities, production of 
hydrogen peroxide, and mitochondrial DNA over-replication (compared to nuclear 
DNA).   
 
5. mTOR signaling pathway. Synaptic dysfunction caused by aberrant protein 
synthesis is believed to be a key pathogenic mechanism for ASD45. The 
PI3K/AKT/mTOR signaling pathway plays central roles in synaptic protein 
synthesis46, and its dysregulation results in many behavioral abnormalities, and may 
contribute to the pathogenesis of ASD4. Specifically, the phosphatase and tensin 
homolog on chromosome 10 (PTEN), the negative repressor of the 
PI3K/AKT/mTOR pathway, may be a significant regulator of this pathway in 
mediating the ASD phenotype. Deletion of PTEN results in autism-like behavioral 
deficits, hyperactivity of PI3K/AKT/mTOR pathway and alterations in synaptic 
scaffolding proteins47. We will therefore also examine biomarkers of this pathway 
in PBMCs, including gene expression of PTEN, AKT, and mTOR by real time 
PCR, and phosphorylated AKT and mTOR by Western blot. 
 
6. Immune Function and Inflammatory Biomarkers. A number of studies have 
reported increases in cytokine expression, immune-related genes, and other 
biomarkers of inflammation and neuroinflammation in individuals with ASD5,34,48. 
Inflammation at birth may have long term detrimental effects on Nrf2 and with 
chronic neuroinflammation, Nrf2 is downregulated in some neurodegenerative 
diseases49. Sulforaphane can attenuate inflammation in a model of spinal cord 
injury by inhibiting the nuclear factor-κB (NF-κB) pathway, and the enzymatic 
activity of the proinflammatory cytokine macrophage inhibitory factor (MIF)50.  
Therefore, in addition to Nrf2 levels, plasma cytokine levels and cytokine gene 
expression, iNOS and COX-2 will be monitored in PBMC as well during the pilot 
study in order to determine their suitability for followup during the main study. 
 
7. Bioavailability of sulforaphane: Our study drug is administered in the form of 
glucoraphanin rich broccoli seed powder with endogenous myrosinase. 

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Glucoraphanin (GR) is rapidly hydrolyzed by the enzyme myrosinase to 
sulforaphane51. Myrosinase is present in plant cells and is normally segregated from the 
glucoraphanin, until the cellular vacuoles are ruptured when the tissue containing 
glucoraphanin is ingested by mouth52,53. Sulforaphane, in turn, is metabolized rapidly 
by initial conjugation with glutathione, and successive steps of hydrolysis of the 
conjugates lead to the ultimate formation of the N-acetylcysteine derivatives 
(mercapturic acids). All of these conjugates are chemically designated dithiocarbamates 
(DTC) and can be quantified by the cyclocondensation reaction developed in our 
laboratory at Johns Hopkins as a complete and accurate measure of the bioavailability 
of sulforaphane54. We will collect a urine sample to perform measurement of 
Dithiocarbamate levels to assess the conversion of glucoraphanin to sulforaphane 
by the activity of endogenous myrosinase. 
 
In consideration of Specific Aim 3, we will carry out studies of all 6 areas during the 
pilot study, in order to determine those that will be most important and repeatable. We 
expect, from previously published studies and our own experience with the effects of 
sulforaphane55-57 that we will find changes in all 6 types of biomarkers above, however 
several may be more robust than others.     
 
The pilot study will comprise of 2 study visits. 
 
* Pilot study visit 1 - Screening visit: This visit will take around 2.5 hours to 
complete. All participants will undergo a pre-screen assessment to determine if basic 
eligibility criteria are met.  Qualified study staff will ask questions related to the above 
described inclusion/exclusion criteria.  The pre-screen may be completed by telephone 
or direct interview.  Some subjects may require a more extensive examination to ensure 
that their autism meets the study’s inclusion criteria. The subject will be enrolled after 
he and/or his legal guardian agree to a comprehensive informed consent. An informed 
consent will then be obtained. Autism diagnosis will be confirmed clinical judgment 
corroborated by administration of the Autism Diagnostic Observational Scale 
(ADOS)29.  Those who have had an ADOS administered by an accredited professional 
within 3 years of study enrollment will not need to repeat this assessment if 
documentation of results can be provided. In that case, we will reconfirm diagnosis 
with a clinician check-list at enrollment (DSM-V checklist of symptoms).  
 
At the visit, vital signs will be obtained. Then clinical laboratory tests (CBC and 
platelets, complete metabolic profile, including liver and renal function tests and 
thyroid stimulating hormone), blood for research laboratory tests (Nrf2, heat shock 
proteins, measures of oxidative stress and mTOR) and a urine sample for urinalysis will 
be obtained. The maximum amount of blood to be drawn at any stage during this study 
will not exceed 2 ml/kg body weight; we will also not exceed the minimal risk 
limitation of 3 ml/kg in an 8 week period. 
 
If the blood tests done at the time of screening visit (liver function tests, renal function 
tests and thyroid function tests) are within study’s acceptable limits (AST/ALT < 2x 
upper limit of normal, serum creatinine < 1.2 mg/dl, TSH within normal limits), we will  

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either mail the study drug to subjects or ask their parents to pick up the drug. We will 
provide the study participants a 14 day supply of study medication (sulforaphane – 
dosage details below). We will provide detailed instructions to 
subjects/parents/guardians on how to take the medication and include these instructions 
along with the medication package, and they will be made aware (and given written 
instructions) on how to recognize any side effects associated with the medication. They 
will be asked to call the study staff in case they have any questions or concerns.  
 
* Pilot study visit 2 – 14 day follow-up visit: The second visit will take ~1 hour to 
complete. At this visit, vital signs will be obtained. Subjects will be asked about any 
adverse effects that they might have experienced. They will be asked to return the 
completed study medication diary and any unused medication. Also, a blood sample for 
research laboratory tests (Nrf2, heat shock proteins, measures of oxidative stress and 
mTOR) and safety labs (liver, renal and thyroid function tests) and a urine sample for 
urinalysis, isoprostanes and sulforaphane bioavailability will be obtained. The 
maximum amount of blood to be drawn at any stage during this study will not exceed 2 
ml/kg body weight; we will also not exceed the minimal risk limitation of 3 ml/kg in an 
8 week period. 
 
Intervention details: Sulforaphane 
Sulforaphane, an isothiocyanate (1-isothiocyanato-4R-(methylsulfinyl)butane), is 
obtained from lyophilized extracts of 3-day-old broccoli sprouts or as its glucosinolate 
precursor glucoraphanin, from broccoli seeds. Both broccoli sprouts and seeds contain 
the glucosinolate precursor glucoraphanin; neither contains any sulforaphane, which is 
produced from glucoraphanin by the action of myrosinase in the gut. Sulforaphane 
induces HSP and Nrf2 with positive effects on redox regulation, DNA damage sensing 
and repair, molecular chaperones, fatty acid and lipid metabolism and energy 
metabolism13,14. Sulforaphane has been shown to suppress lipopolysaccharide induced 
inflammation in rat microglia15 and may regulate neuroinflammation in degenerative 
disorders of the CNS through its induction of Nrf216. Sulforaphane and related 
compounds are hormetic drugs that induce a general “cell-protective” response, as 
demonstrated in vitro in sickle cell disease as well as X-linked adrenoleukodystrophy17,18, 
fragile X syndrome and spinal muscular atrophy19. Sulforaphane crosses the blood brain 
barrier and is bioavailable orally20,21. Recent evidence shows that dysregulation in the 
mTOR/AKT signaling and cell survival pathway is present in animal models of ASD and 
tuberous sclerosis with ASD4. The lack of suppression of mTOR leads to excessive 
synapse formation due to failure of autophagy and pruning of synapses during brain 
development, which may be improved by administration of rapamycin22. Sulforaphane 
has been shown to induce cell cycle arrest and apoptosis in primary lymphoblasts from 
children with acute lymphoblastic anemia (ALL), and does so, in part, through its 
inhibition of the AKT and mTOR survival pathways23. Sulforaphane, therefore, has at 
least several possible modes of action that may benefit ASD through common cellular 
mechanisms that underlie its heterogeneous phenotypes.  
 
Dosage and administration:  
 

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Sulforaphane (SF) will be administered in an approximate dosage of 1 µmol SF/lb (2.2 kg 
µmol/kg) body weight. This dosage roughly approximates the dosage that was used in 
our previous clinical trial of sulforaphane in male adolescents and adults with autism. The 
sulforaphane will be supplied as glucoraphanin (GR)-enriched broccoli seed extract 
tablets (manufacturing details follow). Each active tablet will contain 125 mg broccoli 
seed powder (source of glucoraphanin, equivalent to 34 µmol GR, which is equivalent to 
at-least ~ 15 µmol SF), 50 mg broccoli sprout extract (source of myrosinase), 15 mg 
ascorbic acid, 55.90 mg microcrystalline cellulose, and 4.10 mg silicon dioxide. 
The total dose per day will depend of study participants’ body weight: 
 30-50 lb 
 
3 tablets (45 µmol/day) 
 50-70 lb 
 
 4 tablets (60 µmol/day)    
 70-90 lb 
 
 6 tablets (90 µmol/day)    
 90-110 lb  
 7 tablets (105 µmol/day) 
 110-130 lb  
 8 tablets (120 µmol/day) 
 
If a child is unable to swallow tablets, parents will be asked to grind the tablets and mix 
the contents of capsules into small cups of applesauce. This method is commonly used to 
dispense medications in children. There has been extensive experience by the Cullman 
Chemoprotection Laboratory and others, in administering sulforaphane and its precursor 
glucoraphanin in a number of forms, both encapsulated and in various types of juice 
(mango, pineapple, lime)58-62. Placebo tablets identical in size and similar in appearance 
to the active tablets will be used.  
 
Route and frequency of administration: The study drug (sulforaphane or placebo) will 
be administered orally, at approximately the same time, once a day, and preferably 
avoiding to take it with a heavy meal.  
 
Duration of intervention: 15 weeks in first (double blind, Phase 1), and 15 weeks in 
open-label (Phase2). 
 
Storage: prior to dispensing the study drug to the participants, the drug will be stored in 
temperature controlled conditions (-20C) at the UMass Memorial Investigational 
Pharmacy. During the study visits, we will directly dispense the medication to patients’ 
parents (except the first visit where we will wait for the screening visit lab results before 
either mailing the study drug or asking the parents to pick it up). The parents will be 
instructed to keep the medication stored in their freezer for the duration of the study.  
 
Concomitant medications allowed: Patients will continue taking previous 
psychopharmaceutical medications they are using to treat autism. However, we will 
request, insofar as possible, that no changes take place in the type or doses of their 
psychopharmaceutical medications for the duration of the study. They will also be 
allowed to continue taking any other medications, such as anti-epileptic medications for 
seizure control.  
 
Study visits narrative:  
The main study will consist of 2 phases of treatment, each lasting 15 weeks per patient. 
Phase 1 is the double blind phase, in which 50% of study participants will receive active 
drug (sulforaphane) and the other 50% will receive active placebo, according to the 

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dosing schedule as detailed above. In Phase 2, from 15 weeks until 30 weeks is an open-
label phase during which all study participants will receive the active drug 
(sulforaphane). All safety laboratory tests will be performed in a CLIA certified 
laboratory at the University of Massachusetts Memorial Medical Center. Phase 3 will be 
from 30 to 36 weeks, without treatment, followed by a final follow up visit at 36 weeks. 
 
The study comprises a total of 6 visits over a period of 36 weeks, with visits spaced 
around 6-8 weeks apart. In addition there will be two telephone surveys between visit 1 
and 2, and between 3 and 4 to detect any possible adverse events. Following is a 
description of procedures that will be performed at each study visit: 
 
1. Screening/baseline visit (double blind Phase 1): This visit will take around 2.5 
hours to complete. All participants will undergo a pre-screen assessment to 
determine if basic eligibility criteria are met.  Qualified study staff will ask 
questions related to the above described inclusion/exclusion criteria.  The pre-
screen may be completed by telephone or direct interview.  Some subjects may 
require a more extensive examination to ensure that their autism meets the study’s 
inclusion criteria. An informed consent will then be obtained. Autism diagnosis 
will be confirmed clinical judgment corroborated by administration of the Autism 
Diagnostic Observational Scale (ADOS)29.  Those who have had an ADOS 
administered by an accredited professional within 3 years of study enrollment will 
not need to repeat this assessment if documentation of results can be provided. In 
that case, we will reconfirm diagnosis with a clinician check-list at enrollment 
(DSM-V checklist of symptoms). A Leiter-3 assessment will also be performed as 
a measure of nonverbal IQ31.  
 
A complete, baseline history, physical and neurological exam will be performed. 
OACIS-S27,28, SRS26, ABC25 and Vineland Adaptive Behavior Scale30 will also be 
performed to quantify disease severity. Also, clinical laboratory tests (CBC and 
platelets, complete metabolic profile, including liver and renal function tests and 
thyroid stimulating hormone), blood for research laboratory tests (Nrf2, heat 
shock proteins, measures of oxidative stress and mTOR) and a urine sample for 
urinalysis will be obtained. The maximum amount of blood to be drawn at any 
stage during this study will not exceed 2 ml/kg body weight; we will also not 
exceed the minimal risk limitation of 3 ml/kg in an 8 week period. 
 
If the blood tests done at the time of screening visit (liver function tests, renal 
function tests and thyroid function tests) are within study’s acceptable limits 
(AST/ALT < 2x upper limit of normal, serum creatinine < 1.2 mg/dl, TSH within 
normal limits), we will either mail the study drug to study participants or ask their 
parents/guardians to pick up the study drug. The subject will be enrolled after he 
and/or his legal guardian agree to a comprehensive informed consent.  
 

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At this visit, the study participant/s will already have been randomly assigned to 
either the sulforaphane or placebo group in the ratio of 1:1 by the study 
statistician. As mentioned above, after the safety labs come back normal, we will 
mail the study participants (or ask their parents to pick up) an 8 week supply of 
study medication (sulforaphane or placebo) that will last until the 7 week visit, 
plus a one week buffer. We will provide detailed instructions to 
subjects/parents/guardians on how to take the medication and include these 
instructions along with the medication package, and they will be made aware (and 
given written instructions) on how to recognize any side effects associated with 
the medication. They will be asked to call the study staff in case they have any 
questions or concerns.  
 
2. 7 week visit (double blind Phase 1): Enrolled subjects will then be asked to 
come for their 7 week (follow-up) study visit, where OACIS-I, SRS, ABC and 
Vineland Adaptive Behavior Scale will be administered. A complete physical 
examination will be performed and vital signs obtained. This visit will take ~ 1.5 
hours to complete. They will be asked about any serious side effects they might 
have experienced. Also, clinical laboratory tests (CBC and platelets, complete 
metabolic profile (including liver function tests, renal function tests, thyroid 
function tests) and research laboratory tests blood - (Nrf2, heat shock proteins, 
measures of oxidative stress and mTOR) and urine (isoprostanes) will be 
obtained. Maximum amount of blood drawn at any stage during this study will not 
exceed 2 ml/kg body weight; we will also not exceed the minimal risk limitation 
of 3 ml/kg in an 8 week period. 
 
At this visit, we will give the study participants a further 7 week supply of study 
medication (sulforaphane or placebo) that will last until the next visit (15 week 
visit). Study participants will be asked to finish the medication from the previous 
lot before starting the latter batch. They will be asked to continue to take the study 
medication unless instructed otherwise. We will provide detailed instructions for 
subjects/parents/guardians on how to take the study medication along with the 
medication package, and they will be made aware (and given written instructions) 
on how to recognize any side effects associated with the study medication. They 
will be asked to call the study staff in case they have any questions or concerns.  
 
If the lab results are found to be abnormal (AST/ALT > 2x upper limit of normal, 
serum creatinine > 1.2 mg/dl, TSH outside normal limits), we will ask the study 
participants to discontinue study medication for 2 weeks. After 2 weeks, we will 
ask them to come for an interim study visit and draw their blood for liver function 
tests, renal function tests and thyroid function tests. If lab values have returned to 
within study’s acceptable limits (AST/ALT < 2x upper limit of normal, serum 
creatinine < 1.2 mg/dl, TSH within normal limits), and as advised by the study 
Data Safety Monitoring Board, we will then ask them to resume the study 
medication.  
 

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After 2 weeks, if the lab tests are still abnormal (AST/ALT > 2x upper limit of 
normal; serum creatinine > 1.2 mg/dl; TSH outside normal limits), then the study 
participants will discontinue study medication permanently (although we will ask 
them continue to be followed up in the study). 
 
3. 15 week visit (end of double blind Phase 1): Enrolled subjects will then be 
asked to come for their 15 week (follow-up) study visit, where OACIS-I, SRS, 
ABC and Vineland Adaptive Behavior Scale will be administered. A complete 
physical examination will be performed and vital signs obtained. This visit will 
take ~ 1.5 hours to complete. They will be asked about any serious side effects 
they might have experienced. Also, clinical laboratory tests (CBC and platelets, 
complete metabolic profile (including liver function tests, renal function tests, 
thyroid function tests), and research laboratory tests blood-  (Nrf2, heat shock 
proteins, measures of oxidative stress and mTOR) and urine sample for 
isoprostanes will be obtained. Maximum amount of blood drawn at any stage 
during this study will not exceed 2 ml/kg body weight; we will also not exceed 
the minimal risk limitation of 3 ml/kg in an 8 week period. 
 
At this visit, we will give the study participants a further 8 week supply of study 
medication (sulforaphane) that will last until the next (22 week) visit plus a one-
week buffer. We will provide detailed instructions for subjects/parents/guardians 
on how to take the study medication along with the medication package, and they 
will be made aware (and given written instructions) on how to recognize any side 
effects associated with the study medication. They will be asked to call the study 
staff in case they have any questions or concerns.  
 
If at this visit lab results are found to be abnormal (AST/ALT > 2x upper limit of 
normal, serum creatinine > 1.2 mg/dl, TSH outside normal limits), we will ask the 
study participants to discontinue study medication for 2 weeks. After 2 weeks, we 
will ask them to come for an interim study visit and draw their blood for liver 
function tests, renal function tests and thyroid function tests. If lab values have 
returned to within study’s acceptable limits (AST/ALT < 2x upper limit of 
normal, serum creatinine < 1.2 mg/dl, TSH within normal limits), and as advised 
by the study Data Safety Monitoring Board, we will then ask them to resume the 
study medication.  
 
After 2 weeks, if the lab tests are still abnormal (AST/ALT > 2x upper limit of 
normal; serum creatinine > 1.2 mg/dl; TSH outside normal limits), then the study 
participants will discontinue study medication permanently (though we will ask 
them continue to be followed-up in the study). 
 
4. 22 week visit (open label Phase 2): Enrolled subjects will then be asked to come 
for their 22 week (follow-up) study visit, where OACIS-I, SRS, ABC and 
Vineland Adaptive Behavior Scale will be administered. A complete physical 

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examination will be performed and vital signs obtained. This visit will take ~ 1.5 
hours to complete. They will be asked about any side effects they might have 
experienced. Also, clinical laboratory tests (CBC and platelets, complete 
metabolic profile (including liver function tests, renal function tests, thyroid 
function tests), and research laboratory tests blood-  (Nrf2, heat shock proteins, 
measures of oxidative stress and mTOR) and urine sample for isoprostanes will be 
obtained. Maximum amount of blood drawn at any stage during this study will not 
exceed 2 ml/kg body weight; we will also not exceed the minimal risk limitation 
of 3 ml/kg in an 8 week period.  
 
At this visit, we will give the study participants a further 7 week supply of study 
medication (sulforaphane) that will last until the next (30 week) visit. We will 
provide detailed instructions for subjects/parents/guardians on how to take the 
study medication along with the medication package, and they will be made 
aware (and given written instructions) on how to recognize any side effects 
associated with the study medication. They will be asked to call the study staff in 
case they have any questions or concerns.  
 
If at this visit lab results are found to be abnormal (AST/ALT > 2x upper limit of 
normal, serum creatinine > 1.2 mg/dl, TSH outside normal limits), we will ask the 
study participants to discontinue study medication for 2 weeks. After 2 weeks, we 
will ask them to come for an interim study visit and draw their blood for liver 
function tests, renal function tests and thyroid function tests. If lab values have 
returned to within study’s acceptable limits (AST/ALT < 2x upper limit of 
normal, serum creatinine < 1.2 mg/dl, TSH within normal limits), and as advised 
by the study Data Safety Monitoring Board, we will then ask them to resume the 
study medication.  
 
After 2 weeks, if the lab tests are still abnormal (AST/ALT > 2x upper limit of 
normal; serum creatinine > 1.2 mg/dl; TSH outside normal limits), then the study 
participants will discontinue study medication permanently (though we will ask 
them continue to be followed-up in the study). 
 
5. 30 week visit (open label Phase 2): Enrolled subjects will then be asked to come 
for their 30 week (follow-up) study visit, where OACIS-I, SRS, ABC and 
Vineland Adaptive Behavior Scale will be administered. A complete physical 
examination will be performed and vital signs obtained. This visit will take ~ 1.5 
hours to complete. They will be asked about any side effects they might have 
experienced. Also, laboratory tests (CBC and platelets, complete metabolic 
profile, including liver function tests, renal function tests, thyroid function tests), 
and research laboratory tests blood - (Nrf2, heat shock proteins, measures of 
oxidative stress and mTOR) and urine sample for isoprostanes will be obtained. 
Maximum amount of blood drawn at any stage during this study will not exceed 2 
ml/kg body weight; we will also not exceed the minimal risk limitation of 3 ml/kg 

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in an 8 week period. At this visit, we will not give the study participants any 
further study medication. 
 
6. 36 week final follow-up visit (Phase 3): This will be the final study visit. 
OACIS-I, SRS, ABC and Vineland Adaptive Behavior Scale will be 
administered. A complete physical examination will be performed and vital signs 
obtained. This visit will take ~ 1.5 hours to complete. Participants and their 
caregivers will be asked about any side effects they might have experienced. Also, 
research laboratory tests for blood - (Nrf2, heat shock proteins, measures of 
oxidative stress and mTOR) and urine sample for isoprostanes will be obtained. 
 
Following is a description of the telephone surveys: 
 
1. Phone survey 1: This phone call will take place between visits 1 and 2 at 
approximately 3 weeks from the study drug start date. This will help us ensure 
that the study participants does not experience any unwanted side effects from 
taking the study drug. This survey will take around 15 minutes to complete. 
During this phone call we will ask them questions about their child’s health since 
starting the study. We will ask them questions about any side effects that your 
child may have experienced since starting the study drug.  
 
2. Phone survey 2: This phone call will take place between visits 3 and 4 at 
approximately 18 weeks from the initial study drug start date. This will help us 
ensure that the study participants does not experience any unwanted side effects 
from taking the study drug. This survey will take around 15 minutes to complete. 
During this phone call we will ask them questions about their child’s health since 
starting the study. We will ask them questions about any side effects that your 
child may have experienced since starting the study drug.  
 
 
Laboratory evaluations: 
We will collect blood samples for safety evaluations as well as for biomarker evaluations.  
 
1. Specimens to be collected, amount, schedule: 
a. Specimen to be collected: blood, urine 
b. Schedule: Blood and urine samples will be collected at screening visit, 7 
week visit, 15 week visit, 22 week visit, 30 week visit and 36 week visit 
c. Amount: Blood ~ 13 ml (so long as it is not exceeding 2ml/kg body weight 
limit) each time (~4 ml for liver, thyroid and renal function tests, ~1.5 ml 
for complete blood counts, ~7.5 ml for biomarker measurement) 
   Urine ~ 5 ml 
2. Evaluations to be made: 
a. Safety evaluations: They will include liver function tests (SGOT/SGPT, 
Total bilirubin), renal function tests (serum electrolytes, serum creatinine), 
thyroid function tests (TSH). 
b. Biomarker measurement: Following biomarkers will be evaluated: 

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1. Determination of Nrf2 Levels. The transcription factor Nrf2 (Nuclear 
factor-erythroid factor 2) regulates a significant fraction of the 
genome (perhaps 4-5%) that codes mostly for cytoprotective gene 
products14,36,37. It orchestrates protective responses to a diversity of 
endogenous and exogenous stresses. Nrf2 levels are substantially 
depressed in ASD to 45% of typically developed children2.  
Determination of PBMC Nrf2 levels and the effects of sulforaphane 
treatment will be one of the primary biomarkers to be examined. 
Effects of sulforaphane treatment on the expression and activities of 
Nrf2-dependent enzymes such as nicotinamide nucleotide quinone 
oxidoreductase 1 (NQO1) and heme oxygenase 1 (HO-1) will be also 
examined in PBMC of autistic children.    
2. Oxidative Stress Biomarkers.  Most of the methods for these analyses 
are described in Rose (2014)38. These biomarker endpoints will be 
examined in PBMC, plasma, and urine, as appropriate. Biomarkers 
we will measure include: (a) levels of oxidized and reduced 
glutathione, and their ratios, (b) reactive oxygen species and their 
generation as determined by fluorescent probes, (c) plasma and urine 
F2 isoprostanes which are considered the most sensitive indicators of 
redox dysfunction39, (d) plasma levels of 3-chlorotyrosine (measure 
of reactive nitrogen species and myeloperoxidase activity) and of 3-
nitrotyrosine (measures of chronic immune activation and oxidative 
protein damage)40, and (e)  urinary levels of 8-
hydroxydeoxyguanosine.   
3. Heat Shock Response Biomarkers. The heat shock response is 
complex and evolutionarily conserved. Much evidence points to the 
neuroprotective role of heat shock proteins (HSPs), and the enhanced 
susceptibility of cells to damage when HSPs are depressed. HSPs are 
involved in sensing and repairing DNA damage, and function as 
chaperones for a number of misfolded proteins41.  Since fever can 
dramatically but temporarily ameliorate abnormal symptoms in a 
substantial fraction of autistic patients8, and fever is associated with 
activation of heat shock proteins42, we will examine expression of 
heat shock proteins, focusing on heat shock factor 1 (HSF-1), HSP70 
and HSP90, which are also upregulated by sulforaphane3,43.  
4. Mitochondrial Function Biomarkers. In the light of persuasive 
evidence for extensive mitochondrial dysfunction in autism2,44, we 
will survey mitochondrial oxygen consumption and evidence for 
increased glycolysis (pyruvate and lactate levels and ratios in 
plasma). Determinations will also include measurements of 
mitochondrial NADH oxidase and pyruvate dehydrogenase activities, 
production of hydrogen peroxide, and mitochondrial DNA over-
replication (compared to nuclear DNA).   
5. mTOR signaling pathway. Synaptic dysfunction caused by aberrant 
protein synthesis is believed to be a key pathogenic mechanism for 
ASD45. The PI3K/AKT/mTOR signaling pathway plays central roles 

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in synaptic protein synthesis46, and its dysregulation results in many 
behavioral abnormalities, and may contribute to the pathogenesis of 
ASD4. Specifically, the phosphatase and tensin homolog on 
chromosome 10 (PTEN), the negative repressor of the 
PI3K/AKT/mTOR pathway, may be a significant regulator of this 
pathway in mediating the ASD phenotype. Deletion of PTEN results 
in autism-like behavioral deficits, hyperactivity of PI3K/AKT/mTOR 
pathway and alterations in synaptic scaffolding proteins47. We will 
therefore also examine biomarkers of this pathway in PBMCs, 
including gene expression of PTEN, AKT, and mTOR by real time 
PCR, and phosphorylated AKT and mTOR by Western blot. 
6. Immune Function and Inflammatory Biomarkers. A number of 
studies have reported increases in cytokine expression, immune-
related genes, and other biomarkers of inflammation and 
neuroinflammation in individuals with ASD5,34,48. Inflammation at 
birth may have long term detrimental effects on Nrf2 and with 
chronic neuroinflammation, Nrf2 is downregulated in some 
neurodegenerative diseases49. Sulforaphane can attenuate 
inflammation in a model of spinal cord injury by inhibiting the 
nuclear factor-κB (NF-κB) pathway, and the enzymatic activity of the 
proinflammatory cytokine macrophage inhibitory factor (MIF)50.  
Therefore, in addition to Nrf2 levels, plasma cytokine levels and 
cytokine gene expression, iNOS and COX-2 will be monitored in 
PBMC as well during the pilot study in order to determine their 
suitability for follow-up during the main study. 
 
 
Following table depicts the study procedures that will be performed at each visit and also 
approximately how long each visit will take: 
 
Visit 
Informed 
consent, 
eligibility 
criteria 
(ADOS,    
Leiter-3) 
Adverse 
event 
reporting 
Medical 
history, 
vitals, 
physical 
exam 
Autism 
symptom 
severity   
(OACIS-S, 
OACIS-I, 
ABC, SRS, 
Vineland 
Adaptive 
Behavior 
Scale)
Blood draw 
(LFTs, 
RFTs, TSH, 
CBC, HSP, 
mTor, 
Nerf), urine 
sample for 
isoprostanes 
Study drug 
dispending 
Visit 
duration 
Double blind phase (50% participants receive sulforaphane and 50% receive placebo) 
Screening/ 
baseline visit 
X 
  
X 
X 
X 
8 weeks 
~ 2.5 hours 
3 week phone 
survey 
 
X 
 
 
 
 
~ 15 
minutes 

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7 week visit 
  
X 
X 
X 
X 
7 weeks 
~ 1.5 hours 
15 week visit 
  
X 
X 
X 
X 
8 weeks 
~ 1.5 hours 
Open label phase (all participants receive sulforaphane) 
18 week 
phone survey 
 
X 
 
 
 
 
~ 15 
minutes 
22 week visit 
  
X 
X 
X 
X  
7 weeks 
~ 1.5 hours 
30 week visit 
  
X 
X 
X 
X 
  
~ 1.5 hours 
36 week visit 
  
X 
X 
X 
X  
  
~ 45 
minutes 
 
 
11) Data and Specimen Banking* 
We plan to store leftover blood and urine samples in -80 C freezer in the Dept. of 
Pediatrics at UMass and the Cullman Chemoprotection Laboratory at Johns Hopkins for a 
period of 3 years after completion of the study. They will be accessed by the P.I. or study 
staff. The samples will continue to be de-identified and labeled with unique study I.D. 
numbers. The link between the study ID and identifiable information will only be 
accessible by the UMass personnel. At the end of 3 years the samples will be forever 
anonymized, and the link between study ID and identifiable information will be 
destroyed. We will make application to the IRB for any further studies to be conducted, 
by ourselves or others, using these specimens.  
12) Data Management* 
 
Statistical Analysis:  The OACIS-I, SRS and ABC data will be analyzed in a mixed 
model ANOVA with fixed effects for visit, treatment group, and their interaction and 
random participant-specific intercepts.  The effect of sulforaphane on mean change in 
OACIS-I, SRS and ABC total scores from baseline will be estimated using linear 
contrasts.  Estimates will be unbiased if any loss to follow-up is missing at random, 
conditional on the observed data and our model assumptions.  Significantly greater 
improvement from baseline in mean scores among participants randomized to 
sulforaphane based on a two-tailed test at alpha = 0.05 will be taken as support of our 
primary hypothesis.  
 
A secondary analysis assessing proportion of participants in both groups who have 
responded to the study drug will be performed by Fisher’s Exact test. This analysis will 
be done by categorizing the OACIS-I, SRS and ABC data. Participants will be considered 
as responders to study drug if they experience either of the following: they are much/very 
much improved (a score of 2 or less) on any subdomain of OACIS-I scale, their total 
ABC scores at 15 weeks decrease by at least 25% from baseline score, and their total 
SRS scores at 15 weeks decrease by at least 25% from their baseline score.  

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Biomarkers (testing mechanism of action of SFN) will be tested both as outcome 
variables in mixed model analyses and as predictors of differences in behavioral 
symptoms using the models described above for behavioral symptoms.  Markers that both 
respond to sulforaphane treatment and predict differences in behavioral symptoms will be 
interpreted as possible mediators of any benefit from sulforaphane treatment.  Safety of 
sulforaphane treatment will be assessed based on absolute rates of liver, thyroid and renal 
toxicity among participants treated with sulforaphane and based on comparison of the 
proportion of participants with such toxicities between the two treatment groups. 
 
Power Calculations: We plan to enroll a total of 50 participants in the study, randomized 
1:1 to sulforaphane and placebo. With OACIS-I, ABC, and SRS assessments at 0, 7 and 
15 weeks our proposed design will have at least 80% power to detect significance in the 
following outcome measures if the following sample sizes are considered: OACIS-I 
average Score: With a total of 40 patients (20 on placebo and 20 on sulforaphane) 
entering this two-treatment parallel-design study, the probability is 80 percent that the 
study will detect a treatment difference at a two-sided 0.05 significance level, if the true 
difference of average OACIS-I score at 15 weeks between two treatments is 4 units. This 
is based on the assumption that the standard deviation of the average OACIS-I score is 
4.9. OACIS-I response rate on aberrant behaviors: With a total of 44 patients (22 on 
placebo and 22 on sulforaphane) entering this two-treatment parallel-design study, there 
will be an 88% chance of detecting a significant difference at a two sided 0.05 
significance level.  This assumes that the response rate at 15 weeks of placebo is 9% and 
the response rate of sulforaphane is 54% on aberrant behavior subscale of OACIS-I. 
OACIS-I response rate on social communication: With a total of 44 patients (22 on 
placebo and 22 on sulforaphane) entering this two-treatment parallel-design study,  there 
will be 82% chance of detecting a significant difference at a two sided 0.05 significance 
level.  This assumes that the response rate of placebo is 7% and the response rate at 15 
weeks of sulforaphane is 46% on the social communication subscale of OACIS-I. 
Change in total ABC score from baseline: With a total of 44 patients (22 on placebo 
and 22 on sulforaphane) entering this two-treatment parallel-design study, the probability 
is 81 percent that the study will detect a treatment difference at a two-sided 0.05 
significance level, if the true difference of change in baseline of total ABC score at 15 
weeks between the two treatments is 19.4 units. This is based on the assumption that the 
standard deviation of the change in ABC score is 22.1. Change in total SRS score from 
baseline: With a total of 48 patients (22 on placebo and 22 on sulforaphane) entering this 
two-treatment parallel-design study, the probability is 81 percent that the study will 
detect a treatment difference at a two-sided 0.05 significance level, if the true difference 
of change from baseline of total SRS score at 15 weeks between two treatments is 18.4 
units. This is based on the assumption that the standard deviation of the change in SRS 
score is 22.0. Thus our proposed sample size of 50 participants, randomized 1:1 to 
sulforaphane and placebo will have more than enough power to detect significantly 
changes in majority of the outcomes listed above. The study will have 80% power to 
detect one or more toxicities expected to occur in at least 5.3% of sulforaphane-treated 
participants.  The study is not powered to compare rates of safety outcome between 
sulforaphane and placebo treatment groups for a relevant non-inferiority threshold.   

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Study identifiers: We will make all efforts to preserve the privacy of the study 
participants. All data collected in the study will be identified by a random code and 
participant initials. We will use the following format to label the study participants: SF-
15-001, SF-15-002 along with participant initials (eg. JD). No personally identifying 
information, such as complete name, hospital medical record number, date of birth etc. 
will be used on study documents.  
 
Good Clinical Practices compliance will be maintained at all times during the study. All 
study staff will be trained in maintaining the study documentation according to the GCP 
guidelines. In addition, any electronic database solution we will use will be GCP 
guidelines compliant. University of Massachusetts Medical School has contracted the 
RedCap clinical trials software database solution. REDCap (Research Electronic Data 
Capture) is a secure web-based application built around HIPAA guidelines to support 
data capture for research studies. It allows users to build and manage online surveys and 
databases or a combination of both quickly. REDCap features automated export 
procedures for seamless data downloads to Excel, PDF, and common statistical packages 
(SPSS, SAS, Stata, R). Also included are a built-in project calendar, a scheduling 
module, ad hoc reporting tools, and advanced features, such as branching logic, file 
uploading, and calculated fields. The REDCap Software sits on a dedicated server behind 
a firewall to eliminate all nonessential access credentials. The data itself is isolated on a 
separate server that sits behind an additional firewall layer for added security. Interaction 
between REDCap and the client is encrypted via SSL. 
 
In addition we will have an independent clinical trials monitor conduct regular visits to 
monitor the study for GCP compliance. This study monitor will have authority to stop a 
research study in progress, remove individuals from study, and take any steps to protect 
the safety and well-being of subjects until the IRB can assess. Please refer to the 
document titled “H00007832_ Research Monitor v2 111815” for further details about the 
roles and responsibilities of the Research Monitor.  
 
In addition to the GCP visits, an independent data safety monitoring board (DSMB) of 3 
persons will be constituted to evaluate the continued safety of sulforaphane. This DSMB 
will consist of one pediatrician (from the department of Pediatrics at UMass Medical 
School), one child psychiatrist (from the department of Psychiatry at UMass Medical 
School) and one biostatistician (from the department of Quantitative Sciences at UMass 
Medical School). This DSMB will meet twice a year and review all safety information 
collected in the past 6 months (see “13: Provisions to Monitor the Data to Ensure the 
Safety of Subjects” below).  
 
Confidentiality: Strict confidentiality will be maintained at all times. As mentioned 
above, all subjects and clinical information associated with each subject will be assigned 
an identification number and will only be referred to by that number and subject initials. 
This number will be the only identifying information on test score sheets, data entry 
forms, and in the data files. Some data will require minimum protected health 
information (i.e. date of birth) recorded on the assessment sheets in order to be properly 

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scored. In this case, we will follow HIPAA limited data set guidelines.  Source 
documents will be maintained by the study coordinator in a centralized, double-locked 
office accessible only by the study coordinator and involved members of the research 
team.  Only initials and study number identify any specimens sent to laboratories outside 
of UMass. 
 
We will collect some identifiable information, such as name, address, date of birth, 
contact information (mailing address, phone numbers, email address etc.) demographics 
(race, ethnicity, parental educational background) but this information will be kept 
separate from the other study files and documents. Documents containing this identifying 
information will be kept in a locked cabinet in a secure study location. Only study staff 
directly involved in interaction with the study participants and the study Principal 
Investigator will have access to this identifiable information. The Principal Investigator, 
or designee, will maintain a personal participant identification list (participant numbers 
with the corresponding names) to enable records to be identified. Other study staff (such 
as those working with biomarkers) whose role will not involve interacting with study 
participants will not have access to this identifiable information. Representatives of 
USAMRMC who may be entrusted with the responsibility of conducting site visits may 
be able to get access to the study records for review.   
 
In terms of subject privacy protection, the researchers are aware of the implementation of 
the Health Insurance Portability and Accountability Act (HIPAA) and its impact on 
clinical research and electronic data collection activities.  The database developed under 
this protocol will conform to national standards and procedures for the electronic storage 
and transmission of information to insure privacy and security of health information.  
This includes conformance to rules for transactions and code sets, the standards for 
privacy of individually identifiable health information and security standards. 
 
Any blood samples sent to the laboratories at Johns Hopkins for analysis of heat shock 
proteins and mitochondrial study and measures of oxidative stress will not contain any 
direct identifiers and samples will be labeled with the subject’s unique ID. Samples may 
be stored for future use with explicit consent of the subject and parents or guardians. 
Without such consent, samples will be destroyed following the study.  
 
Data capture, verification, and disposition: Data will be primarily captured on paper 
records. Majority of the study instruments (such as Aberrant Behavior Checklist, Social 
Responsiveness Scale, Autism Diagnostic Observation Schedule, Vineland, Leiter scale) 
are provided as paper booklets and they will be used as the primary data capture devices. 
Additional information, such as physical examination findings, medical history records 
etc. will also be captured in paper records. After the study is complete, we will keep the 
study records for future regulatory purposes for a period of 7 years, at which time all 
study records will be destroyed. However the documents containing identifiable 
information will not be stored after the study ends; they will be destroyed after study is 
completed.  
 

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In addition, we will use a secure computer database to transfer the paper data records to 
an electronic format. University of Massachusetts Medical School has contracted the 
RedCap clinical trials software database solution. REDCap (Research Electronic Data 
Capture) is a secure web-based application built around HIPAA guidelines to support 
data capture for research studies. It allows users to build and manage online surveys and 
databases or a combination of both quickly. REDCap features automated export 
procedures for seamless data downloads to Excel, PDF, and common statistical packages 
(SPSS, SAS, Stata, R). Also included are a built-in project calendar, a scheduling 
module, ad hoc reporting tools, and advanced features, such as branching logic, file 
uploading, and calculated fields. The REDCap Software sits on a dedicated server behind 
a firewall to eliminate all nonessential access credentials. The data itself is isolated on a 
separate server that sits behind an additional firewall layer for added security. Interaction 
between REDCap and the client is encrypted via SSL. To ensure accuracy of transfer of 
data from paper to electronic format and to minimize data entry errors, all data that will 
entered to RedCap will be double entered by two independent study staff members. After 
the study is complete and all data has been collected and entered into the database, the 
database will be locked out from any further editing.  
 
Storage of laboratory samples: The following storage specifications will be met: 
a. For safety evaluations: The blood tubes/vacutainers will be labeled with study 
participant’s unique ID number and shipped right away to the UMass Clinical 
Research Center’s lab for analysis.  
b. For biomarker evaluations: The vacutainer will be labeled with the ID number, 
and the blood processed within 2 hours of collection for isolation of monocytes. 
The monocytes so isolated will be labeled with the unique study ID number and 
frozen in an appropriate culture media at -80C for a period not exceeding 3 
months in the -80 freezer at UMass Memorial’s Clinical Research Center. These 
samples will then be shipped to Johns Hopkins University (Lewis B. and Dorothy 
Cullman Cancer Chemoprotection Center, and Department of Pharmacology and 
Molecular Sciences, The Johns Hopkins University School of Medicine, 
Baltimore, MD 21205) for analysis of biomarkers (details below). The tube 
containing urine will be labeled with the ID number and stored at -80C until 
shipment.  
 
Shipping of laboratory samples and laboratories performing evaluations:  
a. For safety evaluations: The samples will be tested at University of Massachusetts 
Memorial Medical Center Clinical Trials Unit Clinical Research Laboratory 
(located at Clinical Trials Unit, UMass Memorial Ambulatory Care Center, 1st 
Floor, 55 Lake Ave N, Worcester, MA 01655). 
b. For biomarker evaluation: These evaluations will be conducted at Lewis B. and 
Dorothy Cullman Cancer Chemoprotection Center, and Department of 
Pharmacology and Molecular Sciences, The Johns Hopkins University School of 
Medicine, Baltimore, MD 21205. 
c. Transportation of samples: The blood and urine samples for biomarker evaluation 
will be transported frozen. To ensure that proper temperature is maintained during 
transport they will be shipped in special Styrofoam containers in dry ice. The 

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person arranging specimen packing and shipment will be trained in shipping and 
transportation of hazardous materials.  
 
13) Provisions to Monitor the Data to Ensure the Safety of Subjects* 
 
Risk management and emergency response: Risks to subjects will be minimized by 
close follow-up and consideration of all reported symptoms and concerns of parents, 
guardians and caregivers. Regular examinations and laboratory testing of blood counts 
and serum chemistries, including liver function tests, renal function tests, and thyroid 
function tests will be performed at the screening visit, 7 week,15 week, 22 week, 30 week 
and 36 week visits, and reviewed by the study team and Data Safety Monitoring Board. 
The PI or physician colleague will be on call 24/7 for any concerns of adverse events 
during the study. Risks of phlebotomy will be reduced by behavioral preparation, use of 
topical anesthetic and carried out by phlebotomists experienced in working with persons 
with autism. Protection of subjects will include confidential treatment of all health related 
information, and all research data will be coded, the reference codes to be maintained 
under lock and key by the PI and study coordinator at all times. 
 
If lab signs of toxicity are found (AST/ALT > 2x upper limit of normal; serum creatinine 
> 1.2 mg/dl; TSH outside normal limits), the patient will discontinue study medication 
for 2 weeks before resuming the study medication, once lab values return to within 
study’s acceptable limits. At the end of 2 weeks, we will do lab tests again. If at that time, 
lab tests are still not within study’s acceptable limits (AST/ALT > 2x upper limit of 
normal; serum creatinine > 1.2 mg/dl; TSH outside normal limits), subjects will 
discontinue permanently the study medication (though they will still continue to be 
followed-up in the study). Lab tests are required at screening, 7-week, 15-week, 22 week, 
30 week and closeout visits:  Hematology and complete metabolic profile (including 
liver, kidney, thyroid function tests); urinalysis for protein and hemoglobin. Possible 
clinical side effects of sulforaphane will be monitored, including: GI disturbances 
(flatulence, soft stool, and large bowel movement), and frequent urination63. Staff 
members will be available 24/7 for emergencies. Maximum amount of blood drawn at 
any stage during this study will not exceed 2 ml/kg body weight; also we will not exceed 
the minimal risk requirements of 3ml/kg in an 8 week period. 
 
If any child in the study has a seizure while taking the medication, the study drug will be 
discontinued and study physicians and Data Safety Monitoring Board (DSMB) will be 
notified. The child will be evaluated by Dr. Zimmerman or his designee, pediatrician or 
family doctor, and treated and followed up appropriately for the duration of the study. We 
will ask the parents to return with the child for repeat safety lab studies and to return 
unused study drug. 
 
In the event of significant shifts in behavior, based on the family’s experience and 
judgment, the child will be examined by the study physician(s) or the child’s pediatrician, 
the study drug will be temporarily discontinued for 2 weeks and DSMB notified. If the 
behavior(s) subside, the medication will be resumed on the previous schedule. Since 

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children with ASD often have cyclic behavior changes without respect to the medications 
they are taking, we will rely primarily on the caregivers’ experience in assessing whether 
the behavior(s) in question are out of the norm for their child. However, we will be 
cautious in evaluating the child for possible medical reasons for the change in behavior 
(e.g., gastrointestinal discomfort, disordered sleep). If adverse behaviors do not subside 
after 2 weeks and no medical cause for the behaviors is found, the study medication may 
be restarted if the DSMB, study physicians and the parents agree. Otherwise, medication 
will not be restarted and we will ask that the family return the remaining study drug 
supply.    
 
We will constitute study drug pause and study drug stop rules based on objectively 
measurable criteria. These criteria appear below: 
 
Subject Drug Pause Criteria: Subjects will be asked to stop taking study medication for 
2 weeks if: 
1. At 7 week visit, subjects reported any serious side effects and subsequent laboratory 
tests are outside of study’s acceptable limits - liver function tests (AST/ALT > 2x 
upper limit of normal); renal function tests (serum creatinine > 1.2 mg/dl); thyroid 
function tests (TSH outside normal limits). 
2. At 15 week visit, subjects reported any serious side effects and subsequent laboratory 
tests are outside of study’s acceptable limits - liver function tests (AST/ALT > 2x 
upper limit of normal); renal function tests (serum creatinine > 1.2 mg/dl); thyroid 
function tests (TSH outside normal limits). 
3. At any time during the study, subjects reported any serious side effects and 
subsequent laboratory tests are outside of study’s acceptable limits - liver function 
tests (AST/ALT > 2x upper limit of normal); renal function tests (serum creatinine > 
1.2 mg/dl); thyroid function tests (TSH outside normal limits). 
4. There is a potential interaction of study medication with another medication a subject 
is taking. We will ask the subject to temporarily stop taking study medication if the 
other drug is a short term medication (e.g., a course of antibiotics, or prednisone).  
5. Intercurrent illness (e.g., infection, asthma, accident or serious injury), requiring 
medical attention (e.g., emergency room visit or hospital admission). All such 
incidents will be reviewed with study physicians and the DSMB as they arise, and we 
will consult with the child’s physician regarding the advisability of pausing the study 
drug and continuing follow up of the child’s illness. 
6. In the event of significant shifts in behavior, based on the family’s experience and 
judgment, the child will be examined by the study physician(s) or the child’s 
pediatrician, the study drug will be temporarily discontinued for 2 weeks and DSMB 
notified. If the behavior(s) subside, the medication will be resumed on the previous 
schedule. Since children with ASD often have cyclic behavior changes without 
respect to the medications they are taking, we will rely primarily on the caregivers’ 
experience in assessing whether the behavior(s) in question are out of the norm for 
their child. However, we will be cautious in evaluating the child for possible medical 
reasons for the change in behavior (e.g., gastrointestinal discomfort, disordered 
sleep). If adverse behaviors do not subside after 2 weeks and no medical cause for the 
behaviors is found, the study medication may be restarted if the DSMB, study 

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physicians and the parents agree. Otherwise, medication will not be restarted and we 
will ask that the family return the remaining study drug supply.    
 
Subject Drug Stop Criteria: At the end of 2 weeks, we will do lab tests again. At that 
time, subjects will discontinue the study medication permanently if: 
1. Subsequent laboratory tests repeated after 2 weeks are outside of study’s acceptable 
limits - liver function tests (AST/ALT > 2x upper limit of normal); renal function 
(serum creatinine > 1.2 mg/dl); thyroid function (TSH outside normal limits). 
2. At any point of the study, extreme exacerbation of behavioral symptoms is noted, as 
above and these behaviors do not subside after 2 weeks of study drug stoppage and no 
medical cause for the change in behavior is apparent (e.g., gastrointestinal discomfort, 
disordered sleep). 
3. If any child in the study has a seizure while taking the medication, the study drug will 
be discontinued and study physicians and Data Safety Monitoring Board (DSMB) 
will be notified. The child will be evaluated by Dr. Zimmerman or his designee, 
pediatrician or family doctor, and treated and followed up appropriately for the 
duration of the study. We will ask the parents to return with the child for repeat safety 
lab studies and to return unused study drug. 
4. At any time, if there is an interaction of study medication with another long term 
medication a subject is taking: In a case in which the other drug is the one which a 
subject is likely to require for a long period of time, subjects will discontinue study 
medication permanently if there is a possibility of adverse effects as a result of an 
interaction with that drug (for example, appearance of extrapyramidal signs in a 
subject taking risperidone).  
 
Data Safety Monitoring Board: An independent data safety management board of 3 
persons will be constituted to evaluate the continued safety of sulforaphane. This DSMB 
will consist of one pediatrician (from the department of Pediatrics at UMass Medical 
School), one child psychiatrist (from the department of Psychiatry at UMass Medical 
School) and one bio-statistician (from the department of Quantitative Sciences at UMass 
Medical School). This DSMB will meet twice a year and review all safety information 
collected in the past 6 months.  
 
14) Withdrawal of Subjects* 
 
See study drug pause and stop criteria above. 
 
15) Risks to Subjects* 
 
Sulforaphane Safety:  
Cruciferous vegetables, including broccoli sprouts, are generally regarded as safe and are 
regular dietary components in many regions of the world. Previous estimates of the daily 
dietary intake of cruciferous vegetables vary regionally, averaging 40 g/day in 
Singapore64, 11 g/day in the United States65, 16 g/day in Canada66, 30 g/day in the UK67, 
and 112 g/day in Japan68. There have been numerous adult human and animal studies 

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assessing the safety of sulforaphane. These studies are summarized in the safety and 
efficacy section of the Investigator Brochure. 
 
Singh et al recently conducted a double blind, placebo controlled, randomized, Phase II 
study of sulforaphane in 40 male adolescents and adults with moderate to severe autism24. 
The participants were dosed according to body weight: 50 µmol (1 capsule) of 
sulforaphane for <100 lb, 100 µmol (2 capsules) for 101-199 lb, and 150 µmol (3 
capsules) for >200 lb. Sulforaphane treatment effectively improved core aberrant 
behaviors of ASD, and was safe and well-tolerated. Notably, none of the laboratory 
results were outside normal ranges at any time point.  Unexpectedly, the sulforaphane 
group gained significantly more weight over the18-week period, compared to placebo. 
Thirty-six adverse events were noted during the trial. Vomiting, increased aggressions, 
abdominal pain, increased flatulence, irritability, constipation, diarrhea, fever, headache 
and exacerbation of seasonal allergies were reported in 12-19 percent of participants on 
sulforaphane; their incidence was the same in the placebo groups (P > 0.10).  Two 
participants had single unprovoked seizures: one after 3 weeks on sulforaphane, with an 
undisclosed history of recent seizures; the other 3 weeks after discontinuing treatment 
and a past (more than 1 year) history of well-controlled seizures on treatment with anti-
epileptic drugs. Although patients with ASD are predisposed to seizures69,70, the 
possibility of seizures as a possible adverse effect of sulforaphane in ASD cannot be 
ruled out. 
 
 
Although there have not been any studies designed explicitly to test the safety of 
sulforaphane in children, the overall weight of evidence as per our previous clinical trial 
of sulforaphane in adolescents with autism and as demonstrated by the numerous clinical 
as well as preclinical studies summarized in the Investigator Brochure strongly suggests 
that sulforaphane treatment is likely to be safe and without any serious adverse effects. 
Although we do not anticipate that children will show signs of toxicity to sulforaphane, it 
is for this reason that we have designed this clinical trial to carefully evaluate children for 
possible toxicity over 30 weeks of treatment. 
 
Foreseeable risks: No severe adverse events have been observed in any trial with 
sulforaphane. In the case of ASD, however, we will be vigilant for any possible 
behavioral regression that could signify an unwanted interaction with concurrent 
medications or cause undue distress for the participant or guardian. Given the delicate 
balance between real events of this sort and those that could be due to a “reverse placebo 
effect” in the case of parental anxiety about new treatment, home reports of exacerbation 
will be confirmed by clinical review. If parental support for continuation is lacking and/or 
clinical review indicates a suspected undesired effect, censorship is the ethical policy 
even if definite proof of mechanism is lacking or it is possible that a worsened behavioral 
outcome would be transient. 
 
A previous randomized, double-blinded phase I study of repeated administration (3 doses 
per day for 8 days) of sulforaphane-containing Broccoli Sprout Extract in healthy 
volunteers found no problems with safety and tolerance71. Another cross-over clinical 

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trial designed to look into bioavailability of sulforaphane reported no serious adverse 
effects58. Another animal study has shown that sulforaphane raised liver enzymes but 
only on very high dose of >500 µmol/day, which is much higher than our proposed 
dose72. We will be vigilant in treating subjects with autism, for behavioral regression that 
could signify an interaction with concurrent medications or other adverse effect. 
 
In our recently completed, randomized, double blind, placebo controlled clinical trial of 
sulforaphane on 40 male adolescents and adults with autism, the study drug was 
remarkably well tolerated and very few adverse effects were noted24. Vomiting, increased 
aggressions, abdominal pain, increased flatulence, irritability, constipation, diarrhea, 
fever, headache and exacerbation of seasonal allergies were reported in 12-19 percent of 
participants on sulforaphane; their incidence was the same in the placebo group (P > 
0.10).  Two participants in our study had single unprovoked seizures: one after 3 weeks 
on sulforaphane, with an undisclosed history of recent seizures; the other 3 weeks after 
discontinuing treatment and a past (more than 1 year) history of well-controlled seizures 
with anti-epileptic drugs. Although patients with autism are predisposed to seizures, we 
cannot rule out the possibility of seizures as a possible adverse effect of sulforaphane in 
ASD. In consideration of this, we will not enroll any participant who has an active history 
of seizure within 1 year of participation.  
 
Based on previously published data and data from our clinical trial, possible clinical side 
effects of sulforaphane include: seizures, insomnia, vomiting, abdominal pain, flatulence, 
constipation, diarrhea, large bowel movement, soft stools, weight gain, exacerbation of 
seasonal allergies and increased urination63. Caregivers will be advised to notify study 
staff should these side effects be experienced.  Staff members will be available 24/7 for 
emergencies.  We will diligently scrutinize each medication a subject is taking and will 
take precautions to monitor and/or discontinue the study medication if there is any sign of 
interaction, such as the appearance of exatrapyramidal signs in a subject taking 
risperidone. Additionally, we are not aware of any risks associated with stopping the 
study drug without any down titration.  
 
Physical risks to subjects from phlebotomies include pain and discomfort, and bruising. 
Subjects and particularly children and mentally impaired subjects may experience pain 
and anxiety and become upset with phlebotomy.  Physical risks will be minimized by 
having phlebotomy performed by appropriately trained and experienced personnel. 
Subjects will be asked to present for phlebotomy in an adequately hydrated state, so that 
it will be easier to identify and successfully puncture veins.  All subjects will be offered 
the use of a local anesthetic (EMLA cream) prior to the blood draw, in order to minimize 
discomfort.  Emotional risks will be minimized by utilizing personnel who are 
experienced in phlebotomy in pediatric and cognitively impaired populations, and 
ensuring that they have training in appropriate behavioral techniques. Maximum amount 
of blood drawn at any stage will not exceed 2 ml/kg body weight; also we will not exceed 
the minimal risk requirements of 3ml/kg in an 8 week period. 
 
There is a small risk to privacy in answering questions.  All the information will be kept 
strictly confidential and every known effort will be utilized to maintain confidentiality. 

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All records will be kept under lock and key or on encrypted and password protected 
digital media.  
There is a small risk of emotional discomfort, distress and/or fatigue from completing the 
surveys and completing the assessment tasks. We will make all efforts to ensure that 
participants do not have to wait an inordinate amount of time while at the study site for 
completing the requisite surveys and assessments.  
 
There is also a small risk of transient local reactions to EMLA cream such as paleness, 
erythema and edema.  
 
Parents and/or guardians will be responsible for ensuring the timely and complete 
adherence to daily medication dosage. They will be asked to keep a log of the time of day 
and the number of capsules taken. They also will monitor for and report any adverse 
events, undesirable psycho-behavioral reactions throughout the duration of the study or 
until censorship, should that occur. Parents/guardians will be trained to ensure that the 
study medication is kept in the original, properly labeled, child-proof containers in a 
place that is secure from access by siblings, school mates, other children, mentally 
impaired adults, or pets.  Any spilled or lost medication will be documented and reported 
so that replacement will ensure adherence to prescribed dosages. Parents/guardians will 
be reminded by telephone at-least 24 hours beforehand of their appointments. Those who 
miss an appointment will be contacted by telephone in order to elicit the cause and 
reschedule within one week.  
 
16) Potential Benefits to Subjects* 
 
Potential benefits of sulforaphane may include demonstrable functional improvements in 
social responsiveness in subjects with autism. Treatment with sulforaphane may also 
increase mitochondrial function and decrease oxidative stress, both of which have been 
subjects of research in autism.  
 
Current treatment standards for children with ASD include various therapy programs, 
including ongoing speech and language, occupational, physical and behavioral therapies. 
Many are also in special school programs with Individual Educational Plans. We 
anticipate that some of the children will be taking prescriptions of antipsychotic, 
antidepressant, antianxiety or stimulant medications. Approximately 10% will have a 
history of epilepsy and may be taking anticonvulsants.  We will carefully evaluate each 
medication a subject is taking for potential interactions with sulforaphane. Subjects will 
continue to participate in their usual therapy programs.   
 
Novelty: This trial of sulforaphane in children with autism provides a novel approach 
with a strong theoretical basis in clinical observations of the effects of fever in autism. 
Preliminary evidence from in vitro models supports the hypothesized cellular effects of 
sulforaphane on heat shock proteins Nrf2, mitochondrial functions, and the mTOR 
pathway.  
 

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There are increasing reports of successful “off target” therapies for genetic disorders that 
modulate cellular metabolic changes rather than directly ameliorating the underlying 
genetic cause 73,74. The capacity of the general cellular stress response pathways to 
reestablish metabolic homeostasis allows treatment that is independent of -- and without 
knowledge of -- the primary genetic lesion. This has particular relevance for the treatment 
of ASD since it is characterized by genetic heterogeneity and interactive metabolic 
pathways.   
 
Relevance to autism: Medical treatments that affect core features of ASD at the cellular 
level are needed. The approach to treatment presented here, based on clinical 
observations of improvements in patients with ASD during fever, and the known 
stimulation of the cellular stress proteome by sulforaphane in vitro, represents a relevant 
and rational approach to therapy. The findings of this study may provide the basis for 
further clinical trials and laboratory investigation at the cellular level in autism.  
 
17) Vulnerable Populations* 
Since our study involves children, we have reviewed the “CHECKLIST: Criteria for 
Research Involving Children”. 
18) Multi-Site Research* 
This is not a multi-site study in the sense that all patient enrollment will be conducted at 
UMass. We are however collaborating with the Cullman Chemoprotection Laboratory at 
Johns Hopkins University and will perform the biomarker assessments for our study. The 
role of Johns Hopkins is limited to biomarker assessment only and storage of any leftover 
samples. Johns Hopkins will obtain its own IRB approval in order to conduct the 
biomarker analysis. We will hold regular phone conferences with Johns Hopkins 
investigators (once in 3 months) and again at any time if there are any changes to the 
biomarkers portion of the study plan and to discuss interim study results (if any 
available). 
19) Community-Based Participatory Research* 
NA 
20) Sharing of Results with Subjects* 
 
Since several of the safety lab test results (such as complete blood counts/liver/thyroid/ 
renal function tests) could potentially be clinically relevant to study participants or their 
primary care providers, upon request (and especially if the test results are 
abnormal/outside of reference range) we will provide a copy of these test results to study 
participants’ health care providers. 

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21) Setting 
 
The study will be conducted at UMass Medical School and UMass Memorial Medical 
Center. The Clinical Research Center and Child and Adolescent NeuroDevelopment 
Initiative (CANDI) at Shriver Center will be the main locations for conducting of study 
visits and procedures. Study visits may occasionally also be conducted in Benedict 
building Pediatric clinic offices if so preferred and convenient to the subjects. Clinical 
offices and laboratories are located in close proximity where ADOS, screening, 
examinations and phlebotomies will take place in this clinical trial. We will provide maps 
and arrange to meet families on the first visit to show them around the facility. There are 
quiet rooms available for resting and calming participants. The clinical laboratory that 
will run the routine lab studies is located in the hospital and will report results the same 
day. Two laboratories are available to us for centrifuging specimens and processing 
PBMCs from blood, and storing blood and urine specimens at -20°C and -80°C until 
shipment on dry ice to Johns Hopkins (Cullman Chemoprotection Lab).  
 
UMass Research Pharmacy with expertise in clinical that will store and dispense 
sulforaphane and placebos, randomize participants and dispense the materials with proper 
labeling. Adequate office, clinic and lab spaces are available for this study through the 
department of pediatrics, along with various services and spaces in the UMMC. Adequate 
computer facilities, messaging and copying services are readily available.  
 
The Cullman Chemoprotection Laboratory at Johns Hopkins School of Medicine, 
Baltimore, MD is a well-established and fully equipped investigational pharmacology 
laboratory. It is well staffed and equipped for all of the proposed research laboratory 
studies we have proposed, both in the pilot study and main clinical trial in this study. This 
laboratory will produce the broccoli seed powder, the source of sulforaphane, under 
approved protocols and certification of purity prior to transfer to UMass for this study. In 
other parts of the lab, clinical samples will be received, in close contact with the staff at 
UMass regarding processing, storage and shipment of samples.  
 
22) Resources Available 
The following is the study organization chart describing various staff involved with study 
procedures and their roles.  
a. Study Organization Chart 
Site 1: University of Massachusetts Medical School, Worcester, MA 
Department: Pediatrics (Division of Pediatric Neurology) 
1. Principal Investigator 
2. PostDoc Associate 
3. Primary Care Physician 
4. Clinical Research Assistant (CRA) 
 

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Department: Child and Adolescent NeuroDevelopment Initiative (CANDI) 
1. Psychologist 
 
Department: Quantitative Health Sciences 
1. Biostatistician 
 
 
Site 2: Johns Hopkins University School of Medicine 
Department: Pharmacology (Lewis B. and Dorothy Cullman Cancer 
Chemoprotection Center) 
1. Johns Hopkins Principal Investigator 
2. Johns Hopkins Co-investigator 
3. Johns Hopkins Co-investigator 
 
Quality Associates Inc.  
1. External Good Clinical Practices (GCP) monitor 
 
b. Study Personnel Description: 
1. Andrew Zimmerman, MD.: Dr. Zimmerman is the Principal Investigator and a 
Professor of Pediatrics and Neurology at UMass Medical School. He is pediatric 
neurologist who has focused his research for over 20 years on novel immunological 
and therapeutic approaches to ASD. In this project, he plans to carry out a clinical 
trial of sulforaphane, a potent but safe natural substance in broccoli, in order to 
modify social and behavioral functioning in children. He will also evaluate 
underlying cellular effects of sulforaphane during the study, in collaboration with 
the collaborators at Johns Hopkins. He solely conceived this project and will be 
responsible for its overall administration and direction. He will supervise 
recruitment, enrollment, study implementation, monitoring of side effects, data 
management and ensure that the research is conducted in line with the ethical 
provisions of the University of Massachusetts Medical School (Aims 1-3). He will 
oversee and assure accurate data collection and analysis. He will present data at a 
national meeting, and prepare manuscripts for publication in peer-reviewed 
journals. 
 
2. PostDoc Associate/Research Coordinator:. He will assist the PI in the planning and 
implementation of all aspects of the study. He is experienced in regulatory affairs 
and ethical considerations and will have a major role in obtaining IRB approval for 
this study and regular reporting to the IRB. He will oversee the day to day operation 
of the study, including recruitment, screening procedures, scheduling, coordinating 
physical examinations, outcome measures and administration of medication. He 
will coordinate efforts of the Primary Care Physician (TBN) for the recruiting, 
screening, physical examination and phlebotomies of subjects at each study visit. 
He will work with our collaborators at Johns Hopkins to supply the Research 

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Pharmacy with drug and placebos, and ensure timely delivery of medication to the 
participants. He will assist with phlebotomies, blood sample preparation and 
shipments to the Cullman Chemoprotection Laboratory at Johns Hopkins. He will 
be responsible for data collection and storage in accordance with FDA guidelines, 
with which he has extensive experience. He will oversee the collection of safety 
data and adverse event reporting for the Data Safety Monitoring Board (DSMB). He 
will also be a liaison to Dr. Maranda, Biostatistician, for data management and 
analysis. 
 
3. Primary Care MD: The primary care MD will assist the PI in supervising and 
assisting with all medical aspects of the study, including review of medical histories 
during recruitment and screening, examinations of participants at each visit, and 
reporting of all potential side effects of treatment. He or she will review 
participants’ pre- and postnatal histories and development, record and rate effects of 
fever on behavior, current and past medications and allergies; record clinical data; 
perform phlebotomies and respond to parents’ calls or emails with respect to 
medical questions or concerns related to the study. This individual will 
communicate with participants’ pediatricians regarding questions about and 
concerns during the study, e.g., intercurrent illnesses, and will report any concerns 
to the PI and DSMB. 
 
4. Psychologist: A psychologist working at the Center for Autism and 
Neurodevelopmental Disorders (CANDO) clinic will assist with performing ADOS, 
Vineland and Leiter-3 assessments.  
 
5. Biostatistician: A biostatistician from Quantitative Health Sciences, will be the 
primary statistician on the team.  This person will have access to specialized 
statistical software, readily available to her for the conduct of this project’s 
statistical analyses. These include SAS, SPSS, StatsDirect and STATA. For this 
project, he/she will assist in the planning for the clinical trial design, data handling 
and storage, and evaluation of outcomes. 
 
6. Clinical Research Assistant: This individual will assist in recruitment and 
scheduling of appointments for screening, reporting and recording data from 
baseline assessments and outcome measures. He or she will work with other 
members of the team to communicate effectively with families in a timely manner, 
in order to assure both safety and compliance with the drug regimen and collection 
of data. The CRA will report any concerns of families to the PI, Research 
Coordinator and Primary Care MD. He or she will communicate with our 
collaborators at Johns Hopkins regarding drug shipments and coordinate with the 
Research Pharmacy, and arrange shipments of specimens to the laboratory in 
Baltimore. 
 
7. Johns Hopkins Principal Investigator and Co-investigator: These persons will assist 
prepare and supply capsules of broccoli seed powder containing standardized 
quantities of glucoraphanin and similar placebo capsules containing 

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microcrystalline cellulose. They will also determine the glucoraphanin content. 
They will be responsible for assurance of purity of the medication. 
 
8. Johns Hopkins Co-investigator: This person will make determinations of biomarker 
levels of oxidative stress, antioxidant capacity, and accumulation of reactive oxygen 
species, inflammatory biomarkers and heat shock proteins. 
 
9. Good Clinical Practices Monitor: This person will conduct yearly site visits to 
ensure that the study is being conducted in compliance with Good Clinical Practices 
and will conduct yearly site visits and audits to ensure GCP compliance.  
 
Our plan for ensuring the standardization of procedures among staff: We will have 
training sessions for all study staff to ensure internal consistency and replicability for 
OACIS scale. We will hold weekly meetings to review study progress, possible 
concerns of families and staff, review of study requirements, and compliance with the 
study plan. We will exchange de-identified data on a regular basis using encrypted 
email with the group at Johns Hopkins. Regular phone contacts and teleconferencing 
will take place, at least biweekly.  
 
All Johns Hopkins investigators are under the jurisdiction of JHU.  
 
23) Prior Approvals 
In addition to obtaining the UMass IRB approval, we will also obtain approval from 
USAMRMC Human Research Protection Office.  An Investigational New Drug (IND) 
approval from the Food and Drug Administration has been obtained. UMass Biosafety 
Committee has provided approval to perform the study. An IRB approval will also be 
obtained from Johns Hopkins University for the purposes of performing biomarker 
analysis at Johns Hopkins.  
 
24) Recruitment Methods 
 
Participants will be enrolled from Massachusetts and nearby New England states. The 
University of Massachusetts (UMass) Memorial Medical Center, Worcester is centrally 
located and has an excellent infrastructure for clinical trials as well as expertise in ASD 
with the Center for Autism and Neurodevelopmental Disorders (CANDO) Clinic. In our 
previous trial (at the Lurie Center, Massachusetts General Hospital for Children in 
Lexington, MA), we were readily able to enroll 44 participants in the span of 2 years 
despite restricting the population to male adolescents and young adults. Expanding the 
population to 50 younger children, both boys and girls, is a reasonable goal and will 
allow us access to an even greater target population than we had in the previous study. 
We will not restrict our study population to any race or ethnicity, and make all efforts to 
have this project meets the intent of the program as a clinical trial in ASD that is novel 

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and holds promise for a nontoxic approach to therapy of known biochemical and 
metabolic abnormalities in ASD.  
 
Patients will also be recruited from the CANDO clinic at UMass Memorial Medical 
Center, developmental pediatrics, pediatric neurology, child psychiatry, genetics and 
general pediatrics clinics at UMass Memorial Medical Center, as well as related clinics in 
the area. Patients suitable for the study will be identified by their treating physicians (not 
study staff members). We will obtain permission from the treating physician to contact 
these patients. The treating physician will be invited to notify their patients of this 
research study by providing their patients with recruiting letters and study flyers. Any 
recruitment letters will include a signed letter from their treating physician. The majority 
of patients at the CANDO Clinic have idiopathic ASD, whereas others have known 
genetic, pre- and postnatal causes of ASD. There are also sizable patient populations with 
ASD in the Reliant Health System and private practices nearby, as well as in community 
schools and treatment centers. 
 
Community based recruitment will also be done, and may include advertisements, 
electronic newsletters, and flyers to primary and subspecialty pediatric clinics within the 
participating institutions, local autism societies and support groups. Appropriate listservs 
and email outlets may be utilized. Advantages of UMass include its central location in 
New England and ready access with minimal traffic congestion, as well as a full-service 
academic medical center and pediatric hospital. 
  
As noted above, participants will be recruited from central Massachusetts and New 
England.  UMass Memorial Medical Center is ideally located and suited for this clinical 
trial of sulforaphane in children with ASD. It is centrally located in New England and is a 
full service academic medical center and children’s hospital, as well as a Clinical 
Research Center. A strong presence in the field of ASD has developed at UMass in recent 
years with the founding of the CANDO Clinic, expansion of pediatric neurology and 
developmental pediatrics. 
 
Participants will be screened by review of verbal histories and records on request as they 
are made available from their referral sources, following parental consent. 
 
Participants will be compensated for their participation in the study. We will provide 
them with a gift card worth $15 for each completed study visit. We will also provide 
them with parking vouchers when families park their vehicle in the UMass Medical 
Center parking lot. Children will be given a small soft toy to keep.  
 
Flyers describing the nature of sulforaphane and how it might help ASD will be used for 
study advertisement. These advertisements will make it clear that it is a research study 
and not a “free treatment program” for ASD, in order to remove any coercive or undue 
inducements for study participation. These flyers will be distributed to various autism 
advocacy centers for display purposes, and may also be distributed by email based 
listservs.  
 

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The recruitment process is similar for both the pilot study as well as the main clinical 
trial. 
 
Screening Procedures:  
Prior to coming for the formal screening visit, all subjects will undergo a pre-screen 
assessment to determine if basic eligibility criteria are met and the study procedures will 
be explained in detail. Qualified study staff will ask questions related to the above 
described inclusion/exclusion criteria. The pre-screen may be completed by telephone or 
direct interview.  If a participant is found to be ineligible for the study, prescreening form 
will be destroyed. 
 
 
At the screening visit, after the consent has been signed by parents/guardians and assent 
signed (if applicable), a complete, baseline medical history, physical and neurological 
exam will be performed. During that visit, ADOS-G, SRS, OACIS-Severity scale and 
ABC-W scales will be performed to confirm eligibility and to quantify disease severity. 
Also, following laboratory blood tests (complete blood count, comprehensive metabolic 
profile: liver function rests, renal function tests, thyroid function test (TSH)) will be 
performed.  Maximum amount of blood drawn at any stage during this study will not 
exceed 2 ml/kg body weight; we will also not exceed the minimal risk limitation of 3 
ml/kg in an 8 week period. 
 
25) Local Number of Subjects 
 
We will enroll 50 participants in the study. We anticipate that 400 to 500 families will be 
screened in order to attract 50 participants. 
 
 
26) Confidentiality  
 
Strict confidentiality will be maintained at all times. As mentioned above, all subjects and 
clinical information associated with each subject will be assigned an identification 
number and will only be referred to by that number and subject initials. This number will 
be the only identifying information on test score sheets, data entry forms, and in the data 
files. Some data will require minimum protected health information (i.e. date of birth) 
recorded on the assessment sheets in order to be properly scored. In this case, we will 
follow HIPAA limited data set guidelines.  Source documents will be maintained by the 
study coordinator in a centralized, double-locked office accessible only by the study 
coordinator and involved members of the research team.  Only initials and study number 
identify any specimens sent to laboratories outside of UMass. 
 
We will collect some identifiable information, such as name, address, date of birth, 
contact information (mailing address, phone numbers, email address etc.) demographics 
(race, ethnicity, parental educational background) but this information will be kept 
separate from the other study files and documents. Documents containing this identifying 
information will be kept in a locked cabinet in a secure study location. Only study staff 

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directly involved in interaction with the study participants and the study Principal 
Investigator will have access to this identifiable information. The Principal Investigator, 
or designee, will maintain a personal participant identification list (participant numbers 
with the corresponding names) to enable records to be identified. Other study staff (such 
as those working with biomarkers) whose role will not involve interacting with study 
participants will not have access to this identifiable information. Representatives of 
USAMRMC who may be entrusted with the responsibility of conducting site visits may 
be able to get access to the study records for review.   
 
In terms of subject privacy protection, the researchers are aware of the implementation of 
the Health Insurance Portability and Accountability Act (HIPAA) and its impact on 
clinical research and electronic data collection activities.  The database developed under 
this protocol will conform to national standards and procedures for the electronic storage 
and transmission of information to insure privacy and security of health information.  
This includes conformance to rules for transactions and code sets, the standards for 
privacy of individually identifiable health information and security standards. 
 
Any blood samples sent to the laboratories at Johns Hopkins for analysis of heat shock 
proteins and mitochondrial study and measures of oxidative stress will not contain any 
direct identifiers and samples will be labeled with the subject’s unique ID. Samples may 
be stored for future use with explicit consent of the subject and parents or guardians. 
Without such consent, samples will be destroyed following the study.  
 
Data will be primarily captured on paper records. Majority of the study instruments (such 
as Aberrant Behavior Checklist, Social Responsiveness Scale, Autism Diagnostic 
Observation Schedule, Vineland, Leiter scale) are provided as paper booklets and they 
will be used as the primary data capture devices. Additional information, such as physical 
examination findings, medical history records etc. will also be captured in paper records. 
After the study is complete, we will keep the study records for future regulatory purposes 
for a period of 7 years, at which time all study records will be destroyed. However the 
documents containing identifiable information will not be stored after the study ends; 
they will be destroyed after study is completed.  
 
In addition, we will use a secure computer database to transfer the paper data records to 
an electronic format. University of Massachusetts Medical School has contracted the 
RedCap clinical trials software database solution. REDCap (Research Electronic Data 
Capture) is a secure web-based application built around HIPAA guidelines to support 
data capture for research studies. It allows users to build and manage online surveys and 
databases or a combination of both quickly. REDCap features automated export 
procedures for seamless data downloads to Excel, PDF, and common statistical packages 
(SPSS, SAS, Stata, R). Also included are a built-in project calendar, a scheduling 
module, ad hoc reporting tools, and advanced features, such as branching logic, file 
uploading, and calculated fields. The REDCap Software sits on a dedicated server behind 
a firewall to eliminate all nonessential access credentials. The data itself is isolated on a 
separate server that sits behind an additional firewall layer for added security. Interaction 
between REDCap and the client is encrypted via SSL. To ensure accuracy of transfer of 

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data from paper to electronic format and to minimize data entry errors, all data that will 
entered to RedCap will be double entered by two independent study staff members. After 
the study is complete and all data has been collected and entered into the database, the 
database will be locked out from any further editing.  
 
 
27) Provisions to Protect the Privacy Interests of Subjects (HIPAA) 
 
In terms of subject privacy protection, we are aware of the implementation of the Health 
Insurance Portability and Accountability Act (HIPAA) and its impact on clinical research 
and electronic data collection activities.  The database developed under this protocol will 
conform to national standards and procedures for the electronic storage and transmission 
of information to insure privacy and security of health information.  This includes 
conformance to rules for transactions and code sets, the standards for privacy of 
individually identifiable health information and security standards. 
 
We will make all efforts to preserve the privacy of the study participants. All data 
collected in the study will be identified by a random code and participant initials. We will 
use the following format to label the study participants: SF-15-001, SF-15-002 along with 
participant initials (eg. JD). No personally identifying information, such as complete 
name, hospital medical record number, date of birth etc. will be used on study documents.  
 
Any blood samples sent to the laboratories at Johns Hopkins for analysis of heat shock 
proteins and mitochondrial study and measures of oxidative stress will not contain any 
direct identifiers and samples will be labeled with the subject’s unique ID. Samples may 
be stored for future use with explicit consent of the subject and parents or guardians. 
Without such consent, samples will be destroyed following the study.  
 
28) Compensation for Research-Related Injury 
No funds have been set aside for any compensation for research related injury. 
 
29) Economic Burden to Subjects 
 
Study participants will not have any costs associated with participating in the study – 
costs for all study related procedures and lab tests will be borne by the study funds. 
Participants will be compensated for their participation in the study. We will provide 
them with a gift card worth $15 for each completed study visit. We will also provide 
them with parking vouchers when families park their vehicle in the UMass Medical 
Center parking lot. Children will be given a small soft toy to keep.  
  
30) Consent Process 
 

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A written consent will be obtained. We will follow the “SOP: Informed Consent Process 
for Research (HRP-802)” for consenting purposes.The consent will be obtained at the 
Clinical Research Center at the UMass Memorial Medical Center. We will allow 
participants/their parents or legal guardians a sufficient time interval (more than at-least 
24 hours) to make up their mind before signing the consent form. After the telephone pre-
screening with parents/guardians, we will mail a copy of the consent and assent form as 
well as study information flyers to their address and encourage them to thoroughly read 
the consent forms and call if they have questions. All potential participants for this study 
will be provided a consent form describing this study and sufficient information for 
subjects and/or their parent or guardian to make an informed decision about their 
participation in this study. 
At the screening visit, the study investigator(s) will again discuss with each subject and 
his or her parents or guardian the nature of the study, its requirements, and its restrictions. 
At that time if they are interested in participating in the study, a written and witnessed 
informed consent will be obtained prior to the performance of any protocol-specific 
procedures. If however they need more time to make up their mind, they will be allowed 
to do so and we will reschedule the study visit to a date when they are ready to participate 
in the study and sign the consent form. Whenever possible, consent from both parents 
will be obtained.  
Since all subjects in this study are children, permission will be obtained from their parent 
or legal guardian and will be documented in writing using the approved Consent Form. 
We will try to obtain the assent of subjects for participation in the research. This assent 
will be done on the approved assent form. If some participants are medically incapable of 
giving assent (those who are severely autistic and incapable of understanding the nature 
of the study) we may not obtain assent from these children. The Principal Investigator 
(who is a pediatric neurologist experienced in evaluating and treating children with ASD) 
will determine whether a patient is medically incapable of giving assent. In these and all 
cases, consent will be obtained from parents or legal guardians.  
The formal consent, or assent, as applicable, using the IRB-approved forms will be 
obtained before that subject is submitted to any study procedure. The form(s) must be 
signed by the subject and/or parent/guardian for the child and the investigator-designated 
research professional witnessing the signatures. Every effort will be made to assure the 
safety of the subjects. A qualified person, such as a licensed physician on the study staff 
will obtain the consent/assent. A research coordinator will not obtain consent/assent from 
the study participants.   
Since majority of instruments (such as the ADOS, Leiter-3) used in the study are in 
English language and validated in English language, we will only enroll English speaking 
participants in the study. 
 
31) Process to Document Consent in Writing 
We will be following “SOP: Written Documentation of Consent (HRP-803).”  
 

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32) Drugs or Devices 
 
Prior to dispensing the study drug to the participants, the drug will be stored in 
temperature controlled conditions (-20C) at the UMass Memorial Investigational 
Pharmacy. During the study visits, we will store the study drug in a locked -20 freezer at 
the Clinical Trials Unit (to which only the study staff will have access to) and directly 
dispense the medication to patients’ parents (except at the first visit where we will wait 
for the safety labs results and then either mail the drug to participants or ask their parents 
to pick it up). The parents will be instructed to keep the medication stored in their freezer 
for the duration of the study.  
 
The firm/entity that will be hired to ensure GCP compliance will also monitor 
compliance with the FDA requirements. 
 
We filed an IND application to the FDA to obtain permission for conducting this clinical 
trial and the FDA has approved the IND (IND number 127062). The IND is held by the 
study PI (Dr. Andrew Zimmerman). We will comply with the Sponsor-investigator 
requirements for IND studies (21 CFR 54, 210, 211, 312). 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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