A Phase II, Randomized, Double-blind, Placebo-controlled Study of Myrosinase-enriched Glucoraphanin, a Sulforaphane Precursor System, in Autism Spectrum Disorder
Phase: PHASE2
Conditions: Autism Spectrum Disorder, Autistic Disorder, Neurodevelopmental Disorder, Childhood Developmental Disorders, Pervasive
Sponsor: University of North Carolina, Chapel Hill

Clinical Protocol: Sulforaphane for ASD 
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Study Title: A phase II, randomized, double-blind, placebo-
controlled study of myrosinase-enriched glucoraphanin, a 
sulforaphane precursor system, in autism spectrum disorder 
 
 
 
 
NCT number 02909959 
Document Date 12/14/2017 
 
 
 
 

Clinical Protocol: Sulforaphane for ASD 
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A phase II, randomized, double-blind, placebo-controlled study of 
myrosinase-enriched glucoraphanin, a sulforaphane precursor 
system, in autism spectrum disorder 
 
 
 
 
 
Regulatory Sponsor: 
Laura C. Politte, M.D. 
Department of Psychiatry 
Carolina Institute for Developmental Disabilities 
University of North Carolina School of Medicine 
101 Renee Lynne Court 
Carrboro, NC 27510   CB#7255 
919-966-5171 
 
Funding Sponsor: 
Supported by a collaborative grant from the UNC Nutrition 
Research Institute 
500 Laureate Way 
Kannapolis, NC 28081 
704-250-5003 
 
Study Personnel: 
Laura Politte, M.D., principal investigator 
Joseph Piven, M.D., co-investigator 
Carol Cheatham, Ph.D., co-investigator 
Mark Weaver, Ph.D., biostatistician 
 
Study Product: 
Sulforaphane (Avmacol®) 
Protocol Number: 
UNC IRB #16-2059 
IND Number: 
131861 
 
Initial version: 
08/05/2016 
Amended: 
[09/23/2016] 
Amended: 
[02/28/2017] 
Amended: 
[03/01/2017] 
Amended: 
[06/08/2017] 
Amended: 
[08/02/2017] 
Amended: 
[08/22/2017] 
Amended: 
[09/13/2017] 
Amended: 
[09/28/2017] 
Amended: 
[12/14/2017] 
 

Clinical Protocol: Sulforaphane for ASD 
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Table of Contents 
 
 
List of Abbreviations .................................................................................................................................. 6 
Study Summary ........................................................................................................................................... 8 
1 
Introduction ......................................................................................................................................... 9 
1.1 
Background ................................................................................................................................. 9 
1.2 
Investigational Agent ................................................................................................................ 11 
1.3 
Preclinical Data ......................................................................................................................... 12 
Clinical Data to Date ............................................................................................................................. 14 
1.4 
Dose Rationale and Risk/Benefits ............................................................................................ 15 
2 
Study Objectives ............................................................................................................................... 17 
3 
Study Design ..................................................................................................................................... 18 
3.1 
General Design.......................................................................................................................... 18 
3.2 
Primary Safety Endpoints ......................................................................................................... 20 
4 
Subject Selection and Withdrawal .................................................................................................... 20 
4.1 
Inclusion Criteria ...................................................................................................................... 20 
4.2 
Exclusion Criteria ..................................................................................................................... 21 
4.3 
Subject Recruitment and Screening .......................................................................................... 21 
4.4 
Early Withdrawal of Subjects ................................................................................................... 22 
4.4.1 When and How to Withdraw Subjects .................................................................................. 22 
4.4.2 Data Collection and Follow-up for Withdrawn Subjects ...................................................... 22 
5 
Study Drug ........................................................................................................................................ 23 
5.1 
Description ................................................................................................................................ 23 
5.2 
Treatment Regimen ................................................................................................................... 23 
5.3 
Method for Assigning Subjects to Treatment Groups .............................................................. 23 
5.4 
Preparation and Administration of Study Drug ........................................................................ 24 
5.5 
Subject Compliance Monitoring ............................................................................................... 24 
5.6 
Prior and Concomitant Therapy ................................................................................................ 24 
5.7 
Packaging .................................................................................................................................. 25 
5.8 
Blinding of Study Drug (if applicable) ..................................................................................... 25 
5.9 
Receiving, Storage, Dispensing and Return ............................................................................. 25 

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5.9.1 Receipt of Drug Supplies ...................................................................................................... 25 
5.9.2 Storage .................................................................................................................................. 26 
5.9.3 Dispensing of Study Drug ..................................................................................................... 26 
6 
Study Procedures .............................................................................................................................. 26 
7 
Statistical Plan ................................................................................................................................... 30 
7.1 
Sample Size Determination....................................................................................................... 30 
7.2 
Statistical Methods .................................................................................................................... 30 
7.3 
Subject Population(s) for Analysis ........................................................................................... 32 
8 
Safety and Adverse Events ............................................................................................................... 32 
8.1 
Definitions................................................................................................................................. 32 
8.2 
Recording of Adverse Events ................................................................................................... 34 
8.3 
Reporting of Serious Adverse Events and Unanticipated Problems ......................................... 35 
8.3.1 Investigator reporting: notifying the study sponsor .............................................................. 35 
8.3.2 Investigator reporting: ........................................................................................................... 35 
8.3.3 Investigator reporting: ........................................................................................................... 36 
8.3.4 Sponsor reporting: Notifying the FDA ................................................................................. 36 
8.4 
Unblinding Procedures.............................................................................................................. 37 
8.5 
Stopping Rules .......................................................................................................................... 37 
8.6 
Medical Monitoring .................................................................................................................. 39 
8.6.1 Internal Data and Safety Monitoring Board  or DSMP ........................................................ 39 
9 
Data Handling and Record Keeping ................................................................................................. 39 
9.1 
Confidentiality .......................................................................................................................... 39 
9.2 
Source Documents .................................................................................................................... 40 
9.3 
Case Report Forms .................................................................................................................... 40 
9.4 
Records Retention ..................................................................................................................... 40 
10 Study Monitoring, Auditing, and Inspecting .................................................................................... 41 
10.1 
Study Monitoring Plan .............................................................................................................. 41 
11 Ethical Considerations ...................................................................................................................... 41 
12 Study Finances .................................................................................................................................. 41 
12.1 
Funding Source ......................................................................................................................... 41 
12.2 
Conflict of Interest .................................................................................................................... 42 
12.3 
Subject Stipends or Payments ................................................................................................... 42 

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13 Publication Plan ................................................................................................................................ 42 
14 References ......................................................................................................................................... 42 
15 Appendix 6A: Schedule of Study Events.......................................................................................... 47 
16 Appendix 6B: Data and Safety Monitoring Plan .............................................................................. 48 
17 Appendix 6C: NC TraCS DSMB Charter ......................................................................................... 58 
18 Appendix 6D: Clinical Studies List .................................................................................................. 65 
 
 
 

Clinical Protocol: Sulforaphane for ASD 
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List of Abbreviations  
 
ABC- Aberrant Behavior Checklist 
ADOS-2- Autism Diagnostic Observation Schedule, 2nd edition 
AE- adverse event 
Akt- protein kinase B 
ALT- alanine aminotransferase 
ARE- Antioxidant response elements 
ASD- autism spectrum disorder 
AST- aspartate transaminase 
ATP- adenosine triphosphate 
BOM- behavioral outcome measures 
BSE- Broccoli Sprout Extract 
CBC- complete blood count 
CGI- Clinical Global Impression Scale 
CIDD- Carolina Institute for Developmental Disabilities 
CMP- comprehensive metabolic panel 
CNS- central nervous system 
CRF- case report form 
DNA- deoxyribonucleic acid 
DSM-5- Diagnostic and Statistical Manual, 5th edition 
DSMB- Data and Safety Monitoring Board 
DTC- dithiocarbamates 
EEG- electroencephalogram 
FDA- Food and Drug Administration 
GLMM- general linear mixed models 
GMP- Good Manufacturing Practice 
GR- glucoraphanin 
GS- glucosinolate 
GSH- reduced glutathione 
GSSG- oxidized glutathione 
GST- glutathione-S-transferase 
HIPAA- Health Insurance Portability and Accounting Act 
HO-1- heme oxygenase-1 
HSF1- heat-shock transcription factor 1 
HSP- heat shock protein 
IDS- Investigational Drug Services 
IF-γ- interferon-gamma 
IL-6- interleukin-6 
iNOS- inducible nitric oxide synthase 
IRB- internal review board 
ITC- isothiocyanate 
IQ- intelligence quotientswi 
Keap1- Kelch-like ECH-associated protein 
LAR- legally authorized representative 
LFTs- liver function tests 
LPS- lipopolysaccharide 
mTOR- mammalian target of rapamycin 

Clinical Protocol: Sulforaphane for ASD 
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NC TraCS- North Carolina Translational and Clinical Science Institute 
NF-KB- nuclear factor-KB 
Nrf2- nuclear factor- erythroid factor 2 (Nrf2) 
NSAID- non-steroidal anti-inflammatory drug 
OACIS- Autism Clinical Impressions Severity and Improvement Scales 
OTU- Operational Taxonomic Unit 
PHI- Protected health information 
PI- Principle Investigator 
PI3K- phosphoinositide-3 kinase 
PBMC- peripheral blood mononuclear cell 
PSA- prostate specific antigen 
PTEN- phosphatase and tensin homolog 
RBSR- Repetitive Behavior Scale-Revised 
RCT- randomized controlled trial 
ROS- reactive oxygen species  
RTI- Research Triangle Institute 
SAE- serious adverse event 
SB-5- Stanford-Binet Intelligence Scales, 5th edition 
SF- sulforaphane 
SRS- Social Responsiveness Scale 
TNF-α- tissue necrosis factor-alpha 
TSC1/2- tuberous sclerosis complex 
TSH- thyroid stimulating hormone 
T3- triiodothyronine 
T4- thyroxine 
UNC- University of North Carolina 
 
 
 

Clinical Protocol: Sulforaphane for ASD 
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Study Summary 
 
Title 
A phase II, randomized, double-blind, placebo-controlled study of 
myrosinase-enriched glucoraphanin, a sulforaphane precursor 
system, in autism spectrum disorder 
 
Short Title 
Sulforaphane in autism spectrum disorder 
Protocol Number 
C15-0560 
Phase 
Phase II 
Methodology 
Randomized, double-blind, placebo-controlled trial 
Study Duration 
24 months 
Study Center(s) 
Single-center: University of North Carolina- Chapel Hill (Carolina 
Institute for Developmental Disabilities) 
Objectives 
(1) To determine if a sulforaphane supplement (Avmacol®) is 
effective for improving social impairment in autism spectrum 
disorder (ASD); (2) To determine if Avmacol® is safe and well-
tolerated in adolescents and young men with ASD. (3) To explore if 
Avmacol® improves behavioral symptoms commonly associated 
with ASD. 
Number of Subjects 
54 subjects 
Diagnosis and Main 
Inclusion Criteria 
Healthy males between ages 13-30 years old with a diagnosis of  
moderate to severe ASD 
Study Product, 
Dose, Route, 
Regimen 
Avmacol® uncoated tablets, each containing 125 mg broccoli seed 
extract (source of glucoraphanin) and 50 mg broccoli sprout extract 
(source of myrosinase) or matched placebo tablets; 3-8 tablets by 
mouth once daily (specific dose determined by weight). 
Duration of 
administration 
12 weeks 
Reference therapy 
Matched placebo tablets 
Statistical 
Methodology 
General linear mixed models will be used to test the primary and 
secondary hypotheses; of interest will be the main effect of 
treatment, treatment x time, and treatment x time x treatment 
phase.  Analyses will include data from all randomized subjects who 
received at least one dose of study medication and participated in at 
least one post-baseline efficacy assessment. 
 

Clinical Protocol: Sulforaphane for ASD 
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1 
Introduction 
 
This document is a protocol for a human research study. This study is to be conducted according 
to US and international standards of Good Clinical Practice (FDA Title 21 part 312 and 
International Conference on Harmonization guidelines), applicable government regulations, and 
University of North Carolina research policies and procedures.  
1.1 Background 
 
Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by severe and 
persistent deficits in social communication and restricted, repetitive behaviors and interests [1]. 
ASD is now estimated to affect 1 in 68 children in the U.S., including 1 in 42 boys [2], and leads to 
substantial, lifelong burden for individuals, families, and communities.  Use of psychiatric 
medication in ASD is high, with 66% of affected individuals taking at least 1 psychotropic 
medication by adolescence [3]. However, no medications are currently available to treat the 
defining clinical features of autism, and pharmacological management is instead limited to 
treating associated behavioral symptoms, such as hyperactivity, self-injury, and aggression.  Only 
risperidone and aripiprazole have FDA approval for use in ASD, specifically for the treatment of 
severe irritability in children [4].  Faced with heavy side effect burden and often limited efficacy, 
patients and their families frequently turn to supplements and alternative treatments that 
promise low toxicity but lack rigorous investigation and oversight.   
 
Developing interventions specifically for ASD has been hindered by heterogeneity in its 
phenotypic expression and implicated genes, epigenetic effects, and molecular pathways; as such, 
no treatments based on an understanding of disease mechanism are presently available.  
However, several emerging lines of research suggest that interrelated metabolic dysfunction and 
immune dysregulation at the cellular level contribute to the pathogenesis of ASD in at least a 
subset of individuals [5, 6].  Preclinical and human studies have documented increased levels of 
oxidative stress [7-9]; reduced antioxidant activity [10-12]; mitochondrial dysfunction [13-15]; 
elevated inflammatory markers [16-19]; evidence of neuroinflammation [20-23]; and synaptic 
dysfunction [24] in association with ASD.  Evidence of cellular dysfunction provides an intriguing 
hypothesis for the systemic phenomena commonly observed in patients with ASD, including 
gastrointestinal problems and improvement in ASD symptoms during fever [25, 26].  Anecdotal 
improvement in core symptoms of autism during fever, particularly repetitive speech and 
behavior, is commonly observed, possibly as a result of upregulation of heat shock proteins (HSPs) 
that play a key role in multiple CNS cellular processes, including synaptic transmission and 
response to metabolic stress [25, 27, 28].   
 
It is hypothesized that this varied evidence of cellular dysfunction may reflect impaired activity in 
common metabolic pathways, the consequence of which is aberrant protein synthesis that 
impedes normal synaptic development and function.  Synaptic dysfunction is believed to be a key 
pathogenic mechanism in ASD [24].  In particular, overactivation of the mammalian target of 
rapamycin (mTOR) intracellular signaling pathway has been implicated in excessive protein 
synthesis and synaptic dysfunction in ASD [29-31].  

Clinical Protocol: Sulforaphane for ASD 
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The Kelch-like ECH-associated protein 1 (Keap1)/nuclear factor-erythroid factor 2 
(Nrf2)/antioxidant response elements (ARE) pathway is a highly efficient system involved in 
maintaining cellular redox balance and reducing oxidative damage through a series of antioxidant 
molecules and detoxifying enzymes.  Nrf2 is a nuclear transcription factor that, when activated by 
oxidative or electrophilic conditions, translocates from the cytoplasm to the nucleus and induces 
expression of a variety of antioxidants and immune mediators involved in limiting cellular damage 
from these conditions [32, 33].  ASD diagnosis in children has been linked to a 45% reduction in 
gene expression of Nrf2 in association with evidence of greater oxidative stress [6].  These 
findings suggest that deficits in the Keap1/Nrf2/ARE pathway may lead to a state of chronic 
inflammation and decreased capacity to respond effectively to conditions promoting oxidative 
stress in autism. While it is not yet clear if these cellular abnormalities are etiological or secondary 
manifestations of another disease-causing mechanism, preliminary studies have suggested that 
treatments aimed at reducing oxidative stress and mitochondrial dysfunction may improve core 
and associated symptoms of ASD [34]. In keeping with this theory, compounds that upregulate 
Nrf2 activity may have the potential to improve symptom burden in ASD. 
 
Sulforaphane (SF) is a naturally-occurring isothiocyanate (ITC) in cruciferous vegetables that 
induces HSP and Nrf2 activity, leading to enhanced redox homeostasis, DNA repair, and fatty acid 
and lipid cellular metabolism [32].  SF has also been shown to (1) suppress lipopolysaccharide 
(LPS)-induced inflammation in rat microglia [35], (2) reduce neuroinflammation in degenerative 
CNS disorders [36], and (3) mitigate reactive oxygen species (ROS)-mediated neurotoxicity in a 
murine model of herpes encephalitis [37] through activation of Nrf2.  In cultured cardiomyoctes 
and prostate cancer cells, SF reduces cellular damage through downregulation of mTOR-mediated 
excessive protein synthesis [38, 39].  
 
Due to its chemoprotective properties, SF has been investigated as a therapeutic agent in clinical 
trials of prostate cancer, breast cancer, exposure to air pollutants, and insulin resistance in type 2 
diabetes with promising results [40-44].  Importantly, SF is bioavailable when taken orally and 
readily crosses the blood-brain barrier [45-47].  SF is attractive as a potential therapeutic 
compound in ASD for its demonstrated capacity to improve redox balance, enhance antioxidant 
capacity, reduce neuroinflammation, support cellular metabolism, and regulate the mTOR 
signaling pathway, all of which have been implicated in autism pathogenesis.  In a recent clinical 
trial, Singh and colleagues [48] first demonstrated that young men with autism treated with oral 
SF for 18 weeks were rated as calmer by caregivers and showed improvement in standardized 
measures of social communication compared with a placebo group.  In this proposed clinical trial, 
we aim to independently replicate this important study and add to the evidence base of SF 
treatment for autism.   
 
Of note, the trial by Singh et al [48] utilized freeze-dried, SF-rich broccoli sprout extracts, an 
investigational product formulated by the Cullman Chemoprotection Center at Johns Hopkins 
University specifically for use in clinical trials.  Hydrolyzed Broccoli Sprout Extract (BSE) is costly, 
difficult to manufacture, and no longer produced in the United States for clinical trials or routine 
clinical use.  Avmacol® is a commercially available nutraceutical that provides a rich source of SF 
through co-delivery of glucoraphanin (GR), a sulforaphane precursor, and myrosinase, the plant 
enzyme that catalyzes conversion of GR to SF.  Avmacol® is readily available as a dietary 

Clinical Protocol: Sulforaphane for ASD 
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supplement, is shelf-stable at room temperature, and offers the potential for post-study 
continuance of therapy.  The proposed study will explore if this SF precursor-enzyme product 
confers similar therapeutic benefit for core deficits in ASD as freeze-dried BSE, which will have 
important broad implications for treatment. 
 
1.2 Investigational Agent 
 
The investigational product, Avmacol® (Nutramax Laboratories, Inc., Edgewood, MD) is a dietary 
supplement containing broccoli seed extract and freeze-dried broccoli sprouts that is 
manufactured under GMP standards and has been commercially available since 2013.  Avmacol® 
provides a rich source of the isothiocyanate (ITC) sulforaphane (1-isothiocyanato-(4R)-
(methylsulfinyl)butane) in the form of glucoraphanin (4-methylsulfinylbutyl-glucosinolate), an 
inert glucosinolate (GS), and myrosinase, the plant enzyme that hydrolyzes glucoraphanin to 
sulforaphane [49].  The chemoprotective properties of cruciferous vegetables (broccoli, 
cauliflower, cabbage, etc.) are largely attributed to their high content of GS, which are converted to 
biologically active ITCs (i.e., sulforaphane) by myrosinase and by the microflora of the 
gastrointestinal tract after ingestion [50]. Myrosinase is present in plant cells and is physically 
segregated from the glucosinolates until the barrier is ruptured via cell damage or chewing.     
 
SF is rapidly conjugated with glutathione by glutathione-S-transferases (GSTs) [51, 52] and 
accumulates in various types of cells through conjugation with cellular glutathione [53, 54]. SF 
conjugates are exported from the cell by a transporter-mediated mechanism [54].   Successive 
steps of hydrolysis of the conjugates lead ultimately to formation of N-acetyl-cysteine derivatives 
(mercapturic acids).  These conjugates are dithiocarbamates (DTC) that are excreted in urine and 
can be quantified through a cyclocondensation reaction developed in the Cullman 
Chemoprotection Center of Johns Hopkins University.  SF activates the Nrf2 pathway, resulting in 
elevated gene transcription via the Antioxidant Response Element (ARE) in the regulatory domain 
of its target genes [55, 56]. 
 
 
 
 
 
 
 

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Each tablet of Avmacol® contains 125 mg broccoli seed powder and 50 mg broccoli sprout extract, 
providing approximately 15 mg (34.4 µmol) of glucoraphanin and enough myrosinase to fully 
hydrolyze the glucoraphanin contained in the tablet.  In the batch of tablets formulated for the 
proposed clinical trial, manufacturing specifications required at least 13 mg glucoraphanin per 
tablet, and recent analysis indicated 17 mg per tablet (see Avmacol® COA in Section 7).  
Manufacturing data from Nutramax, Inc. and bioavailability studies of similar compounds 
containing GR and myrosinase suggest a bioconversion to SF rate in the range of 40-50% [47]; it is 
expected that each Avmacol® tablet with therefore supply approximately 15 µmol SF following 
bioconversion.  Exact amounts of bioavailable SF will varies somewhat from individual to 
individual depending upon endogenous enzymatic conversion following ingestion. 
 
Healthy human volunteers ingesting 200 µmol of SF from a broccoli sprout extract preparation 
demonstrated rapid absorption, first-order metabolism, and urinary excretion of metabolites 
(DTCs), yielding a mean half-life of 1.77 ± 0.13 hr. and volume of distribution of 59.9 ± 7.0 liters 
[57].  Despite the relatively short half-life, SF is believed to have a longer duration of action, as 
induced phase 2 enzymes have half-lives measured in days [56].  SF has been shown to cross the 
blood-brain barrier [58].  Urinary metabolites (DTCs) are renally excreted and can be quantified 
using a cyclocondensation reaction. 
 
1.3 Preclinical Data 
 
A number of animal studies have investigated the biological effects and toxicology of oral broccoli 
sprouts and SF, demonstrating overall in vivo safety and beneficial biological effects, namely via 
induction of phase 2 antioxidant enzymes, which have chemoprotective properties.  Rodents 
treated with relatively high doses of SF and broccoli sprout extracts have not demonstrated any 
significant change in body weight or evidence of organ toxicity [59-62].  SF has also shown little 
cytotoxicity in vitro, though hepatotoxity in rats has been demonstrated at extremely high 
concentrations (>500 µM) [63].  
 
Numerous studies have associated ASD with cellular abnormalities, including: (1) redox imbalance 
and oxidative stress [9, 10, 64]; (2) mitochondrial dysfunction [13-15]; (3) immune 
dysregulation/ neuroinflammation [20-23, 65]; (4) symptomatic improvement with fever/ heat 
shock response [25, 27]; and (4) aberrant mTOR expression and its effects on synaptic 
transmission [29-31].  It is hypothesized that these varying types of cellular dysfunction may 
involve a number of related, interacting metabolic pathways [11], though the relationship 
between them has not been fully characterized.  Preclinical data has identified a role for SF and its 
ability to upregulate the Keap 1/Nrf2/ARE pathway in modulating each of these areas of cellular 
dysfunction, suggesting SF could have therapeutic benefit in autism.   
 
Upregulation of antioxidant enzymes via activation of the Keap 1/Nrf2/ARE pathway has 
important implications for ASD, which has been associated with reduced levels of Nrf2 expression 
and oxidative phosphorylation capacity in cultured cells [6]. Lower concentrations of reduced 

Clinical Protocol: Sulforaphane for ASD 
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glutathione (GSH), the primary intracellular antioxidant, higher levels of oxidized glutathione 
(GSSG), and reduced GSH/GSSG redox ratios have also been identified in plasma, peripheral blood 
mononuclear cells (PBMCs), lymphoblastoid cell lines, brain tissue, and mitochondria of 
individuals with autism in comparison to healthy controls [9, 64, 66, 67].  SF has been found to 
have neuroprotective effects against oxidative stress through Nrf2 induction and the resulting 
increased expression of antioxidant enzymes and elevation of GSH [68-73]. In a rat model of spinal 
cord contusion, SF readily crossed the blood-brain barrier, increasing phase 2 enzyme expression 
and reducing inflammation at the site of the injury [74].  Similarly, in a murine model of herpes 
encephalitis, SF stimulated Nrf2 activity and antioxidant enzyme (heme oxygenase-1, glutathione 
peroxidase 1) expression in astrocytes, inhibiting ROS-mediated neurotoxicity [37].  Animal 
models of neurodegenerative diseases suggest that SF can have a neuroprotective effect by 
limiting damage from oxidative stress and inflammation [36, 75]. 
 
Evidence of mitochondrial dysfunction in individuals with ASD has been demonstrated in brain 
mass resonance spectroscopy imaging [76], cell lines derived from children with ASD [14], and in 
blood cell [13, 77] and brain samples [15] of children with ASD, and this dysfunction may play a 
critical role in disease pathogenesis in a subset of individuals [11, 78, 79].  In particular, 
impairment in fatty acid oxidation is believed to be one of the major contributors to the impaired 
mitochondrial phenotype [80], supported by elevated levels of acyl-carnitines [81] and decreased 
levels of free carnitine [82] in some individuals with ASD.  Emerging evidence suggests that Nrf2 
plays a role in supporting mitochondrial function and metabolism, possibly through improving the 
efficiency of fatty acid oxidation [80, 83].  In a rodent hippocampal model, SF was associated with 
increased adenosine triphosphate (ATP) synthesis and decreased vulnerability to induced 
seizures [84].  SF also induced mitochondrial and peroxisome biogenesis in cultured fibroblasts 
[85]. 
 
Inflammation and immune dysregulation has been well-documented in animal models and 
individuals with ASD, both peripherally and within the central nervous system [5, 11, 22, 65].  ASD 
is associated with elevated rates of microglial activation in murine models, post-mortem studies, 
and brain imaging [20, 21, 23, 86] as well as heightened expression of proinflammatory cytokines 
in the CNS, particularly interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and interferon-
gamma (IF-γ) [16-19].  SF has been shown to modulate neuroinflammation in animal models [35, 
37, 74].  The beneficial effect of SF on inflammation is mediated at least in part through inhibition 
of the nuclear factor-ΚΒ (NF- ΚΒ) pathway, resulting in decreased expression of numerous 
proinflammatory factors [80].  Nrf2-deficient mice demonstrate increased microglial activation 
and release of proinflammatory cytokines in response to LPS challenge [87].  When treated with 
SF, mice exposed to LPS show attenuated neuroinflammation, as evidenced by reduction in 
microglial production of the proinflammatory cytokines inducible nitric oxide synthase (iNOS), IL-
6, and TNF-α [87]. 
 
Common anecdotal reports and observational studies suggest that some individuals with ASD 
experience a substantial but temporary improvement in autistic symptoms (primarily stereotypic 
behavior and in appropriate speech) during fever [25], unrelated to the severity of fever or autism.  
Though the mechanism of this “fever effect” is not entirely clear, direct thermal effects as well as 
indirect effects, including increased expression of heat shock proteins (HSPs) and activation of 
cellular stress responses, may be involved [85, 88].  HSPs have a cytoprotective effect during 

Clinical Protocol: Sulforaphane for ASD 
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thermal stress, functioning as molecular chaperones for misfolded proteins and playing a role in 
cellular repair [88].  In addition, HSPs are central to synaptic transmission and may improve long-
range cerebral cortical connectivity that is depressed in ASD [80, 89].  SF has extensive effects on 
HSPs [85, 90], including induction of the HSP heme oxygenase 1 (HO-1, or Hsp32) [91], activation 
of heat-shock transcription factor 1 (HSF1), and upregulation of Hsp27, which increases 
proteasomal activity [85]. SF may therefore ameliorate features of autism, in part, by inducing 
expression of cytoprotective HSPs [80]. 
 
Finally, dysfunctional synaptic transmission caused by aberrant protein synthesis is believed to 
play a key pathogenic role in ASD [24], potentially due to overactivation of the phosphoinositide-3 
kinase/protein kinase-B/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathway 
[31, 92].  Mutations in genes upstream of mTOR (e.g., phosphatase and tensin homolog (PTEN), 
tuberous sclerosis complex (TSC1/2)) lead to overactivity of the mTOR pathway, resulting in 
excessive protein synthesis, abnormal synaptic function, and ASD phenotypes [80].  Synaptic 
plasticity requires a balance between protein synthesis and degradation [93], and deficits in 
autophagy, a major protein degradation pathway downstream of mTOR signaling, are also 
associated with overactivation of the mTOR pathway and ASD dendritic spine pathology [31].  SF 
has been shown to downregulate mTOR signaling and associated excessive protein synthesis in 
breast cancer [94], cardiomyocyte [38], and prostate cancer cell lines [39], as well as induce 
autophagy in neuronal cells and the brains of mouse models for Huntington’s disease [95, 96].  
mTOR signaling and protein synthesis are also inhibited in acute lymphoblastic leukemia cells 
treated with SF [97].  Taken together, these findings suggest that SF modulates aberrant mTOR 
signaling activity and autophagy, which may have a therapeutic effect for ASD. 
 
 
Clinical Data to Date 
 
Sulforaphane (SF), derived from freeze-dried broccoli sprouts, was recently investigated as a 
treatment for symptoms of ASD in a randomized, double-blind, placebo-controlled trial of 45 male 
adolescents and young adults (ages 13-30 years old) with moderate to severe autism (clinical 
trials identifier NCT01474993) [48].  Fifteen participants were randomized to placebo and 29 to 
SF; each group was treated for 18 weeks, followed by a 4-week discontinuation period and final 
follow-up at 22 weeks.  Forty participants (14 on placebo, 26 on SF) completed the trial; of those 
participants, 32 (80%) had a reported history of behavioral improvement during fever episodes.  
The primary outcome was change in symptoms of autism as measured by change in Social 
Responsiveness Scale (SRS) total scores; additional measures of interest included change in 
caregiver-completed Aberrant Behavior Checklist (ABC) scores and clinician-rated Autism Clinical 
Impressions Severity and Improvement Scales (OACIS-Severity and OACIS-Improvement).  SRS 
scores improved significantly more in the SF group compared with the placebo group (-15 units vs 
-3 units; p=0.02).  Considering the 4 non-completers as non-responders, 31% (9/29) SF 
participants vs 0% placebo participants demonstrated a 25% decrease in total SRS score from 
baseline (p=0.018).  SF participants also demonstrated significantly greater improvement in ABC 
total and OACIS-Improvement scores, particularly in the domains of social interaction (p=0.003), 
aberrant/ abnormal behavior (p=0.007), repetitive/ stereotypical behavior (p=0.08), and verbal 
communication (p=0.008).  SF was well-tolerated with no serious adverse events.  Two subjects 

Clinical Protocol: Sulforaphane for ASD 
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with a history of seizures had single unprovoked seizures during the course of the study (one after 
3 weeks on SF, one 3 weeks after discontinuing SF), and the possibility of seizure as potentially 
associated with SF could not be ruled out. 
 
Based on available data from Phase I human clinical trials of SF-rich compounds, SF appears to be 
extremely well-tolerated with minimal risk aside from mild gastrointestinal symptoms, which may 
include bloating, nausea, and loose stools. 
 
A Phase I study of orally administered SF- and GR-containing BSE (75 µmol glucosinolates, 300 
µmol glucosinolates, or 75 µmol isothiocyanates daily) for 7 days in 12 healthy volunteers did not 
demonstrate any clinical adverse events [51].  Two of 12 subjects assigned to active treatment 
demonstrated an increase in plasma ALT exceeding the upper limit of normal, though ALT levels 
rose for all subjects during the course of the study (including those assigned to placebo control), 
and the increase was not clearly attributable to consumption of BSE.  Three of 12 subjects (2 
active treatment, 1 placebo) demonstrated TSH levels exceeding the upper limit of normal during 
or after the dosing period, though elevated TSH was not associated with clinical symptoms or 
T3/T4 abnormalities.   
 
Egner et al. [46] conducted a cross-over trial in 50 participants, randomized to ingest a SF-rich 
beverage (150 µmol/day) for 7 days in a first or second phase.  Two of 50 participants complained 
of bitter taste or nausea, and no other adverse events were reported.  No abnormal serum 
chemistry values were reported after 7 days.  In a study of healthy participants ingesting a 
beverage containing 600 µmol of glucoraphanin and 40 µmol of sulforaphane, the intervention 
was well-tolerated with no laboratory evidence of effect on liver and kidney function [43]. 
 
A study of orally administered broccoli sprout homogenates (25 to 300 g) in 57 subjects over 3 
separate days found no adverse effects aside from mild digestive effects [98].  Similarly, in a RCT 
of 20 men with recurrent prostate cancer treated with sulforaphane 200 µmol/ day for 20 weeks, 
no serious adverse events occurred, though some reported mild gastrointestinal upset [40]. 
 
A randomized, placebo-controlled trial of the sulforaphane product Prostaphane® in 81 men with 
rising prostate specific antigen (PSA) values following radical prostatectomy for prostate cancer 
represents the highest daily dosing of sulforaphane to date (338 µmol) [99].  No serious adverse 
events were reported, though gastrointestinal bloating was experienced somewhat more often in 
the SF group (N=17 vs. N=10), and one participant in the SF group withdrew from the study due to 
bowel discomfort.  The differences in adverse effects between the two groups were not 
statistically significant (p=0.149).  Furthermore, no significant between-group differences were 
found for body weight, BMI, blood counts, and chemistries. 
1.4 Dose Rationale and Risk/Benefits 
The Avmacol® dosing regimen for the current study was designed to deliver a slightly higher 
concentration of SF as that administered in a previously reported clinical trial of SF for ASD [48].  
In the randomized, placebo-controlled trial by Singh et. al [48], participants randomly assigned to 
the active treatment group received capsules containing SF-rich broccoli sprout extracts that 
provided approximately 50 µmol SF/cap.  Dosing was weight-based as follows: 

Clinical Protocol: Sulforaphane for ASD 
- 16 - 
 
 
 
50 µmol SF (1 cap) daily for weight < 100 
 
100 µmol SF (2 caps) daily for weight 101-199 lb.  
 
150 µmol SF (3 caps) daily for weight ≥ 200 lb.   
 
Using this dosing schedule, participants receiving SF demonstrated significant improvement in 
social impairment as measured by change in Social Responsiveness Scale (SRS) total scores and 
significant improvement in related challenging behaviors as measured by the Aberrant Behavior 
Checklist (ABC) as compared with placebo over the course of the 16-week active treatment period.  
Between group differences were first observed by week 4 of treatment.  These doses of SF were 
well-tolerated by participants and were not significantly associated with adverse events, change in 
blood pressure or heart rate, or change in routine laboratory values.  Unexpectedly, SF treatment 
was associated with minor but significant weight gain, and 2 participants with a history of 
seizures had single, unprovoked seizures (one taking SF, one taking placebo). 
 
The currently proposed clinical trial is designed to independently replicate the study by Singh and 
colleagues [48], to support or refute these findings.  Avmacol® delivers SF in the form of its 
glucosinolate precursor, glucoraphanin (GR; derived by broccoli seed extract), and the active 
enzyme myrosinase (derived from broccoli sprouts), which hydrolyzes GR to SF upon ingestion.  
Bioavailability studies have demonstrated approximately 40-50% endogenous conversion of GR to 
SF upon ingestion of compounds containing GR enriched with myrosinase in healthy participants 
[47].  Each tablet of Avmacol®, formulated for use in clinical trials, contains approximately 15 mg 
(34.4 µmol) of glucoraphanin.  Assuming 45% endogenous conversion of GR to SF (which will vary 
somewhat from individual to individual), each tablet will therefore supply approximately 15.5 
µmol SF/tablet. The weight-based Avmacol®/ placebo dosing schedule for the current study is as 
follows: 
 
 
3 tablets (approx. 46.5 µmol SF) if <100 lb 
 
5 tablets (approx. 77.5 µmol SF) if 100-125 lb 
 
6 tablets (approx. 93 µmol SF) if 126-175 lb 
 
7 tablets (approx. 108.5 µmol SF) if 176-199 lb 
 
8 tablets (approx. 124 µmol SF) if ≥ 200 lb 
 
Based on previous studies of SF in human subjects, this dosing regimen is expected to be very 
well-tolerated with minimal to no adverse effects.  This dosing regimen is also expected to deliver 
a slightly higher weight-based dose of SF than the trial conducted by Singh et al [48] after 
accounting for the 70% bioconversion rate (approximate) when purified SF is administered orally 
(i.e., an oral dose of 50 µg SF derived from freeze-dried BSE is expected to provide 35 µg SF 
following ingestion and bioconversion).   
 
If a participant experiences significant adverse events that affect tolerability, may reasonably be 
related to taking the investigational product, and do not meet the protocol definition for a serious 
AE, the dose may be reduced by approximately 30% according to the following dose modification 
schedule: 
 
 

Clinical Protocol: Sulforaphane for ASD 
- 17 - 
 
Weight 
category 
Prescribed weight-
based dose of 
glucoraphanin 
Prescribed # 
of tablets 
70% of 
weight-based 
dose (ie, 30% 
reduction) 
Prescribed # 
tablets for 
reduced dose 
(rounded to 
nearest 15 mg 
increment)* 
<100 lb 
45 mg 
3 
31.5 mg 
2 
100-125 lb 
75 mg 
5 
52.5 mg 
3 
126-175 lb 
90 mg 
6 
63 mg 
4 
176-199 lb 
105 mg 
7 
73.5 mg 
5 
≥ 200 lb 
120 mg 
8 
84 mg 
6 
 
*Rounded down if equidistant between two 15 mg dosing increments 
 
If the dose is reduced during the course of the study, the participant will continue the lowered 
dose for the remainder of the study.  If adverse events do not improve within 14 days of lowering 
the dose, the investigational product may be permanently discontinued at the discretion of the 
treating study physician, participant, and participant’s guardian. If a participant exhibits extreme 
exacerbation of behavioral symptoms (such as self-injurious behavior or aggression) that do not 
improve substantially following a dose reduction and are determined “probably” or “definitely” 
related to taking the investigational product, the investigational product may be permanently 
discontinued sooner than 14 days post-reduction, and the DSMB will be notified.  As individuals 
with ASD often have cyclical changes in behavior independent of medication effects, the study staff 
will rely on caregivers’ experience with the participant to determine if any observed behavior 
changes are within the expected range for that individual.  If the investigational product is stopped 
permanently, the participant and his caregiver will be asked to complete the remainder of the 
study visits, return any unused study drug, and repeat safety lab studies. 
2 
Study Objectives 
 
Primary Objective:  
The primary objective of this clinical trial is to determine if Avmacol® is superior to placebo for 
improving social communication impairment in adolescent and young adult men with moderate to 
severe autism spectrum disorder (ASD), as measured by change in Social Responsiveness Scale-2 
(SRS-2) [100] total scores. 
 
Secondary Objectives: 
Secondary objectives include: (1) assessing the safety and tolerability of Avmacol® in subjects 
with ASD; and (2) characterizing the effects of Avmacol® on behavioral challenges commonly 
associated with ASD, including hyperactivity, irritability, repetitive behavior, and inappropriate 
speech. 
 
 
 
 

Clinical Protocol: Sulforaphane for ASD 
- 18 - 
 
Exploratory Objectives: 
In a subset of 30 participants, exploratory objectives include: (1) Assessment of gut micobiota 
diversity and relative abundance of microbial species from fecal samples collected before and 
after 12 weeks of treatment with sulforaphane or placebo. (2) Explore the relationship 
between changes in microbiota composition and behavioral outcomes in young men treated with 
sulforaphane or placebo. 
3 
Study Design 
3.1 General Design 
 
We propose to conduct a Phase II, randomized, double-blind, placebo-controlled trial of Avmacol®, 
a rich source of sulforaphane, for the treatment of social impairment in individuals with ASD.  
Fifty-four male subjects with autism spectrum disorder (ASD) between ages 13 and 30 years old 
will be randomized in a 1:1 ratio, stratified by age category (13-18 and 19-30 years old), to receive 
either Avmacol® or identical placebo tablets.  Participation will be limited to males to closely 
approximate the sample used in the SF for ASD clinical trial by Singh et al. [48] and to account for 
possible mechanistic differences in ASD between males and females (ASD is 4 times more common 
in males). Stratification by age will account for possible age-based differences in response to 
treatment (adolescence vs. adulthood) and allow for secondary analysis by age group (adolescent 
vs. adult).   
 
The length of study participation from baseline to study termination for each individual subject is 
16 weeks, including a 12-week active treatment phase (Avmacol® or placebo), followed by a 4- 
week blinded discontinuation phase.  Participants will be monitored with clinic visits at regular 
intervals (baseline and weeks 4, 8, 12, and 16) for improvement in a well-characterized, caregiver-
rated measure of social impairment (Social Responsiveness Scale-2 (SRS-2) [100], the primary 
outcome) as well as secondary measures of (1) repetitive behaviors associated with ASD 
(Repetitive Behavior Scale- Revised; RBSR) [102]; (2) challenging behaviors commonly associated 
with developmental disabilities (Aberrant Behavior Checklist; ABC) [103]; (3) clinician-rated 
functional severity (Clinical Global Impression Scale- Severity; CGI-S) [104]; and clinician-rated 
functional improvement (Clinical Global Impression Scale- Improvement; CGI-I) [104]. 
 
Vital signs and reports of adverse events will be monitored at each study visit (Baseline, Week 4, 
Week 8, Week 12, Week 16) for tolerability and safety monitoring.  A phone call with the subject’s 
caregiver at Week 2 of the treatment phase will also be used to monitor for early adverse events.  
Physical exam and phlebotomy for routine laboratories (Complete Blood Count (CBC); 
Comprehensive Metabolic Panel (CMP); Liver Function Tests (LFTs), and Thyroid Stimulating 
Hormone (TSH)) will be conducted at Baseline and Week 12 for the purposes of safety monitoring.  
Fecal samples will be collected at home in the 48 hr prior to Baseline and Week 12 visits for 
microbiota analyses. 
 
 
 
 

Clinical Protocol: Sulforaphane for ASD 
- 19 - 
 
Schematic Diagram of Study Events: 
 
  N=54 
 
 
 
 
BOM1 
 
BOM1 
 
AE Review2 
BOM1 
 BOM1 
BOM1 
 
Confirm ASD 
Randomization 
  
AE Review2 
 AE Review2 
AE Review2 
Cognitive screen        AE Review2 
Blood chem3  
Blood chem3                Saliva collection             
 
 
Hematology 
 
 
Hematology 
Stool collection 
  
 
 
Physical Exam 
Physical exam 
 
 
  
 
 
Stool collection 
 
1 Behavioral outcome measures (SRS-2, ABC, CGI, RBSR) 
2 Adverse Events Review 
3 Blood chemistries (Comprehensive Metabolic Panel, Liver Function Tests, Thyroid Stimulating Hormone) 
 
Primary Study Endpoints 
 
The primary study endpoint will be percent change in mean SRS-2 total scores from Baseline to 
Week 12, the end of the active treatment phase. 
 
Secondary Study Endpoints 
 
 Percent change in mean SRS-2 total scores from Week 12 (the end of the active treatment 
phase) to Week 16 (the end of the discontinuation phase) 
 Percent change in mean SRS-2 subscale scores (Social Awareness, Social Cognition, Social 
Communication, Social Motivation, and Restricted Interests/ Repetitive Behaviors) from 
Baseline to Week 12 and from Week 12 (the end of the active treatment phase) to Week 16 
(the end of the discontinuation phase) 
 Change in mean CGI-Severity score from Baseline to Week 12 and from Week 12 (the end 
of the active treatment phase) to Week 16 (the end of the discontinuation phase) 
 Proportion of participants with CGI-Improvement (CGI-I) scores of 1 (very much 
improved) or 2 (much improved) in each treatment arm at Weeks 12 and Week 16 
 Percent change in mean RBSR total scores from Baseline to Week 12 and from Week 12 
(the end of the active treatment phase) to Week 16 (the end of the discontinuation phase). 
 Percent change in mean ABC subscale scores (Social Withdrawal, Hyperactivity, 
Inappropriate Speech, Stereotypy, and Irritability) from Baseline to Week 12 and from 
Week 12 (the end of the active treatment phase) to Week 16 (the end of the 
discontinuation phase) 
 Proportion of participants in each treatment arm meeting responder status, as defined by 
10 point or greater reduction in SRS-2 total score AND CGI-I score of 1 (very much 
improved) or 2 (much improved). 
 Proportion of participants in each group meeting partial responder status, as defined by 10 
point or greater reduction in SRS-2 total score AND CGI-I score of 3 (minimally improved). 
Screening 
Week 0: 
Baseline 
Week 2: 
Phone Call 
Week 4: 
Visit #1 
Week 8: 
Visit #2 
Week 12: 
Visit #3 
Week 16: 
Termination 
Visit 
Daily Treatment with Avmacol® or Placebo 
BOM1 
AE Review2 

Clinical Protocol: Sulforaphane for ASD 
- 20 - 
 
 
3.2 Primary Safety Endpoints 
 
 CBC with differential, actual values and percent change in values from Screening visit to 
Week 12 visit. 
 CMP (Na, K, Cl, CO2, BUN, Cr, Ca, Mg, Ph, glucose), actual values and percent change in 
values from Screening visit to Week 12 visit. 
 LFTs (Total bilirubin, AST, ALT), actual values and percent change in values from Screening 
visit to Week 12 visit. 
 TSH, actual value and percent change in value from Screening visit to Week 12 visit. 
 Vital signs (weight, height, blood pressure, and heart rate), actual values at each time point 
measured and percent change in values from Baseline to Week 12 visit (the end of the 
active treatment phase) 
 Physical Examination, general assessment of health at Screening visit and at Week 12 visit 
(the end of the active treatment phase) 
 Mean (%) relative abundance of microbial phyla and genera in fecal samples at Baseline 
and Week 12. 
 Percent change in Bacteroidetes to Firmicutes (B:F) ratio in fecal samples from Baseline to 
Week 12. 
 Change in alpha diversity ("within-sample") metrics (the absolute number of operational 
taxonomic units (OTUs), Chao1 estimator, and Shannon index) in microbiota from Baseline 
to Week 12. 
 Change in beta diversity ("between-sample") metrics (Bray-Curtis dissimilarity, Weighted 
Unifrac distance, and Unweighted Unifrac distance) in microbiota from Baseline to Week 
12. 
 
4 
Subject Selection and Withdrawal 
4.1 Inclusion Criteria 
 Males between ages 13-30 (inclusive) at the time of the consent 
 Subject is capable of giving written informed consent or has a legally authorized 
representative (LAR) with sufficient intellectual capacity to provide written informed 
consent on the patient’s behalf.  For subjects that do not have sufficient capacity to provide 
informed consent, written informed assent will be obtained when developmentally 
appropriate. 
 Subject has a reliable informant (parent or caregiver) who has sufficient past and current 
knowledge of the subject and will oversee the administration of study medication and 
accompany the subject to each study visit. 
 Subject and caregiver have reliable means of transportation to attend study visits. 
 Primary diagnosis of Autism Spectrum Disorder (ASD) severity level 2 or 3, confirmed by 
Diagnostic and Statistical Manual-5 (DSM-5) criteria (as assessed by the PI, a physician 
experienced in the diagnosis of ASD) and meeting the autism cut-off score of 8 or greater 
on the Autism Diagnostic Observation Schedule-2 (ADOS-2) (conducted by a master’s level 

Clinical Protocol: Sulforaphane for ASD 
- 21 - 
 
sub-investigator with extensive experience in administration of the ADOS-2) at the time 
screening 
4.2 Exclusion Criteria 
 Caregiver is unable to proficiently speak and understand English 
 The subject is unable to ingest the study tablets orally, either by swallowing whole or 
crushing and mixing in a small amount of a soft food (e.g., applesauce, yogurt). 
 Chronic medical illness that is not stable, would pose a risk to the participant if he 
participates in the trial, or would otherwise confound results (e.g. autoimmune disorders). 
 A history of seizure within the 12 months preceding study enrollment 
 Changes to psychopharmacological medications (e.g., stimulants, antidepressants, 
anxiolytics, antipsychotics) in the 2 weeks preceding study enrollment 
 Significant changes to non-pharmacological treatments in the 4 weeks preceding study 
enrollment (e.g., starting a new behavioral therapy program) 
 Chronic treatment with anti-inflammatory agents (e.g., ibuprofen, NSAIDs, corticosteroids)  
 Clinically significant laboratory abnormalities at Screening visit (e.g., AST/ALT> two times 
the upper normal limits; serum creatinine > 1.2 mg/dl, TSH outside normal limits) 
 Clinically significant findings on physical examination that investigator determines could 
increase risk of harm from participating in the study 
 Participated in another clinical interventional trial or received an investigational product in 
the 30 days preceding study enrollment 
 Previous therapeutic trial of sulforaphane or participation in a clinical trial in which 
sulforaphane was the investigational agent 
 
4.3 Subject Recruitment and Screening 
 
Subjects will primarily be recruited from the Carolina Institute for Developmental Disabilities 
(CIDD) Research Subject Registry, which includes more than 6000 families that have expressed an 
interest in research participation and have consented to be contacted about potential studies.  
Potential subjects will be contacted directly via email or letter by the Registry staff with 
information about the trial, including contact information of study staff.  Participants will also be 
recruited from the clinical practices of physicians, psychologists, and allied health staff at the 
Carolina Institute for Developmental Disabilities as well as from local referring providers.  
Electronic and written advertisements will be disseminated to local community autism support 
agencies (e.g., Autism Society of North Carolina, Autism Resource Centers).  All recruitment 
materials will first be approved by the University of North Carolina IRB. 
 
Potential subjects expressing an interest in study participation will go through an initial telephone 
screening process to assess eligibility for study participation (e.g., previous autism diagnosis, 
male, ages 13-30, able to be accompanied by a caregiver who can serve as a primary informant, 
not taking chronic anti-inflammatory medication, free of major medical illness, no clinical seizures 
in the previous 12 months).  Before the initial clinic screening visit, the caregiver and subject will 
be sent a copy of the informed consent form for review.   
 

Clinical Protocol: Sulforaphane for ASD 
- 22 - 
 
Written informed consent by a caregiver will be obtained before proceeding with all screening 
assessments.  Whenever possible, informed assent will also be obtained from the participant.  The 
study will pilot a tablet-based electronic decisional capacity tool developed at Research Triangle 
Institute (RTI) International to enhance participation in the assent process for individuals with 
developmental disabilities.  Prior to study enrollment, participants will undergo diagnostic 
confirmation of autism spectrum disorder at the screening visit by review of DSM-5 Autism 
Spectrum Disorder (ASD) criteria (by the study PI) and by Autism Diagnostic Observation Schedule-
2 administration (by a master’s level clinician experienced in ADOS-2 administration for research 
purposes).  Screening requirements will also include routine laboratory testing (CBC, electrolytes, 
LFTs, TSH), medical history, and physical examination to identify any exclusionary factors.  
Screening laboratory values and physical exam findings must be within normal limits or deemed 
not clinically significant by the study physician prior to study enrollment.  Subjects with serious 
medical illnesses that are poorly controlled, that may increase risks associated with study 
participation, or that may otherwise confound results will not be enrolled.   
4.4 Early Withdrawal of Subjects 
4.4.1 When and How to Withdraw Subjects 
 
Subjects will be withdrawn from study participation only if the subject or his legal authorized 
representative revokes consent for inclusion of subject data in group analyses. 
 
Reasons for treatment withdrawal prior to the expected completion for a subject may include: 
 
 The subject experiences a serious adverse event (SAE) that the study physician determines 
is likely related to the study treatment or that presents a safety risk for ongoing 
participation. 
 The subject experiences a clinical seizure during the course of the active treatment phase, 
either by EEG confirmation or by strongly suggestive clinical history. 
 The subject requires significant dose adjustments to concomitant psychopharmacological 
medications (e.g. stimulants, antidepressants, mood stabilizing agents, benzodiazepines) or 
starts a new psychopharmacological medication due to significantly worsened behavioral 
disturbance.   
 The subject requires a new chronic medication that may potentially interact with the study 
medication. 
 
No withdrawal symptoms or adverse effects are anticipated from abrupt discontinuation of the 
study medication.  However, if a patient needs to discontinue the study treatment for one of the 
aforementioned reasons, every effort will be made to bring the subject in promptly for a clinic visit 
to perform safety measures, including physical examination and laboratory measures.  As 
described below, study personnel will request that subjects who prematurely stop treatment 
complete the remaining study visits and procedures as outlined in the protocol. 
4.4.2 Data Collection and Follow-up for Withdrawn Subjects 
 

Clinical Protocol: Sulforaphane for ASD 
- 23 - 
 
If a subject withdraws from treatment prematurely, every effort will be made to obtain the 
subject/ caregiver’s permission to collect data through the end of the planned subject follow-up 
period (week 16 visit).  The data collected will be included in the final data set under intention-to-
treat analysis, provided the participant took at least 1 dose of study medication and attended at 
least 1 post-baseline assessment.    
 
If a subject appears lost to follow-up, study personnel will attempt to reach the subject’s caregiver 
by all provided telephone numbers and email addresses on two occasions each.  If the caregiver 
cannot be reached by phone or email, a certified letter will be sent requesting that the caregiver 
contact the study personnel to further discuss the subject’s involvement in the trial.  A second 
certified letter will be sent two weeks after the first letter was sent if the subject’s caregiver has 
not responded within those two weeks.  If the caregiver does not respond to the second letter, the 
subject will be considered lost to follow-up.  All attempts to contact the subject’s caregiver will be 
documented in a communication log. 
5 
Study Drug  
5.1 Description 
The investigational agent is supplied as a white, flecked, uncoated tablet.  Each active tablet will 
contain 125 mg broccoli seed extract powder (equivalent to approximately 36 µmol GR), 50 mg 
broccoli sprout extract powder (source of myrosinase), 15 mg ascorbic acid, 7.5 mg 
carboxymethylcellulose, 6.5 mg silicon dioxide, and 171 mg microcrystalline cellulose.  The 
placebo tablets will be similar in shape, size, and color. 
5.2 Treatment Regimen 
Participants will take 3-8 Avmacol® or placebo tablets once daily by mouth for a total of 12 weeks, 
with dosing regimen based on weight as follows: 
 
3 tablets (approx. 46.5 µmol SF) if <100 lb 
5 tablets (approx. 77.5 µmol SF) if 100-125 lb 
6 tablets (approx. 93 µmol SF) if 126-175 lb 
7 tablets (approx. 108.5 µmol SF) if 176-199 lb 
8 tablets (approx. 124 µmol SF) if ≥ 200 lb 
 
The tablets may be crushed and mixed with a soft food, such as applesauce, if needed.  Participants 
will be instructed to avoid taking the tablets with a heavy meal. 
 
5.3 Method for Assigning Subjects to Treatment Groups 
 
Enrolled participants will be stratified for age (13-18 and 19-30 years) to account for possible 
effects of age on treatment response.  The goal is to recruit twenty-seven 13-18 year-olds and 
twenty-seven 19-30 year-olds to allow for separate analysis of adolescents and adults 
(exploratory).  Individuals will be randomly assigned to treatment group within age strata by 
generating random numbers for each group for each entry (i.e., 1-27 for younger group and 1-27 

Clinical Protocol: Sulforaphane for ASD 
- 24 - 
 
for older group).  The larger half of the random numbers within each group will be assigned to 
receive the treatment and the smaller half to the placebo condition.  These two lists – listed in 
their original order (1-27 for younger and 1-27 for older) will be provided to UNC Investigational 
Drug Services (IDS) pharmacists.  They will consult the list and provide the appropriate 
medication to each individual depending on the order in which they present to the pharmacy 
within their age group.  Study personnel will remain blinded to treatment condition for the 
duration of the study. 
5.4 Preparation and Administration of Study Drug 
 
Avmacol® and placebo tablets will be shipped in bulk to UNC IDS (984-974-0469) in 500 count 
bottles, which will be stored at 2-8⁰C in the refrigerator until needed.  Prior to a participant’s 
study visit, a 5-week supply of the investigational drug (Avmacol® or placebo) will be packaged in 
a smaller, labeled bottle to be picked up by study staff.  The label will not indicate whether the 
bottles contain Avmacol® or placebo tablets.  The study personnel will verify the number of tablets 
dispensed and record on the drug accountability form.  The bottle of investigational drug will be 
stored in a small refrigerator used exclusively for this purpose at the study site until the 
designated visit.  The investigational drug will be dispensed to the subject’s caregiver at the time 
of the study visit (Baseline, Week 4, and Week 8 visits) in a freezer bag for transport with 
instructions to store the bottle in a home refrigerator.  Subjects and caregivers will be advised to 
avoid taking the tablets with a heavy meal and to take by mouth at approximately the same time 
every day.  If a subject is unable to swallow pills, the tablets may be crushed with a pill crusher or 
mortar and pestle and mixed with a soft food, such as applesauce.  Of note, investigators at the 
Cullman Chemoprotection Center and others have successfully administered sulforaphane and its 
precursor, glucoraphanin, in a number of different formulations, both encapsulated and in various 
types of juices [43, 44, 46, 47, 105]. 
5.5 Subject Compliance Monitoring 
Subjects’ caregivers will be provided with a medication log to track daily dosing of the study 
medication.  At Week 4, Week 8, and Week 12 visits, subjects and their caregivers will be 
instructed to return the medication log and any unused tablets to study staff, who will count the 
remaining tablets and address any discrepancies between remaining tablets and the medication 
log with the families.  Compliance percentages will be recorded on a drug compliance form.  For 
frequent missed doses, the study physician will discuss the importance of adherence to the 
recommended dosing regimen and will identify any potential barriers to compliance, providing 
support when possible.   
5.6 Prior and Concomitant Therapy 
Participants will be permitted to continue their usual regimen of medications, psychotropic and 
otherwise, during the study with the exception of chronic anti-inflammatory medications (e.g., 
ibuprofen, naproxen, corticosteroids), which may confound the treatment effects.  Concomitant 
medication doses must be stable for 4 weeks prior to study enrollment and remain constant for 
the duration of the study, to the extent possible.   

Clinical Protocol: Sulforaphane for ASD 
- 25 - 
 
5.7 Packaging 
Avmacol® will be shipped in bulk from Nutramax Laboratories, Inc. (2208 Lakeside Blvd, 
Edgewood, MD 21040) to UNC IDS in boxes of bottles containing 500 tablets of Avmacol© each.  
The bottle labels will include: product name (Avmacol®), manufacturer (Nutramax Laboratories, 
Inc.), Quantity, Lot Number, Expiration Date, Study Name (Avmacol® for autism spectrum 
disorder), Institution (UNC), PI (Politte), IRB number, “Investigational Study Material,” and 
“Caution: New Drug Limited by Federal Law to Investigational Use Only.”  For an example of 
labelling, please see section 7.3.7 of the IND application. 
 
Matched placebo tablets will be manufactured and shipped from the University of Maryland 
Department of Pharmaceutical Sciences (20 N. Pine St., Baltimore, MD 21201) to UNC IDS in boxes 
of HDPE plastic bottles containing 200 tablets each.  The bottles will be individually labeled as 
described above.   
5.8 Blinding of Study Drug (if applicable) 
Subjects will be randomized by IDS pharmacy personnel using a random numbers list, and 
treatment assignment will not be disclosed to study staff.  Avmacol® and placebo tablets will be 
similar in shape, size, and color, and the labels on bottles dispensed to study staff and families will 
not indicate treatment assignment.  Blinding of treatment assignment for the study team and 
subjects will be maintained until after the conclusion of all data collection.  In the event of a 
serious adverse event, a physician monitor who is not directly associated with the study (ie, an 
independent medical monitor) will determine if the SAE is potentially related to taking the study 
drug.  If a possible association is suspected, the pharmacy will disclose treatment status to the 
medical monitor, and the event will be reviewed by the medical monitor and Data Safety 
Monitoring Board (DSMB) to determine if treatment discontinuation is warranted.   
 
Receiving, Storage, Dispensing and Return 
5.8.1 Receipt of Drug Supplies 
Avmacol® will be shipped in bulk from Nutramax Laboratories, Inc. (2208 Lakeside Blvd, 
Edgewood, MD 21040) to UNC IDS (IDS Pharmacy, Room 3122N, UNC Hospitals, 101 Manning 
Drive, Chapel Hill, NC 27514) in boxes of bottles containing 500 tablets of Avmacol® each.  
Matched placebo tablets will be shipped in bulk from the University of Maryland Department of 
Pharamceutical Sciences (20 N. Pine St., Baltimore, MD 21201) to UNC IDS in boxes of bottles 
containing 200 placebo tablets each. 
 
Upon receipt of the investigational drug/ placebo, an inventory will be performed and a drug 
receipt log filled out and signed by the IDS personnel accepting the shipment. IDS staff will count 
and verify that the shipment contains all of the items noted in the shipment inventory.  Any 
damaged or unusable study drug in a given shipment (active drug or placebo) will be documented 
in the study files.  The PI will notify the manufacturer of any damaged or unusable study drug that 
was supplied to the study site. 

Clinical Protocol: Sulforaphane for ASD 
- 26 - 
 
5.8.2 Storage 
The investigational drug (Avmacol®) and placebo tablets will be stored at 2-8⁰C in the refrigerator  
in UNC Investigational Drug Services (IDS) pharmacy prior to being dispensed to study staff in the 
week prior to the designated study visit. The study drug will be stored on site at the CIDD in a 
refrigerator used expressly for study drug storage until the study visit, at which time it will be 
dispensed to the participant’s caregiver by the study team.  Caregivers will be asked to store the 
bottle of study drug in a home refrigerator as a precautionary measure to preserve the integrity of 
the compound, though the manufacturer of Avmacol® reports stability at room temperature.   
5.8.3 Dispensing of Study Drug 
Study drug will be dispensed by UNC IDS to study staff in labeled bottles containing a 5-week 
supply (4 week supply plus 1 week buffer) of active drug or placebo tablets prior to baseline, week 
4, and week 8 study visits.  The study drug will be stored on site at the CIDD in a refrigerator used 
expressly for study drug storage until the study visit, at which time it will be dispensed to the 
participant’s caregiver by the study team.  Caregivers will be asked to transport the investigational 
drug in a freezer bag and store the bottle of study drug in a home refrigerator as a precautionary 
measure to preserve the integrity of the compound, though the manufacturer of Avmacol® reports 
stability at room temperature.  Caregivers will be asked to record each administered dose on a 
medication log and to return the medication log and any unused study drug to study staff at the 
next visit.  Study drug reconciliation will be performed at week 4, week 8, and week 12 study visits 
to document drug assigned, drug consumed, and drug remaining.  This reconciliation will be 
logged on the drug accountability form, and signed and dated by the study personnel and study 
physician.   
 
Return or Destruction of Study Drug 
 
At the completion of the study, there will be a final reconciliation of drug shipped, drug consumed, 
and drug remaining.  This reconciliation will be logged on the drug reconciliation form, signed and 
dated.  Any discrepancies noted will be investigated, resolved, and documented prior to 
destruction of unused study drug by IDS.  Drug destroyed on site will be documented in the study 
files. 
6 
Study Procedures 
 
6.1 Pre-Screen Phone Call: 
Prior to screening, potential participants will undergo a pre-screening assessment by telephone or 
direct clinical interview with study staff to determine if basic eligibility criteria are met.  A written 
copy of the informed consent document will be provided by mail, electronically, or in person for 
the participant’s caregiver to read prior to the screening visit.   
 
6.2 Screening Visit: 
The screening visit will take approximately 3 hours to complete.  At screening, we will first obtain 
written informed consent from participants or their Legally Authorized Representatives (LAR).  
We anticipate that most subjects will not have capacity to provide informed consent themselves 
due to cognitive and/or communication deficits associated with ASD, though every effort will be 

Clinical Protocol: Sulforaphane for ASD 
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made to increase their participation in the consent/ assent process.  For individuals under 18 
years of age or those unable to consent due to significant cognitive or communication impairment, 
written assent for participation will be sought.  An electronic, tablet-based interactive consent tool 
will be piloted to enhance participation in the consent process. 
 
After written informed consent is obtained, diagnosis of autism spectrum disorder (ASD) will be 
confirmed by two measures: clinical review of the Diagnostic and Statistical Manual-5 (DSM-5) [1] 
ASD criteria and Autism Diagnostic Observation Schedule-2 (ADOS-2) total score [106].  The 
ADOS-2 is a semi-structured assessment of communication, social interaction, and repetitive 
behavior that is widely regarded as the gold standard of ASD diagnosis [106].  The ADOS-2 will be 
performed by a trained master’s-level clinician, and DSM-5 diagnostic criteria for ASD will be 
assessed by a study physician with extensive experience in diagnosing ASD.  ADOS-2 
administration will be videotaped to permit clinician scoring after administration; the digital 
recording will be stored on a secure server than will be accessible only by authorized study 
personnel with a password.  Individuals must meet criteria for a diagnosis of ASD based on both 
DSM-5 criteria (severity level 2 or 3) and ADOS-2 total score (≥8, exceeding the “autism” cut-off) 
to be eligible for participation.  An abbreviated cognitive assessment using the Wechsler 
Abbreviated Intelligence Scales (WASI) two non-verbal subtests will be [107] obtained for the 
purposes of data analysis, though there is no cut-off intelligence quotient (IQ) score for study 
participation.   A medical history will be obtained by the study physician, including current 
medical conditions, past medical history, surgical history, psychiatric history, past and current 
psychotropic medications, allergies, and current non-pharmacological treatments for ASD (e.g., 
speech therapy, occupational therapy, behavioral therapy).  Caregivers will complete the Social 
Responsiveness Scale, Second Edition (SRS-2) [100], Repetitive Behavior Scale-Revised (RBSR) 
[102], and Aberrant Behavior Checklist- Community Edition (ABC) [103] for baseline 
characterization of social communication deficits, repetitive behaviors, and behavioral challenges.  
The study physician will complete the Clinical Global Impression Severity Scale (CGI-S) [104], a 7-
point rating scale of overall symptom severity.  Baseline bloodwork will include a comprehensive 
metabolic profile (CMP), liver function tests (LFTs), thyroid stimulating hormone (TSH), and 
complete blood count (CBC) with white blood count differential.  Participants will be given an 
OMNIGene-Gut stabilization kit to take home and use in the collection of a stool sample in the 48 
hr prior to the scheduled Baseline visit.  The research team will instruct the participant and 
caregiver in the collection of the stool sample and ask them to return it at the Baseline visit. 
 
6.3 Baseline Visit (Week 0): 
The baseline visit will take approximately 1.5 hr to complete.  Prior to the baseline visit, the study 
physician will review safety labs drawn at the screening visit (CBC, CMP, LFTs, TSH) to ensure 
eligibility for participation.  For individuals who meet all inclusionary and exclusionary criteria as 
described in sections 4.1 and 4.2, a baseline visit will be scheduled within 2 weeks of screening.  
Clinical outcome measures will again be obtained from caregivers, including the SRS-2, ABC, and 
RBSR.  The study physician will complete the CGI-S to establish baseline clinical severity of ASD 
prior to intervention.  Vital signs (height, weight, blood pressure, heart rate, and temperature) will 
be obtained by study staff.  An adverse event log for baseline symptom assessment will be 
completed by the study physician.  With specific written informed consent, participants will 
provide a saliva sample (approximately 2.6 mL total), either through expectoration into a special 
collection tube or through cheek swab (approximately 5 swabs).  The de-identified saliva sample 

Clinical Protocol: Sulforaphane for ASD 
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will be stored in the UNC Biospecimen Repository for use in a possible future study of genetic 
markers associated with clinical response to sulforaphane.  Participation in the  storage of genetic 
material is not required for participation in the primary clinical trial.  Participants will also return 
the stool sample in the OMNIGene-Gut stabilization kit, which is stable at room temperature for up 
to 60 days.  The kit will be labeled with the date and participant PIN, securely packaged in a 
Styrofoam container, and mailed to the Cullman Chemoprotection Center at JHU (director, Dr. Jed 
Fahey) for storage in cryovials at -80⁰C until all study samples have been collected. 
 
Participants will be randomly assigned at baseline to receive either the active compound 
(Avmacol®) or identical placebo tablets.  UNC IDS will be provided with a random table of 
numbers for randomization of participants, and both study staff and participants will remain 
blinded to treatment assignment.  Study staff will retrieve the investigational product in labeled 
bottles from IDS and stored on site in a refrigerator at 2-8⁰C prior to the baseline visit.  A 5-week 
supply of investigational drug will be dispensed to the participant and their caregiver in a labeled 
bottle by study staff at the visit, and they will be instructed to transport the bottle home in a 
freezer bag (supplied by study staff) and store the bottle in their home refrigerator to ensure 
compound stability (a precautionary measure; the manufacturer of Avmacol® reports stability at 
room temperature).  They will be given detailed instructions for taking the medication and 
completing the medication log, which will be returned and reviewed at the participant’s next visit.  
Caregivers will also be instructed to return any remaining tablets to the study staff at the 
following visit to determine adherence to the dosing protocol. 
 
6.4 Week 2 Telephone Call: 
The week 2 telephone call is expected to take approximately 20 minutes.  The study physician will 
contact the subject’s caregiver 2 weeks after the baseline visit to assess tolerability and general 
response.  The adverse event log will be completed by the physician.  Caregivers will be asked to 
report any changes in concurrent medications or non-pharmacological treatments since the 
previous study visit. 
 
6.5 Week 4 Follow-Up Visit: 
The week 4 visit will take approximately 1 hour.  The subject and his caregiver will return to the 
clinic for a follow-up visit that will include measurement of vital signs (height, weight, blood 
pressure, heart rate, and temperature), caregiver completion of behavioral outcome measures 
(SRS-2, RBSR, and ABC), and clinician-rated CGI-Severity and CGI-Improvement scores.  The 
adverse event log will also be completed by the study physician.  Caregivers will be asked to 
report any changes in concurrent medications or non-pharmacological treatments since the 
previous study visit.  The medication log and returned tablets will be reviewed/ counted for 
treatment compliance, and the next 5-week supply of investigational drug will be dispensed to the 
participant and his caregiver.  Caregivers will be instructed to administer any unused tablets from 
the previous bottle before starting the new supply. 
 
6.6 Week 8 Follow-Up Visit: 
The week 8 visit will take approximately 1 hr. to complete.  The patient and his caregiver will 
return to the clinic for a follow-up visit that will include measurement of vital signs (height, 
weight, blood pressure, heart rate, and temperature), caregiver completion of behavioral outcome 
measures (SRS-2, RBSR, and ABC), and clinician-rated CGI-Severity and CGI-Improvement scores.  

Clinical Protocol: Sulforaphane for ASD 
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The adverse event log will also be completed by the study physician. Caregivers will be asked to 
report any changes in concurrent medications or non-pharmacological treatments since the 
previous study visit.   The medication log and returned tablets will be reviewed/ counted for 
treatment compliance, and the next 5-week supply of investigational drug will be dispensed to the 
participant and his caregiver.  Caregivers will be instructed to administer any unused tablets from 
the previous bottle before starting the new supply.  Participants will be given a new OMNIGene-
Gut stabilization kit for collection of a stool sample at home in the 48 hr prior to the scheduled 
Week 12 visit.  The research team will remind the participant and his caregiver of how to collect 
the sample and instruct to return it at the next visit. 
 
 
6.7 Week 12 Follow-Up Visit: 
The week 12 visit marks the end of the active treatment phase of the study and will take 
approximately 2 hr. to complete.  The patient and his caregiver will return to the clinic for a 
follow-up visit that will include measurement of vital signs (height, weight, blood pressure, heart 
rate, and temperature), physical exam by the study physician, and repeat blood draw for safety 
monitoring (CMP, LFTs, TSH, and CBC with differential).  Caregivers will complete behavioral 
outcome measures (SRS-2, RBSR, and ABC), and clinicians will assign CGI-Severity and CGI-
Improvement scores.  At this phase-ending visit, responder status will be assigned to those with a 
10 point or greater reduction on the SRS-2 and  CGI-Improvement score of 1 or 2 (very much or 
much improved).  Partial responder status will be assigned to participants with a 10 point or 
greater reduction on the SRS-2 and a CGI-Improvement score of 3 (minimally improved).  The 
adverse event log will also be completed by the study physician. Caregivers will be asked to report 
any changes in concurrent medications or non-pharmacological treatments since the previous 
study visit.    The medication log and returned tablets will be reviewed/ counted for treatment 
compliance, and any remaining investigational drug will be collected by study staff and returned 
to IDS for disposal.  Participants will also return a stool sample in the OMNIGene-Gut stabilization 
kit.  The kit will be labeled with the date and participant PIN, securely packaged in a Styrofoam 
container, and mailed to the Cullman Chemoprotection Center at JHU (director, Dr. Jed Fahey) for 
storage in cryovials at -80⁰C until all study samples have been collected. 
 
 
6.8 Week 16 Follow-Up Visit/ Study Termination: 
The week 16 visit will take approximately 1 hr. to complete.  Participants and their caregivers will 
return for a follow-up visit 4 weeks after discontinuing the investigational treatment.  The 
assessment will include measurement of vital signs (height, weight, blood pressure, heart rate, and 
temperature), caregiver completion of behavioral outcome measures (SRS-2, RBSR, and ABC), and 
clinician-rated CGI-Severity and CGI-Improvement scores.  The adverse event log will also be 
completed by the study physician.  Caregivers will be asked to report any changes in concurrent 
medications or non-pharmacological treatments since the previous study visit.  Treatment 
assignment (Avmacol® or placebo) will be obtained by study staff from IDS and shared with 
participants and their caregiver.  Participants who were assigned to take placebo will have the 
opportunity to receive a 3-month supply of Avmacol® at no cost, supervised by the study 
physician under routine clinical care following study exit.  Monetary compensation for study 
participation will be provided to participants and their caregiver in the form of 2 checks at the end 
of each clinic visit, as described in section 12.3 below. 

Clinical Protocol: Sulforaphane for ASD 
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7 
Statistical Plan 
7.1 Sample Size Determination 
We calculated power for comparing groups on change in SRS-2 total score (the primary outcome 
measure), using a standard approach for linear mixed models (Littell RC, Milliken GA, Stroup WW, 
Wolfinger RD, Schabenberger O. SAS for mixed models [electronic resource]. Cary, N.C.: SAS 
Institute, Inc.; 2006). We assumed that the mean differences observed in the trial by Singh et al. 
represented the true values (i.e., we assumed mean differences of 7, 20, 18.4, and 0.4 at weeks 4, 8, 
12 and 16, respectively), and we assumed a constant standard deviation of 20, consistent with that 
observed by Singh et al. Furthermore, we assume within-participants across time correlation of 
0.5 (a conservative value) and we assume no more than 15% loss distributed over the 16 weeks of 
follow-up.  Under these assumptions, randomizing 27 participants to each group will provide at 
least 80% power for an overall test comparing groups across all time points using a two-sided 4 
degree of freedom test at the 0.05 significance level.   
 
For the exploratory microbiota analysis, we aim to include a subset of 30 subjects in the sample. In 
a recent randomized cross-over trial of the effects of broccoli consumption on microbiota 
composition in 18 healthy adults [Kaczmarek 2017*], participants consuming broccoli exhibited 
an increase in the ratio of Baceteroidetes to Firmicutes by 37% from baseline to end, compared to 
a 5% reduction during the control period (p=0.02). With 15 participants per group, we will have 
>80% power to detect a similar change in the ratio of Bacteroidetes to Firmicutes. 
 
* Kaczmarek JL, et al. Broccoli consumption impacts the human gastrointestinal microbiota. The FASEB 
Journal, 2017. 31(1 suppl): p.965. 
 
7.2 Statistical Methods 
General linear mixed models (GLMM) will be used to test the primary and secondary hypotheses 
in longitudinal analyses: 
  
Primary Hypotheses 
Compared to control participants, treated participants will show larger changes over time on SRS-
2 total scores from Baseline to Week 12, and differences will not be maintained after treatment 
discontinuation at Week 12 through the end of the follow-up period at Week 16.   
 
GLMM analyses will describe individual patterns of change over time on the SRS-2 Total score. 
Individual intercepts and slopes will be estimated as random-effects variables, and will be allowed 
to correlate.  The SRS-2 scores from Baseline, Week 4, 8, 12, and 16 will be modeled as a function 
of treatment, time, time x treatment and will include covariates of age-stratum and the SRS-2 total 
score from screening.  In addition to time, we will add a time-varying variable representing 
treatment phase (0=treatment, 1=follow-up period) and we cross this variable with treatment, 
time, and treatment x time.  Time will be centered at Week 12.  Of interest will be the main effect 
of treatment (i.e., treatment impact at end of active treatment), treatment x time (i.e., treatment 
impact on change over time during the active treatment phase), and treatment x time x treatment 
phase (i.e., treatment differences in change over time during follow-up period).  We will use an 

Clinical Protocol: Sulforaphane for ASD 
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unstructured covariance matrix. We will first test for any differences between groups across all 
four groups using a 4 degree of freedom linear contrast of the model parameters. If this overall 
test is significant at the 5% level, we will then test for differences separately at each time point. 
We will also report estimated mean differences along with 95% confidence intervals. 
 
 
Secondary Hypotheses 
Compared to control participants, treated participates will show larger changes over time on CGI 
Severity and RBSR scores from Baseline to Week 12 and will show reduction in those gains after 
discontinuation through the end of the follow-up period at Week 16. 
 
Compared to control participants, treated participates will show larger changes over time on 
subscales when treatment impacts were observed on the total scores on the SRS-2, CGI Severity, 
and RBSR from Baseline to Week 12 and will show reduction in those gains after discontinuation 
through the end of the follow-up period at Week 16.   
 
Similar GLMM analyses will be conducted to examine the longitudinal assessments of CGI Severity 
and RBSR and of subscales if the total scores show treatment impacts. 
 
Exploratory Hypotheses  
We hypothesize that treatment with sulforaphane will result in greater diversity of gut 
microbial species and a higher ratio of Bacteroidetes to Firmicutes spp. compared to placebo, and 
that these alterations in gut microbiota will be positively associated with behavioral 
improvements in the sulforaphane treatment group. 
 
We will utilize Genotek's validated wet-lab and metagenomic data analytic pipeline for the 
batched sample processing and data analysis. Microbial DNA will be extracted and amplified using 
PCR, and 16S rRNA gene sequencing will be performed using the Illumina MiSeq platform. A 
curated taxonomic database will be used to assign a taxonomic classification to the sequencing 
reads. The mean (%) relative abundance of microbial phyla and genera will be calculated for the 
sulforaphane and placebo groups pre- and posttreatment.  The percent change in Bacteroidetes to 
Firmicutes (B:F) ratio from baseline to endpoint will be compared between treatment groups 
using Analysis of Covariance (ANCOVA), with baseline B:F ratio included as a covariate. 
 
Microbiota diversity and relative abundance will be assessed using conventional within sample 
("alpha diversity") and between-sample ("beta diversity") metrics. Three metrics will be used to 
assess change in alpha diversity over time: the absolute number of operational taxonomic units 
(OTUs), Chao1 estimator, and Shannon index. We will use ANCOVA to test for significant 
differences in alpha diversity from baseline to endpoint within each treatment group. Beta 
diversity will be assessed with 3 metrics: Bray-Curtis dissimilarity, Weighted Unifrac distance, and 
Unweighted Unifrac distance. These indices will be used to determine the variance in an individual 
over time and the mean dissimilarity between individuals in each group. Weighted Unifrac 
distance will be used for Principal Coordinates Analysis (PCoA). We will create a PCoA plot to 
visually identify whether samples from each treatment group cluster together at different time 
points (before and after treatment). This can help determine whether any changes identified 
during beta diversity analysis are directed changes or random noise. 

Clinical Protocol: Sulforaphane for ASD 
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We will utilize our behavioral data collection to determine if changes in microbiota diversity and 
relative abundance are associated with behavioral improvement. Linear mixed models will be 
used to determine if microbiota composition changes over time covary with change in behavioral 
outcome scores. 
7.3 Subject Population(s) for Analysis 
Study analysis will include all available data from all randomized subjects who received at least 
one dose of study medication and participated in at least one post-baseline efficacy assessment 
(i.e., intent-to-treat population).  Missing outcome data will not be imputed and will be assumed to 
be missing at random. 
8 
Safety and Adverse Events 
8.1 Definitions 
Unanticipated Problems Involving Risk to Subjects or Others 
Any incident, experience, or outcome that meets all of the following criteria:  
 Unexpected in nature, severity, or frequency  (i.e. not described in study-related documents 
such as the IRB-approved protocol or consent form, the investigators brochure, etc) 
 Related or possibly related to participation in the research (i.e. possibly related means there 
is a reasonable possibility that the incident experience, or outcome may have been caused 
by the procedures involved in the research, 
 Serious (as defined below)  “Serious” is different than “severe” as reported in the CTC 
criteria that applies a grade to the AE. 
 
Adverse Event 
An adverse event (AE) is any symptom, sign, illness or experience that develops or worsens in 
severity during the course of the study.  Intercurrent illnesses or injuries will be regarded as 
adverse events.  Abnormal results of diagnostic procedures are considered to be adverse events if 
the abnormality: 
 results in study withdrawal 
 is associated with a serious adverse event 
 is associated with clinical signs or symptoms 
 leads to additional treatment or to further diagnostic tests 
 is considered by the investigator to be of clinical significance 
 
Serious Adverse Event 
Adverse events are classified as serious or non-serious.  A serious adverse event is any AE that is:  
 fatal 
 life-threatening 
 requires or prolongs hospital stay 
 results in persistent or significant disability or incapacity 
 a congenital anomaly or birth defect 
 an important medical event 

Clinical Protocol: Sulforaphane for ASD 
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Important medical events are those that may not be immediately life threatening, but are clearly 
of major clinical significance.   They may jeopardize the subject, and may require intervention to 
prevent one of the other serious outcomes noted above.  For example, drug overdose or abuse, a 
seizure that did not result in in-patient hospitalization, or intensive treatment of bronchospasm in 
an emergency department would typically be considered serious.  
 
All adverse events that do not meet any of the criteria for serious should be regarded as non-
serious adverse events.  
 
Based on previous clinical trials of sulforaphane in humans [43, 48, 51], we do not anticipate any 
significant adverse events with Avmacol®, though the following gastrointestinal symptoms may 
potentially occur with greater frequency: 
 
 Nausea and vomiting 
 Abdominal pain 
 Large or soft stools 
 Constipation 
 Diarrhea 
 Weight gain 
 
In a randomized, controlled trial of sulforaphane for autism spectrum disorder conducted by 
Singh et al. [48], 2 subjects with a history of seizures had single unprovoked seizures during the 
course of the study (one after 3 weeks on sulforaphane, one 3 weeks after discontinuing 
sulforaphane), and the possibility of seizure as a potential adverse effect of sulforaphane could not 
be ruled out.  In our proposed study, a history of clinical seizure in the year prior to screening is an 
exclusionary criterion for safety reasons. 
 
One subject enrolled in this trial experienced a worsening of behavioral symptoms that may or 
may not have been directly study related. 
 
 
Adverse Event Reporting Period 
The study period during which adverse events must be reported is defined as the period from the 
initiation of any study procedures to the end of the study treatment follow-up.  For this study, the 
study treatment follow-up is defined as 30 days following the last administration of study 
treatment.  
 
Preexisting Condition 
A preexisting condition is one that is present at the start of the study.  A preexisting condition will 
be recorded as an adverse event if the frequency, intensity, or the character of the condition 
worsens during the study period. 
 
General Physical Examination Findings 

Clinical Protocol: Sulforaphane for ASD 
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At screening, any clinically significant abnormality will be recorded as a preexisting condition.  At 
interval study visits and at the end of the study, any new clinically significant 
findings/abnormalities that meet the definition of an adverse event will also be recorded and 
documented as an adverse event.  
 
Post-study Adverse Event 
All unresolved adverse events will be followed by the investigator until the events are resolved, 
the subject is lost to follow-up, or the adverse event is otherwise explained.  At the last scheduled 
visit, the study physician will instruct each subject to report any subsequent event(s) that the 
subject, or the subject’s personal physician, believes might reasonably be related to participation 
in this study.   
 
 
Abnormal Laboratory Values 
A clinical laboratory abnormality will be documented as an adverse event if any one of the 
following conditions is met:  
 The laboratory abnormality is not otherwise refuted by a repeat test to confirm the 
abnormality 
 The abnormality suggests a disease and/or organ toxicity 
 The abnormality is of a degree that requires active management; e.g. change of dose, 
discontinuation of the drug, more frequent follow-up assessments, further diagnostic 
investigation, etc. 
 
Hospitalization, Prolonged Hospitalization or Surgery 
Any adverse event that results in hospitalization or prolonged hospitalization will be documented 
and reported as a serious adverse event.  Any condition responsible for surgery will be 
documented as an adverse event if the condition meets the criteria for an adverse event.  
 
Neither the condition, hospitalization, prolonged hospitalization, nor surgery are reported as an 
adverse event in the following circumstances: 
 Hospitalization or prolonged hospitalization for diagnostic or elective surgical procedures 
for a preexisting condition.  Surgery should not be reported as an outcome of an adverse 
event if the purpose of the surgery was elective or diagnostic and the outcome was 
uneventful. 
8.2 Recording of Adverse Events 
At each contact with the subject, the investigator will seek information on adverse events by 
specific questioning and, as appropriate, by examination.  Information on all adverse events will 
be recorded immediately in the source document, and also in the appropriate adverse event log of 
the case report form (CRF).  All clearly related signs, symptoms, and abnormal diagnostic 
procedures results will be recorded in the source document, though grouped under one diagnosis. 
 
All adverse events occurring during the study period will be recorded in the adverse event log.  
The clinical course of each event will be followed until resolution, stabilization, or until it has been 
determined that the study treatment or participation is not the cause.  Serious adverse events that 

Clinical Protocol: Sulforaphane for ASD 
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are still ongoing at the end of the study period will be followed up, either by phone call or clinic 
visit, to determine the final outcome.  Any serious adverse event that occurs after the study period 
and is considered to be possibly related to the study treatment or study participation will be 
recorded and reported immediately to the FDA, UNC Biomedical IRB, and DSMB. 
8.3 Reporting of Serious Adverse Events and Unanticipated Problems 
The study PI will immediately report any serious adverse events to the FDA, UNC Biomedical IRB, 
and DSMB meeting the following 3 criteria: 
 related to study participation, 
 unexpected, and  
 serious or involve risks to subjects or others  
(see definitions, section 8.1).   
 
If the report is supplied as a narrative, the minimum necessary information to be provided at the 
time of the initial report includes: 
 Study identifier 
 Study Center 
 Subject number 
 A description of the event 
 Date of onset 
 Current status 
 Whether study treatment was discontinued 
 The reason why the event is classified as 
serious 
 Investigator assessment of the association 
between the event and study treatment 
 
8.3.1 Investigator reporting: notifying the study sponsor 
 
The proposed trial is investigator-initiated, and thus the investigator is also the sponsor.  Any 
study-related unanticipated problem posing risk of harm to subjects or others, and any type of 
serious adverse event, will be reported to the FDA within 7 calendar days using FDA Form 3500A 
for mandatory reporting.  SAEs meeting the criteria outlined in the beginning of section 8.3 will 
also be reporting to the UNC Biomedical IRB by the study PI via IRBIS (the IRB information system 
for UNC) within 7 calendar days of becoming aware of the adverse event.  Significant new 
information on ongoing serious adverse events will be provided promptly to the FDA and UNC 
IRB.  A written report of the SAE will also be provided to the DSMB for committee review within 
14 calendar days of the event. 
8.3.2 Investigator reporting:   
For reportable deaths, the initial submission to the UNC IRB will be made by contacting the IRB 
Director or Associate Director. The AE/Unanticipated Problem Form is required as a follow up to 
the initial submission. 
 
Other Reportable events: 
For clinical drug trials, the following events are also reportable to the UNC IRB: 

Clinical Protocol: Sulforaphane for ASD 
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 Any adverse experience that, even without detailed analysis, represents a serious unexpected 
adverse event that is rare in the absence of drug exposure (such as agranulocytosis, hepatic 
necrosis, Stevens-Johnson syndrome). 
 Any adverse event that would cause the sponsor to modify the investigator’s brochure, protocol 
or informed consent form, or would prompt other action by the IRB to assure protection of 
human subjects. 
 Information that indicates a change to the risks or potential benefits of the research, in terms of 
severity or frequency. For example:  
– Safety monitoring indicates that a particular side effect is more severe, or more frequent 
than initially expected. 
– A paper is published from another study that shows that an arm of your research study 
is of no therapeutic value. 
 Change in FDA safety labeling or withdrawal from marketing of a drug, device, or biologic 
used in a research protocol. 
 Breach of confidentiality 
 Change to the protocol taken without prior IRB review to eliminate apparent immediate 
hazard to a research participant. 
 Incarceration of a participant when the research was not previously approved under 
Subpart C and the investigator believes it is in the best interest of the subject to remain on 
the study. 
 Complaint of a participant when the complaint indicates unexpected risks or the complaint 
cannot be resolved by the research team. 
 Protocol violation (meaning an accidental or unintentional deviation from the IRB approved 
protocol) that in the opinion of the investigator placed one or more participants at 
increased risk, or affects the rights or welfare of subjects.  If a large number of protocol 
deviations are identified, or if they are deemed to affect patient safety or the overall efficacy 
of the trial, the protocol deviations will also be reported to the DSMB.   
8.3.3 Investigator reporting:  
Not applicable, as this is a single-site study. 
8.3.4 Sponsor reporting: Notifying the FDA 
 
The study PI (study sponsor) will promptly complete a written IND safety report for any adverse 
event meeting the following criteria: 
 
 Within 7 calendar days 
Any study event that is: 
– associated with the use of the study drug 
– unexpected,  
– fatal or life-threatening, and  
 
 Within 15 calendar days 

Clinical Protocol: Sulforaphane for ASD 
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Any study event that is: 
– associated with the use of the study drug, 
– unexpected, and 
– serious, but not fatal or life-threatening 
-or- 
– a previous adverse event that was not initially deemed reportable but is later found to fit 
the criteria for reporting (reporting within 15 calendar days from when event was 
deemed reportable). 
 
Additional reporting requirements 
The study PI (sponsor) will identify in IND safety reports all previous reports concerning similar 
adverse events and analyze the significance of the current event in light of the previous reports. 
 
Reporting Process 
Adverse events will be submitted on FDA Form 3500A or in a narrative format, along with FDA 
form 1571.  If supplied as in a narrative format, the minimum information to be supplied is noted 
above at the beginning of section 8.3.  
 
8.4 Unblinding Procedures 
The study team will make every effort to maintain blinding of treatment assignment for both 
subject and investigator for the entire duration of the individual subject’s study participation.  
Treatment unblinding will be limited to the occurrence of a serious adverse event (SAE) that the 
independent medical monitor determines is possibly related to study treatment and study 
participation.  In the event of such an unanticipated problem, study staff will contact UNC IDS via 
the on-call pager (919-216-9727) to report the occurrence of the event and request that treatment 
assignment status be disclosed to the independent medical monitor.  The study staff and 
participant and caregiver will remain blinded to treatment.  The treatment status will be faxed by 
IDS to the medical monitor and relevant medical personnel involved in treating the subject (e.g., 
emergency room physician, primary care physician).  A narrative description of the SAE and 
treatment status will be documented in the CRF, and the FDA and UNC IRB will be promptly 
notified as specified above in Section 8.3.1. 
8.5 Stopping Rules  
 If a participant experiences significant adverse events that affect tolerability, may 
reasonably be related to taking the investigational product, and do not meet the protocol 
definition for a serious AE (including non-extreme behavioral changes), the dose may be 
reduced by approximately 30% according to the following dose modification schedule: 
 
 
 
 
 
 
 

Clinical Protocol: Sulforaphane for ASD 
- 38 - 
 
 
 
 
Weight 
category 
Prescribed weight-
based dose of 
glucoraphanin 
Prescribed # 
of tablets 
70% of 
weight-based 
dose (ie, 30% 
reduction) 
Prescribed # 
tablets for 
reduced dose 
(rounded to 
nearest 15 mg 
increment)* 
<100 lb 
45 mg 
3 
31.5 mg 
2 
100-125 lb 
75 mg 
5 
52.5 mg 
3 
126-175 lb 
90 mg 
6 
63 mg 
4 
176-199 lb 
105 mg 
7 
73.5 mg 
5 
≥ 200 lb 
120 mg 
8 
84 mg 
6 
 
*Rounded down if equidistant between two 15 mg dosing increments 
 
 If the dose is reduced during the course of the study, the participant will continue the 
lowered dose for the remainder of the study.  If adverse events do not improve within 14 
days of lowering the dose, the investigational product may be permanently discontinued at 
the discretion of the treating study physician, participant, and participant’s guardian. If a 
participant exhibits extreme exacerbation of behavioral symptoms (such as self-injurious 
behavior or aggression) that do not improve substantially following a dose reduction and 
are determined “probably” or “definitely” related to taking the investigational product, the 
investigational product may be permanently discontinued sooner than 14 days post-
reduction, and the DSMB will be notified.  As individuals with ASD often have cyclical 
changes in behavior independent of medication effects, the study staff will rely on 
caregivers’ experience with the participant to determine if any observed behavior changes 
are within the expected range for that individual.  If the investigational product is stopped 
permanently, the participant and his caregiver will be asked to complete the remainder of 
the study visits, return any unused study drug, and repeat safety lab studies. 
 If a participant has a seizure while taking the study medication, the study medication will 
be stopped, and the DSMB will be notified.  The participant will be evaluated by the study 
PI and the participant’s primary care physician, treated, and monitored appropriately for 
the duration of the study.  The participant and his caregiver will be asked to return to 
complete the remainder of the study visits, return any unused study drug, and repeat safety 
lab studies. 
 If the participant newly requires a long-term medication that is thought to interact 
adversely with the study medication, the study medication will be stopped.  The participant 
and his caregiver will be asked to return to complete the remainder of the study visits, 
return any unused study drug, and repeat safety lab studies. 
 If a participant experiences a serious adverse event that is thought to be related to taking 
the study medication (as determined by an independent medical monitor), the study 
medication will be permanently discontinued, and the appropriate overseeing regulatory 
organizations will be notified (FDA, IRB, and DSMB). The participant and his caregiver will 

Clinical Protocol: Sulforaphane for ASD 
- 39 - 
 
be asked to return to complete the remainder of the study visits, return any unused study 
drug, and repeat safety lab studies. 
 Potential reasons for stopping the entire study include: occurrence of a grade 3(severe) or 
grade 4 (life-threatening) SAE in more than one participant that an independent medical 
monitor and DSMB believe is likely related to active treatment; new information from other 
clinical trials that indicate serious risks associated with oral administration of a 
sulforaphane product; the death of a participant that is determined to be related to the 
study intervention; and insufficient funds to complete study tasks. 
8.6 Medical Monitoring 
It is the primary responsibility of the Principle Investigator to oversee the safety of the study.  This 
safety monitoring will include careful assessment and appropriate reporting of adverse events as 
noted above, as well as the construction and implementation of a data and safety-monitoring plan 
(DSMP) (see section 9 Auditing, Monitoring and Inspecting).  A medical monitor (ie, a physician 
not directly affiliated with the study) will be retained to review any SAEs and determine if the SAE 
may reasonably be related to treatment status. 
8.6.1 Internal Data and Safety Monitoring Board  or DSMP   
The North Carolina Translational and Clinical Science (NC TraCS) Institute Data and Safety 
Monitoring Board (DSMB) will provide additional safety and oversight for the proposed trial. The 
NC TraCS DSMB will consist of a chair, an ethicist, an epidemiologist, a biostatistician, and one or 
more clinical researchers, each serving three year terms (please see Attachment 6C, NC TraCS 
DSMB Charter, for a listing of current Committee members).  The DSMB will review all SAEs and 
any seizure events, which the study PI will submit in writing to the DSMB chair within 7 calendar 
days of the event.  The DSMB will also review safety information (abnormal baseline and end-of-
treatment screening labs; unanticipated problems; significant physical exam findings) in a face-to-
face meeting after every 10 participants have completed the study.  The study PI will supply this 
information in a written summary to the DSMB prior to the meeting and will participate in the first 
part of the committee meeting to review the findings.  The study PI will also complete the Annual 
Data Report form, provided by the UNC Biomedical IRB, and submit to the DSMB annually for 
review of all adverse event reports.  All data presented to the DSMB will be de-identified to protect 
the confidentiality of participants.  Due to the relatively small size and anticipation of minimal risk 
associated with this study, no interim data analysis is planned.   
The DSMB staff or chair will prepare minutes of each meeting within ten (10) working days of 
each meeting.  These minutes will be circulated and approved at the next regularly scheduled 
meeting of the DSMB.  The DSMB will report their findings to the UNC Biomedical IRB, as 
appropriate, and will make any recommendations directly to the IRB and to the study PI. 
9 
Data Handling and Record Keeping 
9.1 Confidentiality 
Information about study subjects will be kept confidential and managed according to the 
requirements of the Health Insurance Portability and Accountability Act of 1996 (HIPAA).  Those 
regulations require a signed subject/ LAR authorization informing the subject of the following:  

Clinical Protocol: Sulforaphane for ASD 
- 40 - 
 
 What protected health information (PHI) will be collected from subjects in this study 
 Who will have access to that information and why 
 Who will use or disclose that information 
 The rights of a research subject to revoke their authorization for use of their PHI.  
 
In the event that a subject revokes authorization to collect or use PHI, the investigator, by 
regulation, retains the ability to use all information collected prior to the revocation of subject 
authorization.  For subjects that have revoked authorization to collect or use PHI, attempts should 
be made to obtain permission to collect at least vital status (i.e. that the subject is alive) at the end 
of their scheduled study period. 
9.2 Source Documents 
Source data is all information, original records of clinical findings, observations, or other activities 
in a clinical trial necessary for the reconstruction and evaluation of the trial.  Source data are 
contained in source documents.  Examples of these original documents, and data records include: 
hospital records, clinical and office charts, laboratory notes, memoranda, subjects’ diaries or 
evaluation checklists, pharmacy dispensing records, recorded data from automated instruments, 
copies or transcriptions certified after verification as being accurate and complete, microfiches, 
photographic negatives, microfilm or magnetic media, x-rays, subject files, and records kept at the 
pharmacy, at the laboratories, and at medico-technical departments involved in the clinical trial. 
 
Source documents will be maintained behind two levels of locked doors (ie, in a locked room on a 
floor with badge-access only).  Only study staff directly involved in data collection, entry, and 
analysis and independent monitoring personnel will have access to source documents. 
9.3 Case Report Forms  
The study case report form (CRF) is the primary data collection instrument for the study.  All data 
requested on the CRF must be recorded.  All missing data must be explained.  If a space on the CRF 
is left blank because the procedure was not done or the question was not asked, write “N/D”.  If 
the item is not applicable to the individual case, write “N/A”.  All entries should be printed legibly 
in black ink.  If any entry error has been made, to correct such an error, draw a single straight line 
through the incorrect entry and enter the correct data above it.  All such changes must be initialed 
and dated.  DO NOT ERASE OR WHITE OUT ERRORS.  For clarification of illegible or uncertain 
entries, print the clarification above the item, then initial and date it. 
9.4 Records Retention 
 
It is the investigator’s responsibility to retain essential study documents for at least 3 years after 
the study is completed and the FDA is notified (in accordance with 21 CFR 312.62).   
 

Clinical Protocol: Sulforaphane for ASD 
- 41 - 
 
10 Study Monitoring, Auditing, and Inspecting 
10.1 Study Monitoring Plan 
This study will be monitored according to the monitoring plan in Appendix 6B.  The investigator 
will allocate adequate time for such monitoring activities.   
 
Auditing and Inspecting 
The investigator will permit study-related monitoring, audits, and inspections by the IRB, 
government regulatory bodies, and University compliance and quality assurance groups of all 
study related documents (e.g. source documents, regulatory documents, data collection 
instruments, study data etc.).  The investigator will ensure the capability for inspections of 
applicable study-related facilities (e.g. pharmacy, diagnostic laboratory, etc.). 
 
Participation as an investigator in this study implies acceptance of potential inspection by 
government regulatory authorities and applicable University compliance and quality assurance 
offices. 
11 Ethical Considerations 
This study is to be conducted according to US and international standards of Good Clinical Practice 
(FDA Title 21 part 312 and International Conference on Harmonization guidelines), applicable 
government regulations and Institutional research policies and procedures. 
 
This protocol and any amendments will be submitted to the UNC Institutional Review Board (IRB), 
in agreement with local legal prescriptions, for formal approval of the study conduct.  The decision 
of the IRB concerning the conduct of the study will be made in writing to the investigator and a 
copy of this decision will be provided to the investigator-sponsor before commencement of this 
study.   
 
All subjects for this study and their caregivers will be provided a consent form describing this 
study and providing sufficient information for subjects to make an informed decision about their 
participation in this study.  The consent form will be submitted with the protocol for review and 
approval by the IRB for the study.  The formal consent of a subject or Legally Authorized 
Representative (LAR), using the IRB-approved consent form, must be obtained before that subject 
undergoes any study procedure.  The consent form must be signed by the subject or LAR, and the 
investigator-designated research professional obtaining the consent. If the subject is unable to 
provide informed consent, every effort will be made to obtain informed assent for participation. 
12 Study Finances 
12.1 Funding Source 
This study is financed through a collaborative grant from the University of North Carolina 
Nutrition Research Institute and with research funds from Dr. Politte’s KL2 Career Development 
Award (NIH/NCATS KL2TR001109). 

Clinical Protocol: Sulforaphane for ASD 
- 42 - 
 
12.2 Conflict of Interest 
Any investigator who has a conflict of interest with this study (patent ownership, royalties, or 
financial gain greater than the minimum allowable by their institution, etc.) must have the conflict 
reviewed by a properly constituted Conflict of Interest Committee with a Committee-sanctioned 
conflict management plan that has been reviewed and approved by the study investigator-sponsor 
prior to participation in this study.  All UNC investigators will follow the University conflict of 
interest policy. 
12.3 Subject Stipends or Payments 
Subjects’ caregivers will be reimbursed $30 per study visit to cover travel expenses.  Subjects will 
also be reimbursed $20 per study visit as a token of appreciation for their participation.  Payment 
will be provided in the form of two separate checks (one to caregiver, one to subject) at the 
conclusion of the specified study visit. 
13 Publication Plan 
The sponsor-investigator will hold primary responsibility for publication of the results of the 
study. 
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Carrasco-Pozo, C., K.N. Tan, and K. Borges, Sulforaphane is anticonvulsant and improves mitochondrial 
function. J Neurochem, 2015. 135(5): p. 932-42. 
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Brose, R.D., et al., Activation of the stress proteome as a mechanism for small molecule therapeutics. 
Hum Mol Genet, 2012. 21(19): p. 4237-52. 
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Takano, T., Role of Microglia in Autism: Recent Advances. Dev Neurosci, 2015. 37(3): p. 195-202. 
87. 
Innamorato, N.G., et al., The transcription factor Nrf2 is a therapeutic target against brain inflammation. J 
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Horowitz, M. and S.D. Robinson, Heat shock proteins and the heat shock response during hyperthermia 
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Wiggins, J.L., et al., Using a self-organizing map algorithm to detect age-related changes in functional 
connectivity during rest in autism spectrum disorders. Brain Res, 2011. 1380: p. 187-97. 
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Gan, N., et al., Sulforaphane activates heat shock response and enhances proteasome activity through 
up-regulation of Hsp27. J Biol Chem, 2010. 285(46): p. 35528-36. 
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Prestera, T., et al., Parallel induction of heme oxygenase-1 and chemoprotective phase 2 enzymes by 
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Shehata, M. and K. Inokuchi, Does autophagy work in synaptic plasticity and memory? Rev Neurosci, 
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Pawlik, A., et al., Sulforaphane inhibits growth of phenotypically different breast cancer cells. Eur J Nutr, 
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Jo, C., et al., Sulforaphane induces autophagy through ERK activation in neuronal cells. FEBS Lett, 2014. 
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Liu, Y., et al., Sulforaphane enhances proteasomal and autophagic activities in mice and is a potential 
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Clinical Protocol: Sulforaphane for ASD 
- 47 - 
 
 
 
15 Appendix 6A: Schedule of Study Events 
 
Visit 
Consent, 
Eligibility 
(ADOS-2, 
DSMI-5 
criteria) 
Medical 
history 
Physical 
Exam 
WASI 
Vital 
signs 
AE 
Review 
Behavioral 
outcome 
measures 
(SRS-2; 
ABC; 
RBSR; CGI-
S,I) 
Safety 
labs 
(CBC, 
Chem, 
LFTs, 
TSH) 
Saliva 
collection 
for 
storage 
Stool 
collection 
for 
microbiota 
analysis 
Visit 
Duration 
Screen 
X 
X 
X 
X 
X 
 
X 
X 
 
 
3 hr 
Week 0 
Baseline 
 
 
 
 
X 
X 
X 
 
X 
X 
1.5 hr 
Week 2 
Phone call 
 
 
 
 
 
X 
 
 
 
 
20 min 
Week 4 
 
 
 
 
X 
X 
X 
 
 
 
1 hr 
Week 8 
 
 
 
 
X 
X 
X 
 
 
 
1 hr 
Week 12 
Treatment 
End 
 
 
X 
 
X 
X 
X 
X 
 
X 
2 hr 
Week 16 
Study End 
 
 
 
 
X 
X 
X 
 
 
 
1 hr 
 
 
 

Clinical Protocol: Sulforaphane for ASD 
- 48 - 
 
16 Appendix 6B: Data and Safety Monitoring Plan 
 
I. 
RISKS TO SUBJECTS 
a. 
Human Subjects Involvement and Characteristics: Approval from the UNC IRB for this 
research project will be obtained prior to any recruitment or involvement of human subjects.  The study 
will include 54 healthy adolescent and young adult males, ages 13-30 years old, with a diagnosis of 
Autism Spectrum Disorder (ASD) severity level 2 or 3, confirmed by review of DSM-5 diagnostic 
criteria for ASD, and exceeding the ASD cut-off score of 8 on the Autism Diagnostic Observation 
Scheduled-2 (ADOS-2).  The study population will be limited to male subjects for two primary reasons: 
(a) we aim to conduct an independent replication of a previous clinical trial of sulforaphane in ASD [1], 
which included only male participants; (b) potential mechanistic differences in ASD between males and 
females may lead to excessive heterogeneity in this relatively small sample size (ASD is 4 times more 
common in males than females).  The proposed study includes adolescents and adults with 
developmental disabilities, both vulnerable populations, rather than healthy volunteers because the 
study intervention, a nutraceutical containing broccoli extracts, may provide specific benefit for social 
deficits observed in ASD, but is not expected to confer behavioral benefits for those without ASD.  The 
intervention also has a well-documented safety profile and is considered low-risk.  Furthermore, 
interventions in children with developmental disorders are likely to have greater impact than similar 
treatments at a later stage of development due to increased neuroplasticity early in development.  The 
study will exclude individuals with significant medical problems and active seizure disorders (defined 
as clinical seizure activity in the 12 months preceding the study) due to theoretical increased risk of 
seizure with sulforaphane treatment [1].  Individuals taking chronic anti-inflammatory agents, such as 
corticosteroids or non-steroidal anti-inflammatory drugs, will also be excluded due to potential 
confounding of results.   
 
Subjects will primarily be recruited from the Carolina Institute for Developmental Disabilities Research 
Subject Registry, which includes more than 6000 families that have expressed an interest in research 
participation and have consented to be contacted about potential studies.  Potential subjects will be 
contacted directly via email or letter by the Registry staff with information about the trial, including 
contact information of study staff.  Participants will also be recruited from the clinical practices of 
physicians, psychologists, and allied health staff at the Carolina Institute for Developmental Disabilities 
as well as from referring providers.  Electronic and written advertisements will be disseminated to local 
community autism support agencies (e.g., Autism Society of North Carolina, Autism Resource 
Centers).  All advertisement materials will first be approved by the University of North Carolina IRB. 
 
Enrolled participants will be stratified for age (13-18 and 19-30 years) to account for possible effects of 
age on treatment response.  The goal is to recruit twenty-seven 13-18 year-olds and twenty-seven 19-30 
year-olds to allow for secondary analysis by age group.  Individuals will be randomly assigned to 
treatment group within age strata by generating random numbers for each group for each entry (i.e., 1-27 
for younger group and 1-27 for older group).  The larger half of the random numbers within each group 
will be assigned to receive the treatment and the smaller half to the control condition.  These two lists – 
listed in their original order (1-27 for younger and 1-27 for older) will be provided to UNC 
Investigational Drug Services (IDS) pharmacists.  They will consult the list and provide the appropriate 
medication to each individual depending on the order in which they present to the pharmacy within their 
age group.  Study personnel will remain blinded to treatment condition for the duration of the study. 
 

Clinical Protocol: Sulforaphane for ASD 
- 49 - 
 
During the course of the study, participants will take 3-8 Avmacol© or placebo tablets once daily by 
mouth for a total of 12 weeks, with dosing regimen based on weight as follows: 
 
3 tablets (approx. 46.5 µmol SF) daily if <100 lb 
5 tablets (approx. 77.5 µmol SF) daily if 100-125 lb 
6 tablets (approx. 93 µmol SF) daily if 126-175 lb 
7 tablets (approx. 108.5 µmol SF) daily if 176-199 lb 
8 tablets (approx. 124 µmol SF) daily if ≥ 200 lb 
 
If unable to swallow the requisite number of tablets whole, subjects will have the option to crush the 
tablets and mix with a soft food, such as applesauce or yogurt, for administration.   
 
If a participant experiences significant adverse events that affect tolerability, may reasonably be related 
to taking the investigational product, and do not meet the protocol definition for a serious AE (including 
behavioral changes that are not severe), the dose may be reduced by approximately 30% according to 
the following dose modification schedule: 
 
Weight 
category 
Prescribed weight-
based dose of 
glucoraphanin 
Prescribed # of 
tablets 
70% of weight-
based dose (ie, 
30% reduction) 
Prescribed # 
tablets for 
reduced dose 
(rounded to 
nearest 15 mg 
increment)* 
<100 lb 
45 mg 
3 
31.5 mg 
2 
100-125 lb 
75 mg 
5 
52.5 mg 
3 
126-175 lb 
90 mg 
6 
63 mg 
4 
176-199 lb 
105 mg 
7 
73.5 mg 
5 
≥ 200 lb 
120 mg 
8 
84 mg 
6 
 
*Rounded down if equidistant between two 15 mg dosing increments 
 
If the dose is reduced during the course of the study, the participant will continue the lowered dose for 
the remainder of the study.  If adverse events do not improve within 14 days of lowering the dose, the 
investigational product may be permanently discontinued at the discretion of the treating study 
physician, participant, and participant’s guardian. If a participant exhibits extreme exacerbation of 
behavioral symptoms (such as self-injurious behavior or aggression) that do not improve substantially 
following a dose reduction and are determined “probably” or “definitely” related to taking the 
investigational product, the investigational product may be permanently discontinued sooner than 14 
days post-reduction, and the DSMB will be notified.  As individuals with ASD often have cyclical 
changes in behavior independent of medication effects, the study staff will rely on caregivers’ 
experience with the participant to determine if any observed behavior changes are within the expected 
range for that individual.  If the investigational product is stopped permanently, the participant and his 
caregiver will be asked to complete the remainder of the study visits, return any unused study drug, and 
repeat safety lab studies. 
 

Clinical Protocol: Sulforaphane for ASD 
- 50 - 
 
b. 
Source of Materials:  Data collection will primarily include clinical and behavioral information, 
including: medical history, medication/treatment history, developmental history, behavioral 
questionnaires/ inventories, clinical assessment for ASD, cognitive evaluation, report of adverse events, 
and physical examination.  Phlebotomy will be performed at screening and at the end of the active 
treatment phase (Week 12 visit) for (a) basic safety lab monitoring (complete blood count, 
comprehensive metabolic panel, liver function tests, and thyroid stimulating hormone).  Stool samples 
will be collection at Baseline and Week 12 for analysis of microbiota diversity and relative abundance. 
 
c. 
Potential Risks: Cruciferous plants, including broccoli sprouts, are widely regarded as safe for 
ingestion and are regular dietary components in many regions of the world.  Based on previous data 
from clinical trials of broccoli sprout extracts (BSE), we anticipate few, if any, adverse effects, as 
summarized below: 
 
A Phase I study of orally administered sulforaphane- and glucoraphanin-containing BSE (75 µmol 
glucosinolates, 300 µmol glucosinolates, or 75 µmol isothiocyanates daily) for 7 days in 12 healthy 
volunteers did not demonstrate any clinical adverse events [2].  Two of 12 subjects assigned to active 
treatment demonstrated an increase in plasma ALT exceeding the upper limit of normal, though ALT 
levels rose for all subjects during the course of the study (including those assigned to placebo control), 
and the increase was not clearly attributable to consumption of BSE.  Three of 12 subjects (2 active 
treatment, 1 placebo) demonstrated TSH levels exceeding the upper limit of normal during or after the 
dosing period, though elevated TSH was not associated with clinical symptoms or T3/T4 abnormalities.   
 
Egner et al. [3] conducted a cross-over trial in 50 participants, randomized to ingest a sulforaphane-rich 
beverage (150 µmol/day) for 7 days in a first or second phase.  Two of 50 participants complained of 
bitter taste or nausea, and no other adverse events were reported.  No abnormal serum chemistry values 
were reported after 7 days.  In a study of healthy participants ingesting a beverage containing 600 µmol 
of glucoraphanin and 40 µmol of sulforaphane, the intervention was well-tolerated with no laboratory 
evidence of effect on liver and kidney function [4]. 
 
A study of orally administered broccoli sprout homogenates (25 to 300 g) in 57 subjects over 3 separate 
days found no adverse effects aside from mild digestive effects [5].  Similarly, in a RCT of 20 men with 
recurrent prostate cancer treated with sulforaphane 200 µmol/ day for 20 weeks, no serious adverse 
events occurred, though some reported mild gastrointestinal upset [6]. 
 
A randomized, placebo-controlled trial of the sulforaphane product Prostaphane® in 81 men with rising 
PSA values following radical prostatectomy for prostate cancer represents the highest daily dosing of 
sulforaphane to date (338 µmol) [7].  No serious adverse events were reported, though gastrointestinal 
bloating was experienced somewhat more often in the SF group (N=17 vs. N=10), and one participant in 
the SF group withdrew from the study due to bowel discomfort.  The differences in symptoms between 
the two groups were not statistically significant (p=0.149).  Furthermore, no significant between-group 
differences were found for body weight, BMI, blood counts, and chemistries. 
 
In a RCT of 45 male subjects with autism, ages 13-30, administered sulforaphane 50-150 µmol/day for 
16 weeks, no significant differences in adverse events or safety labs (blood counts, chemistries, liver 
function tests, TSH) were reported [1].   Two subjects with a history of seizures had single unprovoked 
seizures during the course of the study (one after 3 weeks on sulforaphane, one 3 weeks after 

Clinical Protocol: Sulforaphane for ASD 
- 51 - 
 
discontinuing sulforaphane), and the possibility of seizure as a potential adverse effect of sulforaphane 
could not be ruled out. 
 
One subject enrolled in the current trial experienced a worsening of behavioral symptoms that may or 
may not have been directly study related. 
 
Finally, there is some evidence from animal studies and in vitro studies suggesting that sulforaphane 
and/or its metabolites can inhibit expression of CYP 1A2, 3A4, and 2D6, which could be a potential 
source of drug-drug interactions if the participant is taking medications that are substrates of these 
enzymes.  The risk of possible drug-drug interactions is not well-established.  Upon reviewing the 
participants concurrent medications at screening and all subsequent visits, the study physician will 
consult an extensive P450 Table to identify possible drug-drug interactions. If the participant develops 
significant adverse effects that appear consistent with increased exposure to a concurrent medication 
metabolized by 1A2, 3A4, or 2D6, the dose of SF will be lowered by 30% as specified in the 
protocol.  If the adverse events persist 14-day post-reduction, SF may be permanently discontinued at 
the discretion of the study physician, participant, and guardian. 
 
In summary, based on available data from human clinical trials of SF-rich compounds, SF appears to be 
extremely well-tolerated with minimal risk aside from mild gastrointestinal symptoms, which may 
include bloating, nausea, and loose stools.  No significant changes in blood counts, serum chemistries, 
liver function, kidney function, or thyroid function are anticipated.  As it is unclear if sulforaphane may 
lower the seizure threshold in those with a history of seizures, our study will exclude those with a 
history of seizure within the previous year.  As with any medication, there is a theoretical risk of allergic 
reaction, and participants and their caregivers will be instructed on how to identify possible signs of 
allergic reaction.  Additional risks include mild physical discomfort, psychological distress, or mild 
bruising and bleeding associated with phlebotomy.  Participants will be offered the use of a local 
anesthetic (EMLA cream) prior to blood draw to minimize physical discomfort, and there is a small risk 
of transient local reaction to EMLA cream, including paleness, erythema, and edema.  Many individuals 
with ASD experience oral sensory aversions, and some could find taking multiple medication tablets on 
a daily basis unpleasant.   
 
Though every effort will be taken to ensure the protection of confidential information, there is a small 
risk to privacy in the event that security measures are breeched.  In addition, participants may experience 
some mild anxiety or psychological distress in answering questions about their psychiatric and medical 
history.   
 
II. 
ADEQUACY OF PROTECTION AGAINST RISKS 
 
a. 
Recruitment and Informed Consent:  Subjects will primarily be recruited from the Carolina 
Institute for Developmental Disabilities Research Subject Registry, which includes more than 6000 
families that have expressed an interest in research participation and have consented to be contacted 
about potential studies.  Potential subjects will be contacted directly via email or letter by the Registry 
staff with information about the trial, including contact information of study staff.  Participants will also 
be recruited from the clinical practices of physicians, psychologists, and allied health staff at the 
Carolina Institute for Developmental Disabilities as well as from referring providers.  Electronic and 
written advertisements will be disseminated to local community autism support agencies (e.g., Autism 

Clinical Protocol: Sulforaphane for ASD 
- 52 - 
 
Society of North Carolina, Autism Resource Centers).  All advertisement materials will first be 
approved by the University of North Carolina IRB.   
 
Participants and their caregivers/ Legally Authorized Representatives (LARs) will be provided with a 
written copy of the Informed Consent document to read thoroughly prior to the initial screening visit.  
Due to social communication and cognitive deficits inherent in ASD, we anticipate that most 
participants, including those 18 years and older, will not be capable of providing adequate informed 
consent.  For subjects unable to provide informed consent, either due to age or cognitive disability, 
every effort will be made to obtain written informed assent.  To enhance the ability of participants to 
participate meaningfully in the consent/ assent process, a novel electronic tablet-based consent tool will 
be utilized during the consent process.  Informed consent from the caregiver or participant will be 
obtained prior to conducting any study-related measures or activities.  Participants or their LARs will 
sign a separate stored specimen consent form for the de-identified storage of a saliva specimen for 
possible future genetic analysis. Subjects will still be allowed to participate in the primary clinical trial 
if they do not consent for specimen storage. 
 
b. 
Protection against risk: Risks to participants will be minimized through frequent follow-up and 
careful consideration of all reported symptoms and concerns of participants and their caregivers.  The 
study PI or a designated physician colleague will be available by pager and/or mobile phone 24/7 for 
any emergencies or urgent concerns about potential adverse events during the course of the study.  
Adverse events will be monitored and recorded at each study visit and with a phone call at week 2 after 
beginning the study intervention.  Safety labs (complete blood count with differential, chemistries, liver 
function tests, and thyroid stimulating hormone) will be obtained at screening and again at the 
completion of the active treatment phase, and the data will be reviewed periodically by the DSMB as 
described below.   
 
The minor risks associated with phlebotomy will be minimized through use of calming behavioral 
strategies, offering topical anesthetic prior to blood draw, and having the blood draw performed by 
professionals with extensive experience in working with individuals with ASD.  The maximum amount 
of blood drawn at any visit will not exceed 2 ml/kg body weight.  To minimize risk of psychological 
distress associated with routine assessment and answering questions about personal history, every effort 
will be made to provide a relaxed atmosphere with as little waiting time as possible; breaks will be 
offered as needed.  Confidentiality will be protected by limiting identifiable personal information on 
source documents, which will be labeled with a designated participant number and initials only.   Paper 
charts will be maintained in a locked file cabinet behind two locked doors and will only be accessed by 
staff directly involved in the study and monitors from regulatory agencies.  Electronic data files will be 
protected by password and encryption, and only study staff directly involved in data collection/ entry 
and monitors from regulatory agencies will have access to them.  Saliva specimens will be stored in a 
locked cabinet and labeled with a personal identifier number rather than patient name, and the key will 
be maintained in an encryption-protected digital file accessed only by study staff and regulatory 
monitors. 
 
If a participant experiences significant adverse events that affect tolerability, may reasonably be related 
to taking the investigational product, and do not meet the protocol definition for a serious AE (including 
non-extreme behavioral symptoms), the dose may be reduced by approximately 30% according to the 
following dose modification schedule: 

Clinical Protocol: Sulforaphane for ASD 
- 53 - 
 
 
 
 
 
 
 
 
Weight 
category 
Prescribed weight-
based dose of 
glucoraphanin 
Prescribed # of 
tablets 
70% of weight-
based dose (ie, 
30% reduction) 
Prescribed # 
tablets for 
reduced dose 
(rounded to 
nearest 15 mg 
increment)* 
<100 lb 
45 mg 
3 
31.5 mg 
2 
100-125 lb 
75 mg 
5 
52.5 mg 
3 
126-175 lb 
90 mg 
6 
63 mg 
4 
176-199 lb 
105 mg 
7 
73.5 mg 
5 
≥ 200 lb 
120 mg 
8 
84 mg 
6 
 
*Rounded down if equidistant between two 15 mg dosing increments 
 
If the dose is reduced during the course of the study, the participant will continue the lowered dose for 
the remainder of the study.  If adverse events do not improve within 14 days of lowering the dose, the 
investigational product may be permanently discontinued at the discretion of the treating study 
physician, participant, and participant’s guardian. If a participant exhibits extreme exacerbation of 
behavioral symptoms (such as self-injurious behavior or aggression) that do not improve substantially 
following a dose reduction and are determined “probably” or “definitely” related to taking the 
investigational product, the investigational product may be permanently discontinued sooner than 14 
days post-reduction, and the DSMB will be notified.  As individuals with ASD often have cyclical 
changes in behavior independent of medication effects, the study staff will rely on caregivers’ 
experience with the participant to determine if any observed behavior changes are within the expected 
range for that individual.  If the investigational product is stopped permanently, the participant and his 
caregiver will be asked to complete the remainder of the study visits, return any unused study drug, and 
repeat safety lab studies. 
 
There is some evidence from animal studies and in vitro studies suggesting that sulforaphane and/or its 
metabolites can inhibit expression of CYP 1A2, 3A4, and 2D6, which could be a potential source of 
drug-drug interactions if the participant is taking medications that are substrates of these enzymes.  The 
risk of possible drug-drug interactions is not well-established.  Upon reviewing the participants 
concurrent medications at screening and all subsequent visits, the study physician will consult an 
extensive P450 Table to identify possible drug-drug interactions.  If the participant develops significant 
adverse effects that appear consistent with increased exposure to a concurrent medication metabolized 
by 1A2, 3A4, or 2D6 and do not improve with a 30% investigational product dose reduction within 14 
days, discontinuing the investigational product may be recommended.   
 

Clinical Protocol: Sulforaphane for ASD 
- 54 - 
 
If a participant has a seizure while taking the study medication, the study medication will be stopped, 
and the study PI and DSMB will be notified.  The participant will be evaluated by the study PI and his 
primary care physician, treated, and monitored appropriately.  The participant and his caregiver will be 
asked to return to complete the remainder of the study visits, return any unused study drug, and repeat 
safety lab studies. 
 
If the participant newly requires a long-term medication that is thought to interact adversely with the 
study medication, the study medication will be stopped.  The participant and his caregiver will be asked 
to return to complete the remainder of the study visits, return any unused study drug, and repeat safety 
lab studies. 
 
If a participant experiences a serious adverse event that is thought to be related to taking the study 
medication (as determined by an independent medical monitor, the study medication will be 
permanently discontinued, and the appropriate overseeing regulatory organizations will be notified 
(FDA, IRB, and DSMB). The participant and his caregiver will be asked to return to complete the 
remainder of the study visits, return any unused study drug, and repeat safety lab studies. 
 
III. 
POTENTIAL BENEFITS OF THE PROPOSED RESEARCH TO THE SUBJECTS AND 
OTHERS 
 
Preliminary evidence suggests that sulforaphane can improve social relatedness and have an overall 
calming effect for some individuals with ASD [1].  Treatment with sulforaphane may also improve 
mitochondrial dysfunction and redox imbalance, both of which have been implicated in autism.  Broader 
benefits for the autism community include new knowledge about a safe and potentially efficacious 
treatment for core symptoms of autism, for which current treatment options are limited. 
 
IV. 
IMPORTANCE OF THE KNOWLEDGE TO BE GAINED 
 
ASD is a neurodevelopmental disorder that is now estimated to affect 1 in 68 children in the U.S., 
including 1 in 42 boys [8], and leads to substantial, lifelong burden for individuals, families, and 
communities.  Use of psychiatric medication in ASD is high with 66% of affected individuals taking at 
least 1 psychotropic medication by adolescence [9]. Faced with heavy side effect burden and often 
limited efficacy, caregivers frequently turn to supplements that promise low toxicity but lack rigorous 
investigation and oversight. To date, no pharmacological agents have indication for the treatment of core 
social communication impairments in autism, and safe, evidence-based, rationally-developed treatments 
for ASD are greatly needed.  Sulforaphane has shown initial promise as a well-tolerated and potentially 
efficacious treatment for ASD in a small clinical trial [1], though this finding warrants replication before 
being adopted into clinical practice more widely.  The form of sulforaphane used in the ASD study by 
Singh et al. [1] is also prohibitively expensive to manufacture on a large scale and no longer produced in 
the United States, and Avmacol© (Nutramax, Inc), a sulforaphane precursor, is a far less costly and 
more readily available alternative.  It is not yet clear if a sulforaphane precursor (and converting 
enzyme) will have benefit similar to direct ingestion of sulforaphane, and the currently proposed study 
will contribute to knowledge about the efficacy and safety of this widely available nutraceutical for the 
treatment of ASD. 
 
 

Clinical Protocol: Sulforaphane for ASD 
- 55 - 
 
V. 
DATA AND SAFETY MONITORING PLAN 
a. 
Definitions of Unanticipated Problems, Adverse Events, and Serious Adverse Events 
1. 
Unanticipated Problems Involving Risk to Subjects or Others is any incident, 
experience, or outcome that meets all of the following criteria:  
 
Unexpected in nature, severity, or frequency  (i.e. not described in study-related 
documents such as the IRB-approved protocol or consent form, the investigator’s brochure, etc.), 
 
Related or possibly related to participation in the research (i.e. possibly related means 
there is a reasonable possibility that the incident experience, or outcome may have been caused 
by the procedures involved in the research, 
 
Serious (as defined below)  “Serious” is different than “severe” as reported in the CTC 
criteria that applies a grade to the AE. 
 
2. 
An adverse event (AE) is any symptom, sign, illness or experience that develops or 
worsens in severity during the course of the study.  Intercurrent illnesses or injuries will be 
regarded as adverse events.  Abnormal results of diagnostic procedures (eg, laboratory values) 
are considered to be adverse events if the abnormality: 
 
results in study withdrawal 
 
is associated with a serious adverse event 
 
is associated with clinical signs or symptoms 
 
leads to additional treatment or to further diagnostic tests 
 
is considered by the investigator to be of clinical significance 
 
3. 
 A serious adverse event is any AE that is:  
 
fatal 
 
life-threatening 
 
requires or prolongs hospital stay 
 
results in persistent or significant disability or incapacity 
 
a congenital anomaly or birth defect 
 
an important medical event (of major clinical significance and may require intervention 
to prevent one of the serious outcomes noted above) 
 
All adverse events that do not meet any of the criteria for serious will be regarded as non-serious 
adverse events.  
 
b. 
Monitoring of adverse events: The study physician will review AEs with the participant and 
family member at each study visit (baseline, week 2, week 4, week 8, week 12, week 16), and patients 
and their families will have 24/7 access to the study PI or a designated covering physician by phone/ 
pager to report possible AEs as needed between visits.  AEs will be recorded on an AE log and assigned 
a numerical grade of 0 (not at all a problem), 1 (mild problem), 2 (moderate problem), and 3 (severe 
problem).  As previously noted, an AE rated as severe (“3”) is distinct from classification of a serious 
AE, defined above.  The study period during which adverse events must be reported is defined as the 
period from the initiation of any study procedures to the end of the study treatment follow-up.  For this 

Clinical Protocol: Sulforaphane for ASD 
- 56 - 
 
study, the study treatment follow-up is defined as 30 days following the last administration of study 
treatment.  
 
 
A preexisting condition is a reported symptom, clinically significant physical exam finding, or 
laboratory finding that is present at the start of the study and will be recorded as an adverse event if the 
frequency, intensity, or the character of the condition worsens during the study period.  At interval study 
visits and at the end of the study, any new clinically significant findings/abnormalities that meet the 
definition of an adverse event will also be recorded and documented as an adverse event.  
 
All unresolved adverse events will be followed by the investigator until the events are resolved, the 
subject is lost to follow-up, or the adverse event is otherwise explained.  At the last scheduled visit, the 
study physician will instruct each subject to report any subsequent event(s) that the subject, or the 
subject’s personal physician, believes might reasonably be related to participation in this study.   
 
c. 
Entity responsible for monitoring: The study PI or a designated Co-Investigator will be the 
primary individual responsible for eliciting, recording, and monitoring AEs, and the study PI will have 
the ultimate responsibility of responding to AEs and reporting serious AEs to monitoring agencies (e.g., 
IRB, DSMB, FDA) in a timely manner.  Additional monitoring entities for this study will include the 
University of North Carolina IRB and the North Carolina Translational and Clinical Science (NC 
TraCS) Institute Data and Safety Monitoring Board (DSMB). 
Any study-related unanticipated problem posing risk of harm to subjects or others, and any type of 
serious adverse event (SAE), will be reported to the FDA within 7 calendar days using FDA Form 
3500A for mandatory reporting.  SAEs will also be reporting to the UNC IRB by the study PI via IRBIS 
(the IRB information system for UNC) as well as the NC TraCS DSMB within 7 calendar days of 
becoming aware of the adverse event.  Significant new information about ongoing serious adverse 
events will be provided promptly to the FDA, UNC IRB, and DSMB. 
 
The NC TraCS DSMB will consist of a chair, an ethicist, an epidemiologist, a biostatistician, and one or 
more clinical researchers, each serving three year terms (please see attached TraCS DSMC Charter for a 
listing of current Committee members).  The DSMB will review all SAEs, which the study PI will 
submit to the DSMB within 7 calendar days of the event.  The DSMB will also review safety 
information (baseline and end-of-treatment screening labs; adverse event forms; significant physical 
exam findings) after every 10 participants have completed the study.  All data presented to the DSMB 
will be de-identified to protect the confidentiality of participants.  Due to the relatively small size and 
anticipation of minimal risk associated with this study, no interim data analysis is planned.  The DSMB 
will report their findings to the UNC Biomedical IRB as appropriate. 
 
Though not expected, any of the following events will also be reported to the UNC IRB: 
 
Any adverse experience that, even without detailed analysis, represents a serious unexpected 
adverse event that is rare in the absence of drug exposure (such as agranulocytosis, hepatic 
necrosis, Stevens-Johnson syndrome). 
 
Any adverse event that would cause the sponsor-investigator to modify the investigators brochure, 
protocol, or informed consent form, or would prompt other action by the IRB to assure protection 
of human subjects. 

Clinical Protocol: Sulforaphane for ASD 
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 
Information that indicates a change to the risks or potential benefits of the research, in terms of 
severity or frequency. For example, safety monitoring indicates that a particular side effect is 
more severe, or more frequent than initially expected. 
 
Breach of confidentiality 
 
Change to the protocol taken without prior IRB review to eliminate apparent immediate hazard to 
a research participant. 
 
Incarceration of a participant when the research was not previously approved under Subpart C 
and the sponsor-investigator believes it is in the best interest of the subject to remain on the study. 
 
Complaint of a participant when the complaint indicates unexpected risks or the complaint cannot 
be resolved by the research team. 
 
Protocol violation (meaning an accidental or unintentional deviation from the IRB approved 
protocol) that in the opinion of the principal investigator placed one or more participants at 
increased risk, or affects the rights or welfare of subjects. 
 
 
VI. 
References 
1. 
Singh, K., et al., Sulforaphane treatment of autism spectrum disorder (ASD). Proc Natl Acad Sci U S A, 
2014. 111(43): p. 15550-5. 
2. 
Shapiro, T.A., et al., Safety, tolerance, and metabolism of broccoli sprout glucosinolates and 
isothiocyanates: a clinical phase I study. Nutr Cancer, 2006. 55(1): p. 53-62. 
3. 
Egner, P.A., et al., Bioavailability of Sulforaphane from two broccoli sprout beverages: results of a short-
term, cross-over clinical trial in Qidong, China. Cancer Prev Res (Phila), 2011. 4(3): p. 384-95. 
4. 
Egner, P.A., et al., Rapid and sustainable detoxication of airborne pollutants by broccoli sprout beverage: 
results of a randomized clinical trial in China. Cancer Prev Res (Phila), 2014. 7(8): p. 813-23. 
5. 
Riedl, M.A., A. Saxon, and D. Diaz-Sanchez, Oral sulforaphane increases Phase II antioxidant enzymes 
in the human upper airway. Clin Immunol, 2009. 130(3): p. 244-51. 
6. 
Alumkal, J.J., et al., A phase II study of sulforaphane-rich broccoli sprout extracts in men with recurrent 
prostate cancer. Invest New Drugs, 2015. 33(2): p. 480-9. 
7. 
Cipolla, B.G., et al., Effect of Sulforaphane in Men with Biochemical Recurrence after Radical 
Prostatectomy. Cancer Prev Res (Phila), 2015. 
8. 
Prevalence of autism spectrum disorder among children aged 8 years - autism and developmental 
disabilities monitoring network, 11 sites, United States, 2010. MMWR Surveill Summ, 2014. 63(2): p. 1-
21. 
9. 
Coury, D.L., et al., Use of psychotropic medication in children and adolescents with autism spectrum 
disorders. Pediatrics, 2012. 130 Suppl 2: p. S69-76. 
 
 

Clinical Protocol: Sulforaphane for ASD 
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17 
Appendix 6C: NC TraCS DSMB Charter 
 
 
 
 
North Carolina Translational and 
Clinical Sciences (TraCS) Institute 
 
 
 
 
 
 
 
 
Data Safety Monitoring Board (DSMB) Charter 
 
 
 
 
 
 
 
 
 
 
 
 
 
Revised 10-19-2015 
 

Clinical Protocol: Sulforaphane for ASD 
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NC TraCS Institute 
 
Data and Safety Monitoring Board Charter 
 
Table of Contents 
 
Introduction 
 
Organization 
 
Overview 
 
 
Committee Structure 
 
DSMB Functions and Activities 
 
Guidelines for Members 
 
Process 
 
 
Eligibility 
 
Reporting 
 
Relation to Biomedical Institutional Review Board and Lineberger Oncology Protocol Review 
 
Committee 
 
Protocol Review and Data Monitoring 
 
 
Monitoring and Reporting Requirements 
 
Data Review by the DSMB 
 
Release of DSMB Recommendations 
 
Appendices 
 
 
Appendix 1:  Current members of the DSMB 
 
Abbreviations 
 
AE 
 
Adverse event 
DSMB  
Data and Safety Monitoring Board, NC TraCS Institute  
DSMC  
Lineberger Data and Safety Monitoring Committee, PRC 
CTRC  
Clinical and Translational Research Center 
IRB 
UNC Biomedical Institutional Review Board 
NIH 
 
National Institutes of Health 
PI 
 
Principal Investigator 
PRC 
 
UNC Lineberger Oncology Protocol Review Committee 
 
TraCS  
North Carolina Translational and Clinical Sciences Institute 
 
UNC 
 
University of North Carolina at Chapel Hill 
 
 
 

Clinical Protocol: Sulforaphane for ASD 
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Introduction 
 
The North Carolina Translational and Clinical Sciences (TraCS) Institute is committed to the safety of patients 
participating in clinical trials at our institution.  In addition, it is committed to data accuracy and protocol 
compliance.  The NC TraCS Institute has established an institutional plan to provide data safety and monitoring 
for selected clinical trials conducted at UNC.  This plan is designed to comply with policies and guidelines 
regarding data and safety monitoring from the National Institutes of Health 
(http://grants.nih.gov/grants/guide/notice-files/not98-084.html; http://nih.gov/grants/guide/notice-files/NOT-OD-00-
038.html).   
 
This charter is for the Data Safety and Monitoring Board (DSMB) of the North Carolina TraCS Institute.  The 
charter contains the following: 
 
A description of the composition and organization of the DSMB 
 
Details the roles and responsibilities of DSMB members 
 
Outlines the responsibilities of the Principal Investigators and Sponsors 
 
Lists important contact persons 
 
Organization 
 
Overview 
The DSMB functions as a committee within the NC TraCS Institute.  The charge to the DSMB is review clinical 
trials to assure patient safety both by evaluating adverse events (AEs) and interim analyses of both safety and 
efficacy. 
 
Committee Structure 
The DSMB Chair (currently Dr. Ross Simpson) has the overall responsibility for the chairing the DSMB committee 
and reporting its findings to the UNC Biomedical IRB.  The Committee will be under the direct supervision of the 
NC TraCS Institute Director or designated representative. The Committee will consist of a chair, an ethicist, an 
epidemiologist, a biostatistician, and one or more clinical researchers.  Committee members will serve for 3-year 
terms, but may be reappointed.  Members will be appointed to the Committee by the Chair, Director or designated 
representative.  A member of the DSMB can be removed due to poor attendance, inadequate demonstration of 
effort, unprofessional conduct and /or failure to act in accordance with the objectives of the DSMB.  A list of 
current DSMB members can be found in Appendix 1.   
 
PROCESS 
 
Eligibility 
A clinical trial is defined as “a prospective study involving human subjects designed to answer specific questions 
about the effects or impact of particular biomedical or behavior interventions; these may include drugs, 
treatments, devices, or behavior or nutritional strategies.”  Studies that include nutritional, behavioral, and 
psychosocial interventions are considered to be clinical trials.  Studies evaluating diagnostics (imaging, etc.) in 
which findings alter the patient’s clinical care are also considered to be clinical trials.  Observational studies, 
epidemiologic studies, studies of diagnostics that do not effect patient care, and studies that do not test 
interventions are not considered to be clinical trials. 
 
DSMB Functions and Activities 
The DSMB is responsible for reviewing data from clinical trials approved by the UNC Biomedical IRB.  
Investigators with commercially or governmentally sponsored trials will normally work with their sponsor’s DSMB.  
The DSMB will review data from the following types of trials, when review is deemed necessary by the Biomedical 
IRB or requested by the Principal Investigator (PI), in order to ensure the safety of participating subjects. 
 
Phase I, I/II, II, and II/III trials when such review is deemed necessary by the UNC Biomedical Institutional 
Review Board and the Principal Investigator.    
 
Phase III clinical trials (single site trials where the PI is a UNC faculty member and for which UNC is the 
sponsor) 

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 
Phase IV clinical trials (single site trials where the PI is a UNC faculty member and for which UNC is the 
sponsor) 
 
Select multicenter clinical trials in which UNC is the coordinating center or the PI of the study is a UNC faculty 
member IF the DSMB determines that it has adequate resources to conduct the monitoring required of the 
study.  
 
The DSMB will meet at least every other month and has the following responsibilities. 
 
Assessing risk and complexity of clinical trials submitted for review 
 
Determining the appropriate level of data and safety monitoring 
 
Reviewing serious adverse events (AEs) reports when requested.   
 
Reviewing yearly data and safety monitoring reports when requested. Recommending appropriate actions 
(closure, increased monitoring, etc.) to the Principal Investigator, UNC Biomedical IRB, and the Director or 
designated representative. 
 
Communicating its finding with the Biomedical IRB  
 
Preparing minutes for all meetings 
 
Preparing an annual summary of activity for review by the Director, NC TraCS Institute 
 
Review of the monthly reports of the UNC Lineberger Oncology Protocol Review Committee (PRC) -Data and 
Safety Monitoring Subcommittee (DSMS) 
 
Assessing ethical and scientific soundness of trials reviewed 
 
DSMB-Investigator Communications 
The DSMB will make available to investigators the following: 
 
An Annual Data Reporting form for annual reporting of all AEs 
 
A Data Safety Monitoring Plan template  
 
Principal Investigators shall make available to the DSMB the following information: 
 
A copy of the approval letter from the UNC Biomedical Institutional Review Board 
 
A Data Safety Monitoring Plan  
 
All adverse event reports using the most current UNC Biomedical Institutional Review Board form 
 
An annual summary of all AEs 
 
Relation to Biomedical Institutional Review Board and Lineberger Oncology Protocol Review Committee 
The DSMB will report its findings and make recommendations to the UNC Biomedical IRB.  For research 
conducted by the Lineberger Cancer Center, the DSMB will report its findings and make recommendations to the 
UNC Lineberger Oncology Protocol Review Committee (PRC).  Trials required to have an independent data and 
safety monitoring board as defined in this charter are expected to use the NC TraCS Institute DSMB instead of 
establishing their own independent board, unless outside expertise not available on the DSMB is necessary to 
adequately monitor the trial.   
 
Notice of a recommendation of early closure or suspension will be reported directly to the Biomedical IRB and the 
Director or designated representative.  As appropriate, such recommendations will be reported to the Director of 
the Lineberger Cancer Center and the Director of the Clinical Translational Research Center.  The Chair of the 
DSMB in conjunction with the Chair of the Biomedical IRB is responsible for seeing that these trials are closed to 
accrual. 
 
Guidelines for Members 
In order for the DSMB to fulfill its responsibilities, the member will observe the following guidelines: 
 
Members are free of apparent conflicts of interest involving financial, scientific, or regulatory matters.  In case 
of any question of conflict of interest, standards used by NIH in determining conflict of interest for advisory 
committee members and investigators shall apply 
 
Members should assess trial objectives and design in an unbiased way. 
 
Members are guided both by pre-specified study performance criteria, such as early stopping rules, and also 
by a masked and, if necessary, unmasked review of all data prior to making decisions 
 
The DSMB members will review data and pertinent procedures in order to be confident that the data on which 
the decisions are based are accurate and complete 

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 
All decisions of the DSMB shall be independent 
 
PROTOCOL REVIEW AND DATA MONITORING 
 
Monitoring and Reporting Requirements 
In collaboration with the Principal Investigator and study biostatistician, the DSMB will set data monitoring and 
reporting requirements before study enrollment begins.  Members will review the interim analysis plan to ensure 
the analyses planned will provide necessary information for DSMB decisions.  Members will participate in 
specification and review of all tables and specifications for data that they will review in accordance with timelines 
established by agreement with the Principal Investigator. 
 
The DSMB will have face-to-face meeting(s) to review and discuss results of interim analysis.  The DSMB may 
also have face-to-face meeting(s) as prompted by unplanned interim analyses deemed necessary because of 
safety concerns.  The DSMB staff or chair will prepare minutes of each meeting within ten (10) working days of 
each meeting.  These minutes will be circulated and approved at the next regularly scheduled meeting of the 
DSMB.   
 
The Principal Investigator and study biostatistician will work with the DSMB for appropriate preparation of reports 
to be viewed at DSMB meetings and resolution of questions arising during data analysis.  If requested by the 
DSMB, the Principal Investigator will turn over to the chairperson of the DSMB evidence of validation of all 
computer programs used to generate reports and analyses for each DSMB meeting.  The documents will be 
made part of the minutes of each meeting.  All formal reports will be circulated to the DSMB members no later 
than one week prior to each DSMB meeting. 
 
Data Review by the DSMB 
DSMB meetings will consist of an open and a closed session.  The open session will provide a forum for 
exchange of information among DSMB members, Principal Investigator, and study biostatistician.  During the 
open session, the Principal Investigator may present a brief summary report of the study progress, including 
enrollment rates, data collection, and data quality.  There will be an opportunity for the Principal Investigator to 
ask advice from the DSMB on any matter concerning conduct of the trial. 
 
Only members of the DSMB will attend the closed session.  During this session, the DSMB will address issues 
regarding the following: 1) safety concerns, 2) efficacy concerns, 3) termination of the trial due to pre-specified 
stopping criteria, and 4) ethical concerns.  The DSMB will be furnished with relevant information by the Principal 
Investigator to make these decisions.  During this session the DSMB members may ask the Principal Investigator 
to provide them with data that is partially unmasked (i.e., treatment A or treatment B without revealing what 
treatment A and B represent) or completely unmasked (i.e., identify treatment group). 
 
In addition to the open and closed sessions, the voting members of the DSMB may meet in an executive session 
at their discretion.  The DSMB will: 
 
Review the protocol and any protocol amendments 
 
Review the interim analysis monitoring plan, make recommendations, and give approval 
 
Review interim analysis reports and meet as a group 
 
Communicate recommendations in writing to the Principal Investigator 
 
If there are any concerns about reviewed material (such as safety, efficacy, ethics, and stopping rules), the DSMB 
will take appropriate action.  This may involve a request for additional information, or a request for an early, 
unscheduled meeting of the DSMB with the Principal Investigator and study biostatistician.  At each meeting that 
includes review of interim data, the DSMB will recommend one of the following actions to the Principal 
Investigator, Biomedical IRB, and when appropriate the NC TraCS Institute Director or designated representative. 
 
Continue the study according to the protocol 
 
Modify the study protocol.  Modifications may include such items as changes in the inclusion/exclusion 
criteria, nature and frequency of safety monitoring, study procedures, study drug/intervention dosing, consent 
form changes, subject re-notification, and any other changes deemed necessary 
 
Discontinue one or more study arms 

Clinical Protocol: Sulforaphane for ASD 
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 
Discontinue the study 
 
The DSMB may request additional data, analyses, or meetings to address specific concerns.  The DSMB may 
hold additional meetings without knowledge of the sponsor or Principal Investigator. 
 
Release of DSMB Recommendations 
The DSMB will refrain from revealing to the sponsor, Principal Investigator, or any other party information that 
would lead to compromising the integrity of the trial unless such a release is required to protect subject safety.  In 
particular, the following guidelines will be followed with respect to the dissemination of the DSMB interim analysis 
results. 
 
Individual patient treatment assignments will not be revealed 
 
Individual center results will not be revealed 
 
The magnitude of treatment differences in efficacy will not be revealed 
 
Study results will not be communicated to investigators 
 
The DSMB will not make any public disclosures of its discussions and decisions 
 
The DSMB will complete a brief report documenting decisions and rationale behind its decisions.  The report 
will be conveyed confidentially to the Principal Investigator and sponsor, who in turn, will forward it to 
appropriate regulatory agencies.  A copy of the report will also be made available to the UNC Biomedical 
Institutional Review Board, in form that does not contain information that could compromise the integrity of the 
clinical trial. 
 
When appropriate a copy of the report will also be made available to the Director of the Lineberger Cancer 
Center and/or the Director of the Clinical Translational Research Center.   
 
All DSMB decisions will be deemed advisory to the Principal Investigator, UNC Biomedical Institutional 
Review Board, and the Director, NC TraCS Institute. 
 

Clinical Protocol: Sulforaphane for ASD 
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Appendix 1  NC TraCS Institute DSMB Members, Year 2013 - 2015 
 
Member 
 
 
 
Department/School 
Email 
 
Ross J. Simpson, Jr., MD, PhD (chair) 
Medicine 
 
rsimpson@med.unc.edu 
 
David Weber, MD, MPH  
 
Medicine 
 
dweber@unch.unc.edu 
 
David Couper, PhD 
 
 
Biostatistics 
 
david_couper@unc.edu 
 
Marcia Van Riper, RN, PhD 
 
School of Nursing 
vanriper@email.unc.edu 
 
Kenneth Ataga, MD 
 
 
Medicine 
 
kenneth_ataga@med.unc.edu 
 
Marie Rape, RN, BSN  
 
NC TraCS Institute 
marie_rape@med.unc.edu 
 
Gretchen Stuart, MD, MPH 
 
Obstetrics/Gynecology gstuart@med.unc.edu  
 
Samantha Meltzer-Brody, MD, MPH 
Psychiatry 
 
samantha_meltzer-brody@med.unc.edu 
 
 
 

Clinical Protocol: Sulforaphane for ASD 
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18 
Appendix 6D: Clinical Studies List 
 
 
Other Clinical Studies with Oral and Topical Broccoli Sprout Extracts 
 
Approved IND Applications: 
 
1.  Andrew Nel (UCLA) entitled: “Effect of broccoli sprout extracts on pulmonary function in 
volunteers exposed to diesel exhaust particles.”  
IND# 76,411 
 
2.  Robert H. Brown (Anesthesiology, Johns Hopkins University School of Medicine): 
NA_00011275, entitled: “The effect of broccoli sprout extract on asthma.”  Approved 3/14/2008 
JHM-IRB5 
IND# 79, 274 
 
3.  Kala Visvanathan (Oncology and Epidemiology, Johns Hopkins Bloomberg School of Public 
Health), entitled:“Effect of broccoli sprout extracts on breast cancer.” 
IND# 103,019 
 
4.  Yuesheng Zhang (Roswell Park Cancer Institute, Buffalo, NY), entitled: “A Phase I pre-
cystectomy trial of broccoli extract in patients with advanced bladder cancer.” 
IND# 103,466 
 
5.  Paul Talalay (Pharmacology, Johns Hopkins University School of Medicine) NA_00018779, 
entitled: “Effects of topical application of sulforaphane-containing broccoli sprout extracts on 
radiation dermatitis during external beam radiation therapy for breast cancer.” 
IND# 104,164 
 
6.  Paul Talalay (Pharmacology, Johns Hopkins University School of Medicine) RPN 03-05-28-03, 
entitled: “Induction of phase 2 enzymes in skin by broccoli sprout extracts.”  Approved renewal 
7/28/2008 JHM-IRB2. 
Amendment to IND# 104,164 
 
7.  Paul Talalay (Pharmacology, Johns Hopkins University School of Medicine) NA_00004897, 
entitled: “Effects of topical application of sulforaphane-containing broccoli sprout extracts to 
human skin on the dose of UVB radiation required to produce erythema.”  Approved 10/11/2007 
JHM-IRB2  
Amendment to IND# 104,164 
 
8.  David W. Lin (Fred Hutchinson Cancer Center, Seattle, WA) entitled: “In Vivo effects of 
sulforaphane supplementation on human prostate.”  
IND# 105,321 
 
9.  Robert Wise (Johns Hopkins Asthma and Allergy Center), entitled: “Broccoli sprout extract for 
antioxidant protection in COPD.” 

Clinical Protocol: Sulforaphane for ASD 
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IND# 109,233 
 
10.  Joshi Alumkal (Oregon Health Sciences University), entitled: “The effects of sulforaphane-rich 
broccoli sprout extracts in patients with biochemical recurrence of prostate cancer.”   
IND# 109,782 
 
11.  Jackilen Shannon (Department of Veterans Affairs Medical Center, Portland, OR), entitled: 
“Chemoprevention of prostate cancer, HDAC inhibition & DNA methylation.” 
IND# 111,736 
 
12.  Thomas W. Kensler (Department of Environmental Health Sciences, Johns Hopkins Bloomberg 
School of Public Health), entitled: “Broccoli Sprout Extract for Protection Against Environmental 
Toxins.” 
IND# 112,038. 
 
13.  John M. Kirkwood (University of Pittsburgh Cancer Institute), entitled: “A Pilot Study 
Evaluation of Sulforaphane in Atypical Nevi—Precursor Lesions: Assessment of STAT1 and STAT3 
Risk Markers of Melanoma.” 
IND# 112,691 
 
14.  Paul Talalay (Pharmacology, Johns Hopkins University School of Medicine) NA_00045538, 
entitled: “Bioavailability of Stabilized Sulforaphane (SF) from Broccoli Sprout Extracts (BSE).” 
IND# 113,228 
 
15.  Andrew Zimmerman (Lurie Family Autism Center, Massachusetts General Hospital), entitled: 
“Broccoli Sprout Extract in Autism.” 
IND# 113,542 
 
16.  Li Tang (Roswell Park Cancer Institute, Buffalo, NY), entitled: “A Pilot Study of Broccoli Sprout 
Extract in Patients with Invasive Breast Cancer.” 
IND# 117,001 
 
17.  Paul Talalay (Pharmacology, Johns Hopkins University School of Medicine) NA_00081276, 
entitled: “Prevention of Alcohol Intolerance.” 
IND# 118,023 
 
18. David Alberts (The University of Arizona Cancer Center), entitled: “Pilot study to evaluate the 
signaling pathway modulation demonstrated by topical sulforaphane by acutely exposing human 
skin to solar simulated light.” 
IND# 118,454  
 
19.  Paul Talalay (Pharmacology, Johns Hopkins University School of Medicine), entitled: 
“Prostaphane.” 
IND# 126,335 
 

Clinical Protocol: Sulforaphane for ASD 
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20.  Andrew Zimmerman (University of Massachusetts Medical School), entitled: “Sulforaphane 
treatment of children with autism spectrum disorder (ASD).” 
IND# 127,062 
 
21.  Hua Jin (University of California, San Diego), entitled: “A 6-month randomized placebo 
controlled study to evaluate sulforaphane add-on effects in treatment of first onset and early stage 
schizophrenia.” 
IND# 127,513 
 
22.  Robert Wood (Johnson Medical School, Rutgers University), entitled: “Sulforaphane in autism. 
A treatment trial to confirm phenotypic improvement with sulforaphane in individuals with 
autism.” 
IND# 127,543 
 
 
 
 
 

Clinical Protocol: Sulforaphane for ASD 
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Johns Hopkins Medical Institutions IRB Approved Trials (Without IND Approval): 
 
 
1.  RPN 04-05-18-01, entitled: “Evaluation of the biological effects of broccoli sprout extract in 
smokers.” Initial approval 10/25/2005 (P.I. J. Brahmer) 
 
2.  RPN 04-07-30-01, entitled: “Quantification of sulforaphane and sulforaphane metabolites in 
human prostate tissue after oral administration of broccoli sprout extracts.” Initial approval 
10/20/2004  (P.I. A.W. Partin) 
 
3.  NA_00035554, entitled: “Broccoli sprout extract (BSE) taste test.” Approved 6/10/2010 (P.I. 
J.W. Fahey) 
 
4.  NA_00085573, entitled: “Conversion of Broccoli Sprout Glucosinolates to Isothiocyanates.” 
Initial approval 8/1/2013 (P.I. P. Talalay) 
 
5.  NA_00008645, entitled: “Gastrointestinal conversion of broccoli sprout glucosinolates to 
isothiocyanates.” Approved 10/25/07 (P.I.  P. Talalay) 
 
6.  NA_00088662, entitled: “Conversion of Broccoli Seed Glucosinolates to Isothiocyanates- Dietary 
Supplements.” Initial approval 12/5/2013 (P.I. P. Talalay) 
 
7.  NA_00009109, entitled: “Dietary intervention to ameliorate Helicobacter pylori infection in a 
high risk population.”  Approved 12/15/08 (P.I. J.W. Fahey) 
 
8.  H.18.02.03.11.81, entitled: “Chemopreventive Efficacy of Broccoli Sprouts, and Cross-Over 
Broccoli Sprouts Trial-Qidong. Study of the effects of broccoli sprout extracts on the urinary 
excretion of aflatoxin N7-guanine adducts and phenanthrene tetraol levels.” Approved 3/10/10 
(P.I. T. W. Kensler) 
 
9.  IRB00077442, entitled: “Bioavailability of Sulforaphane from a Glucoraphanin-Rich Broccoli 
Supplement (Avmacol).”  Initial approval 9/22/2015 (P.I. J. W. Fahey) 
 

Clinical Protocol: Sulforaphane for ASD 
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Clinical Trials of Broccoli Sprout Extract Registered in www.ClinicalTrials.gov 
 
1. NCT00252018. The Effect of Broccoli Sprouts as a Nutritional Supplement in the Prevention of 
Cardiovascular Disease. [C. Torp-Pedersen, Department of Cardiology, Bispebjerg Hospital, 
Denmark] The purpose of this study is to investigate whether a daily intake of dried broccoli sprouts 
will improve the endothelial function of the participants as measured by Flow mediated dilation 
(FMD). The dried sprouts are chosen because broccoli sprouts are known as containing large 
amounts of the glucosinolate glucoraphanin which in vitro and in animal models has been shown to 
have a positive effect on the endothelium as measured by NO release. 
Status: Completed 
 
2. NCT00255775. Broccoli Sprout Extract in Preventing Lung Cancer in Smokers [J.Brahmer, Johns 
Hopkins.] The purpose of this study is to investigate the effectiveness broccoli sprout extract has in 
preventing lung cancer in smokers.  
Status: Completed 
 
3.  NCT00607932.  Brassica Vegetables or Indole-3-Carbinol in Treating Patients With PSA 
Recurrence After Surgery for Prostate Cancer [Vanderbilt University].   Eating a diet high in 
vegetables may lower the risk of some types of cancer. Brassica vegetables (such as cabbages, kale, 
broccoli, Brussels sprouts, and cauliflower) and indole-3-carbinol (a substance found in cruciferous 
vegetables) may help lower the risk of prostate cancer recurrence. This randomized clinical trial is 
studying the side effects and how well Brassica vegetables work compared with indole-3-carbinol in 
treating patients with PSA recurrence after surgery for prostate cancer. 
Status: Completed 
 
4. NCT00621309. Sulforaphane as an Antagonist to Human PXR-mediated Drug-drug Interactions. 
[D.L. Eaton, University of Washington, Seattle, WA.]  The purpose of this project is to determine if 
sulforaphane (SFN) can be used to block adverse DDIs that occur when drugs bind to and activate the 
PXR receptor and subsequently induce CYP3A4 activity. This project will determine whether SFN can 
prevent the drug Rifampin from binding to PXR and increasing CYP3A4 activity in humans following 
oral administration of SFN (broccoli sprout extract). 
Status: Unknown 
 
5.  NCT00836186.  Cytokine Expression During Radiation for Breast Cancer [Sidney Kimmel 
Comprehensive Cancer Center].  To assess the magnitude and frequency of changes in 
chemo/cytokine expression in women receiving radiation treatment. To asses the impact of 
race/ethnicity on the magnitude and frequency of changes in chemo/cytokine expression during 
radiation therapy for breast cancer. And finally to assess the interaction between radiation-induced 
chemo/cytokine expression changes, and race/ethnicity, with respect to normal tissue reactions to 
radiation and tumor-associated outcomes. 
Status: Unknown 
 
6. NCT00843167. Broccoli Sprout Extract in Treating Women Who Have Had a Mammogram and 
Breast Biopsy. [J. Shannon, Knight Cancer Institute, Oregon Health and Science University, 
Portland, OR.] This randomized phase II trial is studying how well broccoli sprout extract works in 
treating women with newly diagnosed ductal carcinoma in situ and/or atypical ductal hyperplasia.  

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Status: Completed, has Results 
 
7. NCT00882115. Broccoli Sprout Extract Effects on Allergic Inflammation in the Nose. [A. Nel, 
UCLA and NIAID].  The purpose of this study is to explore the effects of broccoli sprout extract on the 
inflammatory process in the nose caused by diesel exhaust particles, which are present in air 
pollution.  
Status: Completed 
 
8. NCT00894712. Topical Application of Sulforaphane-containing Broccoli Sprout Extracts on 
Radiation Dermatitis. [R.Zellars and P. Talalay, Johns Hopkins]  The purpose of this study is to 
investigate the protective effects of topical sulforaphane-containing broccoli-sprout extracts (BSE) 
on radiation-induced dermatitis in women undergoing external-beam radiation therapy for breast 
cancer. This investigation will employ ionizing rather than ultraviolet radiation. The objective is to 
determine and quantify the effect of topical BSE on radiation-induced skin erythema.  
Status: Active, Not Recruiting 
 
9. NCT00946309. Effects of Sulforaphane on Normal Prostate Tissue. [D.W. Lin, Fred Hutchinson 
Cancer Research Center, Seattle, WA]  The purpose of this study is to identify the biological effects of 
a high-sulforaphane broccoli sprout extract in normal prostate tissue. The investigators hypothesize 
that consumption of high-sulforaphane broccoli sprout extract every other day will inhibit growth of 
prostate cancer cells. 
Status: Enrolling by Invitation 
 
10. NCT00982319. Study to Evaluate the Effect of Sulforaphane in Broccoli Sprout Extract on 
Breast Tissue. [K. Visvanathan, Johns Hopkins.]  The purpose of this research is to examine the effect 
of a broccoli sprout preparation on specific factors in breast tissue that are related to breast cancer 
risk and to assess whether sulforaphane a key component of broccoli sprouts increases the levels of 
protective enzymes in breast tissue. In addition, the investigators will also examine how acceptable 
the broccoli sprouts preparation is to the study participants. 
Status: Completed 
 
11. NCT00994604. The Effects of Broccoli Sprout Extract on Obstructive Lung Disease [R. B. 
Brown, Johns Hopkins.] The purpose of this study is to examine whether broccoli sprout extract can 
effect lung function measurements in individuals with asthma and COPD. 
Status: Completed 
 
12. NCT01008826. Cross-Over Broccoli Sprouts Trial. [T.W. Kensler, Johns Hopkins University.] 
The study hypothesis tested is that broccoli sprouts are effective at altering the urinary levels of 
metabolites of the hepatocarcinogen aflatoxin B1 and of the air-borne pollutant phenanthrene in 
residents of Qidong, PRC, where exposures are unavoidable and high. The study will evaluate which of 
two formulations of broccoli sprouts beverage, glucoraphanin-rich  
 
 

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or sulforaphane-rich, exhibits the best bioavailability and is most effective at modulating the 
biomarkers. 
Status: Completed 
 
13. NCT01108003. Broccoli Sprout Extract in Treating Patients With Transitional Cell Bladder 
Cancer Undergoing Surgery. [Y. Zhang, R. Pili, Roswell Park Cancer Center, Buffalo, NY]. This phase 
I trial is studying the side effects of broccoli sprout extract in treating patients with transitional cell 
bladder cancer undergoing surgery. 
Status: Terminated 
 
14. NCT01114399. Diet and Vascular Health Study. [Institute of Food Research, Norwich, Norfolk, 
United Kingdom]. The aim of this study is to examine the effects of a diet rich in broccoli on 
cardiovascular disease risk using biochemical indicators such as blood lipid profiles, most notably 
cholesterol; markers of inflammation as well as established physiological measurements such as 
Pulse wave velocity (PWV), Augmentation index (AIx) and Ambulatory Blood Pressure Measurements 
(ABPM). Broccoli contains compounds known as glucosinolates which are metabolised to 
isothiocyanates when consumed. The major glucosinolate in broccoli is known as glucoraphanin 
which produces the isothiocyanate sulforaphane. The glucosinolates are thought to be the principal 
component in broccoli that may reduce CVD risk. The investigators will use a standard cultivar of 
broccoli and a cultivar that has enhanced levels of glucosinolates ('HG broccoli'). This broccoli has 
been used in previous intervention studies (e.g. ClinicalTrials.gov NCT00535977). Volunteers will be 
asked to consume 400g of standard broccoli, HG broccoli or peas each week over a 12 week period in 
a double blinded (for the broccoli) parallel study. The volunteers recruited will, according to the Joint 
British Societies (JBS 2) Guidelines on the prevention of cardiovascular disease (CVD) in clinical 
practise, have a 10-20% (mild to moderate) risk of developing cardiovascular disease or having a 
cardiovascular (CV) event in the next 10 years. 
Status: Completed 
 
15.  NCT01183923.  Dietary Interventions in Asthma Treatment: Sprouts Study. [E. Matsui, Johns 
Hopkins University].  The main objectives are to test the effect of broccoli sprouts (BS) on 
biomarkers of oxidative stress (OS), inflammation, basophil activation, and clinical outcomes in 
mouse allergen-induced asthma by: (1) determining if BS improves lung function and airways 
symptom responses in mouse-sensitized adults with asthma undergoing environmental mouse 
allergen challenge (EMAC), (2) examining the effect of BS on OS, inflammation, and basophil 
activation, and (3) examining the effect of BS on changes in OS, inflammation, and basophil 
activation after EMAC. 
Status: Suspended 
 
16.  NCT01228084.  Sulforaphane in Treating Patients with Recurrent Prostate Cancer. [J. Alumkal, 
OHSU Knight Cancer Institute, Portland, OR].  This phase II trial studies how well sulforaphane 
works in treating patients with recurrent prostate cancer. The primary objective is to determine the 
proportion of patients who achieve a 50% decline in prostate-specific antigen (PSA) levels within 20 
weeks of sulforaphane treatment. 
Status: Completed, has Results 
 

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17. NCT01265953. Chemoprevention of Prostate Cancer, HDAC Inhibition and DNA Methylation. 
[Jackilen Shannon, Portland VA Medical Center, Portland, OR]. The objective of the study is to 
identify mechanisms by which compounds found in cruciferous vegetables alter gene expression via 
epigenetic modifications (changes in gene expression) and may prevent prostate cancer 
development. 
The investigators have found that sulforaphane (SFN), an isothiocyanate found in cruciferous 
vegetables, inhibits histone deacetylase (HDAC) activity in human colorectal and prostate cancer 
cells. 
Status: Unknown 
 
18.  NCT01129466.  Effects of 2 Different Broccoli Sprout Containing Supplements on Nasal Cells 
in Healthy Volunteers (Broccosprout).  [T. Noah, University of North Carolina, Chapel Hill, NC].  To 
compare the change in nasal cell HO-1 expression induced by two different preparations of 
sulforaphane (SFN)-containing nutritional supplements. In this pilot study, subjects will be 
randomized to receive either the equivalent of 4 ounces/day of fresh broccoli sprouts in food and tea, 
or the equivalent amount as broccoli sprout homogenates, for 3 consecutive days. Nasal lavage and 
blood samples will be obtained before, during and after this 3-day period. After at least a 2-week 
washout interval, the protocol will be repeated with the alternate supplement, in a crossover design. 
The primary analysis will test the hypothesis that broccoli sprouts in food will increase HO-1 
expression to a similar degree as the equivalent amount of sprouts in homogenates. 
Status: Completed 
 
19.  NCT01269723.  Effects of Sulforaphane (SFN) on Immune Response to Live Attenuated 
Influenza Virus in Smokers and Nonsmokers.  [T. Noah, University of North Carolina, Chapel Hill, 
NC].  The purpose is to learn about short term responses to live attenuated influenza virus (LAIV, 
administered via Flumist® vaccine) between smoking and nonsmoking volunteers treated with 
broccoli sprout homogenates ("shake") or a placebo homogenate. Subjects will be seen for a 
screening visit (inc. HIV test) and then randomly assigned to receive 1 of 2 homogenates. Nasal 
lavage (NL), blood samples and nasal biopsies will performed prior to and after study interventions. 
Status: Completed 
 
20. NCT01315665.  Effect of Sulforaphane in Broccoli Sprouts on Nrf2 Activation. [JF Chmiel, 
Rainbow Babies and Children’s Hospital, Cleveland, OH].  The purpose of this study is to investigate 
the effect of sulforaphane from macerated broccoli sprouts in humans and to evaluate less invasive 
methods of assessing potential anti-inflammatory drugs in cystic fibrosis.  
Status: Completed 
 
21. NCT01335971.  Broccoli Sprout Extracts Trial (BEST). [R. Wise, Johns Hopkins University]. 
This study will investigate whether ingestion of sulforaphane by chronic obstructive pulmonary 
disease (COPD) patients increases Nrf2 activity and expression of downstream antioxidants in 
alveolar macrophages and bronchial epithelial cells? Accordingly, the investigators are conducting a 
placebo-controlled randomized proof of principle trial of two oral doses of sulforaphane, 25 and 150 
micromoles, for 4 weeks in 90 COPD patients. Collections of alveolar macrophages by BAL, bronchial 
epithelial cells by endobronchial brushings will be performed at baseline and 4 weeks. Other 
biospecimens will include nasal epithelial cells, PBMCs, and expired breath condensate. The goal is to 

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establish a safe and tolerable dose of sulforaphane that effects in vivo antioxidants via Nrf2, in order 
to have a novel candidate treatment for longer-term efficacy trials. 
Status: Unknown 
 
22. NCT01344330.  Cruciferous Vegetable Intake and Histone Status in Screening Colonoscopy 
Patients [Texas A&M University].  This research study will assess cruciferous vegetable intake in 
patients presenting for screening colonoscopy and correlate intake with histone status and histone 
deacetylace (HDAC) expression in tissue biopsy specimens and peripheral blood mononuclear cells 
(PBMCs). The investigators will also measure sulforaphane (SFN) metabolites in blood as a 
biomarker of cruciferous vegetable intake. 
Status: Recruiting 
 
23.  NCT01357070.  Effect of Broccoli Sprout on Blood Levels of Sulforaphane to Reduce 
Responsiveness of Immune System.  [PC Evans, Imperial College London, UK].  With particular 
attention to its role in reducing atherosclerosis, investigators want to test whether the consumption 
of a brocco-sprout smoothie containing sulforaphane can protect white blood cells from becoming 
activated in the presence of an experimental stress and how long this protective effect lasts for. To do 
this, the investigators will be analyzing inflammatory changes in blood samples taken at different 
times during the study. 
Status: Unknown 
 
24.  NCT01437501.  Broccoli Sprout Intervention in Qidong, P.R. China. [Johns Hopkins Bloomberg 
School of Public Health].  This study is a 12 week placebo-controlled Phase II broccoli 
sprout intervention to be conducted in Qidong, P.R. China. One thousand two hundred people from 
the farming townships will be screened and three hundred eligible individuals will be enrolled in the 
study. Participants will be randomized into two treatment groups: one will receive a juice beverage 
containing glucoraphanin- and sulforaphane-rich broccoli sprout extract, pineapple juice, lime juice, 
and water and the other will receive a placebo beverage containing pineapple juice, lime juice and 
water. Participants will drink their assigned beverage every evening and provide biweekly urine 
samples and monthly blood samples. The principal endpoints of this study are pharmacokinetic 
evaluation of elimination of glucoraphanin/sulforaphane and their metabolites in urine and 
pharmacodynamic evaluation through measures of urinary levels of exposure biomarkers for dietary 
and air-borne toxins, which are known to be high in this population. 
Status: Completed, has Results 
  
25. NCT01474993.  Sulforphane-rich Broccoli Sprout Extract for Autism. [Lurie Autism Center, 
Cambridge, MA]. The primary objectives of this study are to answer whether there is evidence of 
measurable effects on social responsiveness (primary outcome) and other behavioral symptoms after 
treatment of autistic male adolescents and adults with orally administered sulforaphane-
rich Broccoli Sprout Extract (efficacy). The secondary objectives of this study are to answer whether 
treatment of male adolescents and adults with autism using orally administered sulforaphane-
rich Broccoli Sprout Extract within a specified dose range is safe (toxicity); treatment with 
sulforaphane-rich Broccoli Sprout Extract is well tolerated (side effects and adverse events); key 
cellular biomarkers support the hypothesized mechanisms (proof of principle). 
Status: Completed, has Results 
 

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26.  NCT01522703.  Effects of Whole Sprouts on Upper Airway Allergic Inflammation [Johns 
Hopkins University].  The primary objective of this study is to determine if broccoli sprouts (BS) 
improves nasal inflammatory, oxidative stress (OS), and symptom responses to nasal mouse allergen 
challenge (NAC) in mouse-sensitized adults perennial allergic rhinitis.The study is a double-blind, 
placebo-controlled, randomized trial to compare BS to placebo in 24 mouse-sensitized adults with 
asthma or perennial rhinitis and who develop rhinitis symptoms with nasal mouse allergen 
challenge. Twenty-four adults (age 18-50) who meet these eligibility criteria will be randomized to 
receive either: (a) BS or (b) placebo. At the baseline NAC, FENO, FEV1, nasal epithelial gene 
expression, urinary OS biomarkers, serum inflammatory and OS biomarkers, basophil activation, and 
symptoms will be assessed both before and after NAC to establish pre-intervention responses to NAC. 
Subjects will eat a sprouts sandwich daily for three days, and then undergo repeat NAC that includes 
measurement of the above outcomes both before and after NAC.   
Status: Completed 
 
27. NCT01543074.  Dietary Histone Deacetylase Inhibitors (HDAC).  [Oregon State University]. 
This pilot, three-week study will help scientists understand more about how the foods people eat can 
modify histone deacetylases, enzymes the body produces naturally. Broccoli sprout extract and garlic 
oil are thought to modify these enzymes. The purpose of this study is to see if taking broccoli sprout 
extract alone, garlic oil alone, or broccoli sprout extract and garlic oil together, can decrease the 
action of histone deacetylase (HDAC) and turn on genes in white blood cells. There will be 80 people 
in this study. 
Status: Terminated, has Results 
 
28.  NCT01568996.  Pilot Study Evaluating Sulforaphane in Atypical Nevi-Precursor Lesions. 
[University of Pittsburg].  This is a pilot study to see if oral administration of freeze dried, 
powdered broccoli sprouts have any effect on whether moles end up becoming melanoma. 
Status: Recruiting 
 
29. NCT01715480.  Effect of Broccoli Sprouts Homogenate on SS RBC. [J.-T. A. Chi  & M. J. Telen, 
Duke University]. The overall purpose of this study is to obtain a better understanding of the 
biological response of red blood cells to sulforaphane contained in fresh broccoli sprouts that have 
been put through a blending process. This study will use commercially available fresh broccoli 
sprouts certified by Brassica Protection Products LLC (BroccoSprouts®). This product can also be 
purchased at some local grocery stores in the produce section. It is believed that NRF2, a 
transcription factor encoded by the NFE2L2 gene, plays a role in the regulation of defense against 
oxidative stress. The detrimental accelerated breakdown of sickle cell disease (SCD) red blood cells 
(SS RBC) is partially due to reduced anti-oxidative capacity. Previous analysis of SS RBC microRNAs 
revealed that a reduced level of NRF2, the master regulator of anti-oxidative stress capacity, 
contributes to reduced resistance to oxidative stress and increased hemolysis; NRF2 also induces fetal 
hemoglobin (HbF), which is known to prevent SS RBC sickling. 
First, erythroid progenitors from normal and SCD subjects will be tested ex-vivo to find out how 
sulforaphane, a natural NRF2 activator, affects the oxidative stress capacity, HbF expression, and 
microRNA expression of red cells. 
Second, a pilot clinical trial will be conducted to determine the safety and physiological effects of 3 
weeks of daily consumption of broccoli sprout homogenate in a cohort of Hb SS/SB0 thalassemia 

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adult SCD patients. During this study, subjects RBCs will be assayed for changes in anti-oxidative 
stress capacity and microRNA composition in mature SCD red blood cells. 
 Status: Completed 
 
30. NCT01625130. Effect of Sulforaphane-rich Broccoli Sprout Homogenate on Ozone Induced 
Inflammation Through Modulation of NRF2 (BroccOz). [M. Hernandez, University of North 
Carolina, Chapel Hill]. Purpose: To determine if modulation of NRF2 with a Sulforaphane enriched 
supplement modifies responses to O3. Participants: Recruitment of up to 70 healthy volunteers, ages 
18-50, for completion of 36 volunteers. Procedures: This is a randomized, placebo controlled 2x2 
crossover study of treatment with an NRF2 modifier versus placebo in healthy volunteers which will 
examine airway inflammation before and 4 hours after a 2 hour 0.4 ppm O3 exposure. Participants 
will be randomized to received either the NRF2 modifier, SFN oral supplement (i.e. broccoli 
sprout shake), or placebo (alfalfa shake) for 3 days followed by a 0.4 ppm O3 exposure for 2 hours. At 
least 2 weeks later subjects will return for a 2nd supplementation treatment (using the alternative 
supplement to that provided initially) followed by an ozone exposure identical to the initial one. 
Status: Completed 
 
31. NCT01726127. Green Vegetables and Women's Health. [S. A. Tanumihardjo & N. Binkley, 
University of Wisconsin, Madison]. The 2010 Dietary Guidelines for Americans recommend that 
individuals consume 4.5 to 5 cups fruits and vegetables daily. However, at current intake levels, fruit 
consumption will have to improve by more than 100% and vegetable intake by 50% to meet this 
recommendation. Importantly, intake of brightly colored fruits and vegetables is even lower when 
potatoes are not considered. It is possible that improved fruit and vegetable intake will have 
beneficial health effects. For example, higher intakes of fruits and vegetables, and particularly 
cruciferous vegetables (e.g., broccoli, Brussels sprouts, cauliflower, etc.), are associated with lower 
rates of many degenerative diseases, including some cancers, yet this group of vegetables may 
continue to be under-consumed due to their strong flavors. A supplement made from these vegetables 
(Cruciferous CompleteTM made by Standard Process Inc. Palmyra, WI) contains a group of 
phytochemicals called glucosinolates that can shift estrogen metabolism in a favorable way. One 
proposed biomarker of chemoprotection from breast cancer is the urinary estrogen metabolite ratio 
of 2- to 16α-hydroxyestrogens (2:16). In the main study, the effects of cruciferous vegetables 
(broccoli or Brussels sprouts), Cruciferous CompleteTM whole food supplements, or placebos on this 
ratio of urinary estrogen metabolites in healthy premenopausal women will be compared over an 
eight-week period. The investigators hypothesize that treatment with daily supplements will increase 
the 2:16 ratio as compared to daily consumption of a combination of Brussels sprouts and broccoli or 
a placebo, suggesting reduced breast cancer risk. 
In a sub-study, the relationships between serum α-carotene, β-carotene, β-cryptoxanthin, lutein and 
lycopene with dietary carotenoid intake as measured by a food frequency questionnaire and body 
composition will be evaluated in healthy premenopausal women. Carotenoids are a family of 
lipophilic compounds found primarily in colorful plant tissues and their concentration in human 
blood reflects dietary intake of carotenoid-rich foods. Carotenoid levels in the blood of healthy 
women do not appear to be influenced by menstrual status, but are inversely associated with body 
fatness. Thus, serum carotenoid concentrations may serve as a functional marker for chronic disease 
risk associated with excess body fat. 
Status: Completed 
 

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32. NCT01753908.  Broccoli Sprout Extract in Treating Patients With Breast Cancer [Roswell Park 
Cancer Institute].  This randomized pilot trial studies broccoli sprout extract in treating patients 
with estrogen receptor-positive breast cancer. Broccoli sprout extract contains ingredients that may 
prevent or slow the growth of certain cancers. 
Status: Recruiting 
 
33.  NCT01716858.  An Open Study of Sulforaphane-rich Broccoli Sprout Extract in Patients with 
Schizophrenia. [Chiba University, Japan].  Accumulating evidence suggests a role of oxidative stress 
in the pathophysiology of schizophrenia. The potent antioxidant sulforaphane (SFN) is an 
organosulfur compound derived from a glucosinolate precursor found in cruciferous vegetables such 
as broccoli, Brussels sprouts and cabbage. The protection afforded by SFN is thought to be mediated 
via activation of the NF-E2-related factor-2 (Nrf2) pathway and subsequent up-regulation of phase II 
detoxification enzymes and antioxidant proteins, through an enhancer sequence referred to as the 
electrophilic responsive element or antioxidant responsive element. Recently, we reported that SFN 
could attenuate behavioral abnormalities in mice after the NMDA receptor antagonist phencyclidine. 
Considering the potent antioxidant effects of SFN, we have a hypothesis that SFN would be a 
potential therapeutic drug for schizophrenia. The purpose of this study is to determine whether SFN-
rich broccoli sprout extract have beneficial effects in patients with schizophrenia. 
Status: Completed 
 
34. NCT01743924. Bioavailability of Chemopreventive and Nutritional Compounds in Broccoli.[M. 
C. M. Sardiña, Universidad Politécnica de Cartagena, Murcia, Spain]. The aim of this study was to 
determine the absorption of isothiocyanates (ITC) after ingestion of kailan-hybrid broccoli through 
the analysis of the correspondent urinary biomarkers. Furthermore, the effect of cooking 
(microwave) on the mentioned metabolic fate of these ITC was studied comparing to the uncooked 
vegetable. 
Status: Completed 
 
35. NCT01845220.  Prevention of Alcohol Intolerance. [Johns Hopkins University]. This study is 
designed to determine whether Asians who are especiallly sensitive to alcohol exposure can be 
protected by boosting their activities of an alcohol disposing enzyme. This will be accomplished by 
administering broccoli sprouts that are rich in an agent that increases protective enzyme activity. 
The test system involves applying alcohol patches to the skin and measuring skin redness.  
Status: Active, Not Recruiting 
 
36. NCT01845493. Sulforaphane Supplementation in Atopic Asthmatics. [M. L. Hernandez, 
University of North Carolina, Chapel Hill]. The investigators will perform a pilot study of daily 
treatment with oral sulforaphane (SFN) for 3 days to determine if Nuclear factor (erythroid-derived 
2)-like 2 (NRF2) induction is possible with this supplementation regimen in individuals with allergic 
asthma. 
Status: Completed 
 
37. NCT01879878. Pilot Study Evaluating Broccoli Sprouts in Advanced Pancreatic Cancer 
[POUDER Trial]. [I. Herr, Heidelberg University, Germany]. The goal of the POUDER trial is to 
determine the feasibility of a randomized controlled trial regarding the application of freeze-

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dried broccoli sprouts rich in sulforaphane and quercetin in patients with advanced pancreatic 
ductal adenocarcinoma that receive palliative chemotherapy. 
Status: Recruiting 
 
38. NCT01927666. The Conversion of ENcapsulated GlucorAphanin, Gut Microbiota Phylogeny 
and gEnotype Study (ENGAGE). [R. Mithen, Institute of Food Research, Norwich,  Norfolk, United 
Kingdom]. The variation in extent of isothiocyanate (ITC) excretion in urine from a capsule delivered 
dose of glucoraphanin will correlate with differences in (a) the gut microbiota, and (b) the genotype 
of key polymorphic genes (GSTM1, GSTT1, and other as yet undetermined candidate genes). 
Our study is a human dietary intervention in which participants will consume one capsule containing 
100mg purified glucoraphanin from broccoli. The levels of glucoraphanin delivered by the capsule 
are similar to one to two portions of broccoli. As this is purified glucoraphanin there is no myrosinase 
enzyme present. All conversion of the glucoraphanin, contained within the capsule, to ITC will 
therefore occur by enzymes found in the gut microbiota. 
The ability of the glucoraphanin in the capsule to be metabolised to ITCs by the gut microflora is 
unknown and will be assessed by measuring ITCs excreted in the urine. The ITCs will be quantified in 
urine using validated analytical methods. 
It has been shown in human dietary intervention studies that the extent of conversion of 
glucosinolates varies greatly. In order to assess possible causative factors for variation in rate of 
glucoraphanin metabolism each participant will provide a faecal sample from which their faecal gut 
microbiota phylogeny will be analysed. 
For a small number of participants a second faecal sample will be requested (a maximum of 3 
participants). It is our aim to select one low, one medium and one high ITC excreter. Ideally the low 
and high excreters would be within the lowest and highest 5% excretion of ITC and the third 
participant would be as close to the mean ITC excretion as possible. The aim would be to culture the 
faecal microbiota over time with repeat dosing of glucoraphanin in order to select for microbiota 
that are able to metabolise glucoraphanin. It is known that the main hydrolysis product of 
glucoraphanin, sulforaphane, has a variety of benefits to human health, however there is no known 
clinical relevance to being a high, medium or low excreter of ITC. 
For each participant a blood sample will also be requested in order that we can assess whether 
genotype affects the rate of ITC excretion in urine. The GSTM1 genotype and other, as yet, 
unidentified candidate genes of each participant will be determined. Whether the genotype  
affects the rate of ITC excretion either alone or in combination with the phylogenetic profile will be 
assessed. 
Status: Active, Not Recruiting 
 
39. NCT01948362. Sulforadex in Healthy Volunteers SAD. [J. Täube, Evgen Pharma, London, 
United Kingdom]. To determine the safety and tolerability of single escalating doses of Sulforadex® 
in healthy male volunteers. 
Status: Completed 
 
40. NCT01950143. Effect of Sulforaphane on Prostate CAncer PrEvention. [R. Mithen, Institute of 
Food Research, Norwich, United Kingdom,]. The biology of prostate cancer is associated with 
changes in genes and metabolites within prostate tissue. There is robust evidence to suggest that a 
diet rich in broccoli can prevent or retard the development of prostate cancer by influencing these 

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changes. This is likely to be due to a natural chemical that is obtained in these vegetables known 
as sulforaphane. 
In this study, we are seeking to provide further evidence that a diet rich in broccoli may prevent 
prostate cancer from developing, and to understand how this may happen. We propose to undertake 
a human intervention study to test the hypothesis that a broccoli-rich diet can alter the metabolism 
and gene expression within prostate tissue of men under active surveillance in a manner that would 
reduce the probability of the emergence and progression of aggressive cancerous clones. Participants 
recruited onto this study will be randomly allocated to one of three dietary groups in which they will 
be required to consume one portion per week of a broccoli soup delivering a different concentration 
of sulforaphane. This will be part of their normal diet for one year. 
Blood, urine and prostate biopsy tissue will be obtained before and after a 12 month intervention 
period. Prostate biopsies will be obtained either though transperineal template biopsies, a technique 
accepted as best clinical practice because it provides better sampling of the prostate, or transrectal 
ultrasound guided biopsy which is currently the standard of care for obtaining biopsies. 
Status: Enrolling by Invitation 
 
41.  NCT02023931.  Broccoli Sprout Extracts in Healthy Volunteers: A Pilot Study of Nrf2 Pathway 
Modulation in Oral Mucosa (BSE) [University of Pittsburgh].  A pilot study has been designed to 
determine (primary objective) if three brief interventions with three oral BSE regimens will alter 
mRNA biomarkers of Nrf2 pathway signaling, including NQO1, GSTs and AKRs, in the oral mucosa of 
healthy subjects. Quantitative distribution data and preliminary effect size for specific Nrf2 targets, 
as measured in serial buccal cell scrapings, will be determined during the course of 3-day exposures 
to three BSE regimens. These data will inform the design of a randomized, phase II chemoprevention 
trial in patients with HPV-negative HNSCC.  Ten healthy volunteers will be recruited for this pilot 
study, Age ≥ 18 years, both male and female: 1) The non-cancer population presenting to the 
University of Pittsburgh Eye and Ear Institute or the Hillman Cancer Center. This may include 
patients with benign disease or their friends/family members, or friends/family members of patients 
with cancer; 2) Professionals, staff, or students at the University of Pittsburgh.  
Status: Completed  
 
42. NCT02055716. Sulforadex in Healthy Human Males MAD. [J. Täubel, Evgen Pharma, London, 
United Kingdom]. To determine the safety and tolerability of multiple doses of Sulforadex® in 
healthy male volunteers over 7 days with qd or bid dosing.  
Status: Completed 
 
43. NCT02300324. The Bioavailability Of Sulforaphane From Broccoli Soups Study (BOBS). [R. 
Mithen, Institute of Food Research, Norwich, Norfolk, United Kingdom]. There is current evidence 
that suggests eating cruciferous vegetables like broccoli, cauliflower, cabbage is beneficial to our 
health as they contain compounds which are thought to reduce the risk of diseases such as cancer 
and cardiovascular diseases. Cruciferous vegetables are able to deliver in our body a group of 
compounds called isothiocyanates (ITCs) that are thought to be responsible of their health-
promoting effects. Sulforaphane(SF) from broccoli is one of the most studied ITCs and its anticancer 
properties have been extensively investigated in in vitro and in vivo models. 
The investigators propose to undertake an intervention study to measure the bioavailability of SF 
from the soups used in another intervention study called ESCAPE. The investigators would like to 
investigate the rate and extent to which SF reaches the systemic circulation and is excreted in urine 

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by measuring SF and its metabolites in plasma and urine samples collected from apparently healthy 
participants after consumption of the three types of broccoli + stilton soups. The three types of soups 
are standard broccoli + stilton soups and two high-glucoraphanin (SF precursor) broccoli + stilton 
soups which are able to deliver different levels of SF. 
Status: Completed 
 
44. NCT02336087. Gemcitabine Hydrochloride, Paclitaxel Albumin-Stabilized Nanoparticle 
Formulation, Metformin Hydrochloride, and a Standardized Dietary Supplement in Treating 
Patients With Metastatic Pancreatic Cancer. [V. Chung, City of Hope Medical Center, Duarte, CA]. 
This pilot phase I trial studies the side effects of gemcitabine hydrochloride, paclitaxel albumin-
stabilized nanoparticle formulation, metformin hydrochloride, and a standardized dietary 
supplement in treating patients with pancreatic cancer that has spread from where it started to 
other places in the body. Drugs used in chemotherapy, such as gemcitabine hydrochloride and 
paclitaxel albumin-stabilized nanoparticle formulation, work in different ways to stop the growth of 
tumor cells, either by killing the cells, by stopping them from dividing, or by stopping them from 
spreading. Metformin hydrochloride, used for diabetes, may also help kill cancer cells. Dietary 
supplements (curcumin, vitamin D, vitamin K2, vitamin K1, B-6, high selenium broccoli sprouts, 
epigallocatechin gallate, L-carnitine, garlic extract, genistein, zinc amino chelate, mixed toxopherols, 
ascorbic acid, D-limonene) can block different targets in the cancer cell simultaneously and may slow 
down cancer growth. Giving gemcitabine hydrochloride, paclitaxel albumin-stabilized nanoparticle 
formulation, and metformin hydrochloride with a dietary supplement may work better in treating 
patients with metastatic pancreatic cancer.  
Status: Completed 
 
45. NCT02404428. Utilizing MRI to Study the Effect of Sulforaphane on Prostate Cancer. [R. 
Mithen, Institute of Food Research, U.K.]. Prostate cancer is a major public health problem and 
there is a strong need of new preventive strategies based on drug and lifestyle interventions. It is now 
well-established that healthy eating patterns and increasing physical activity can prevent or delay 
prostate cancer progression. Intake of cruciferous vegetables (e.g. broccoli, cabbage, 
cauliflower, Brussels sprouts, kale) has been associated with decreased risk of prostate cancer 
progression; however the underlying biological mechanisms remain unknown. The investigators 
propose to undertake a pilot study on a group of men with early prostate cancer on active 
surveillance to determine whether a diet rich in broccoli will induce changes in tumor size and blood 
flow measured by conventional Magnetic Resonance Imaging (MRI) techniques. Men with early 
prostate cancer on active surveillance who have visible cancer lesions on MRI will be recruited onto 
this double-blinded randomized intervention and they will be asked to eat one portion 
of broccoli soup per week for 6 months. The investigators will test two varieties of broccoli(standard 
and 'Beneforte extra' broccoli) that are able to deliver two different levels of sulforaphane (SF), an 
active compound extensively studied for its potential anticancer properties. This study will involve 
MRI scans, blood and urine collection before and after a 6 month intervention period. This study 
design will not only allow us to observe diet-induced changes within the prostate but also at the 
systemic level. In addition, participant's lifestyle (habitual diet and physical activity) will be assessed 
by food diaries and exercise questionnaires. 
Status: Enrolling by Invitation 
 

Clinical Protocol: Sulforaphane for ASD 
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46. NCT02561481. Sulforaphane Treatment of Children With Autism Spectrum Disorder (ASD). [A. 
W.  Zimmerman, University of Massachusetts, Worcester]. ASD is a diverse disorder starting in early 
childhood and characterized by social communication impairment as well as restricted interests and 
repetitive behaviors. It affects 1:68 children and is an enormous medical and economic problem for 
which there is no established, mechanism-based treatment. Sulforaphane is an isothiocyanate 
derived from broccoli, and has potent activity in transcriptionally up-regulating genes that control 
mechanisms whereby aerobic cells protect themselves against oxidative stress, mitochondrial 
dysfunction, and inflammation. 
This study is a clinical trial of oral sulforaphane (as broccoli seed powder) in 50 boys and girls (3-12 
years) with ASD in 3 phases over 36 weeks. In Phase 1, 25 children will receive active drug and 25 will 
receive placebo for 15 weeks; in Phase 2, all children will receive sulforaphane from 15-30 weeks; in 
Phase 3, children will receive no treatment for 6 weeks. Study visits will take place at screening, 7, 15, 
22, 30 and 36 weeks, when the Ohio Autism Clinical Clinical Impressions Scale - Severity and 
Improvement (OACIS-S and OACIS-I), Aberrant Behavior Checklist (ABC) and Social Responsiveness 
Scale (SRS) will be recorded. Children will be monitored with physical examinations and for toxicity 
with clinical laboratory studies and examine possible biomarkers: Nuclear factor-erythroid factor 2 
(Nrf2), oxidative stress and mitochondrial function, the mechanistic target of rapamycin (mTOR) 
pathway and cytokine expression. In addition, prior to the main clinical trial, a pilot study will be 
carried out in 10 children with ASD, 6-12 years of age, who will receive sulforaphane, 2.2 
micromoles/kg daily for 14 days. Blood and urine samples before and at the end of treatment will be 
collected, 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. 
Status: Recruiting 
 
47. NCT02592954. Effect of Broccoli Sprout Extract on Keratinocyte Differentiation in Normal 
Skin. [B. Cohen, Johns Hopkins University]. Adult participants will apply a broccoli sprout extract-
jojoba oil compound to one arm every night under occlusion for 1 week. Jojoba oil alone will be 
applied to the other arm. At the end of 1 week, a 6mm punch biopsy will be taken from both arms and 
analyzed via polymerase chain reaction (PCR) and immunohistochemistry for differences in various 
skin proteins.  
Status: Recruiting 
 
48. NCT02614742. SFX01 After Subarachnoid Haemorrhage. [D. Bulters, Evgen Pharma, London, 
United Kingdom]. This is a Safety, Tolerability, Pharmacokinetic and Pharmacodynamic Study of 
SFX-01 in Subarachnoid Haemorrhage, with exploratory evaluations of efficacy.  
Status: Not Yet Recruiting  
 
49. NCT02654743. Open Label of Clinical Trial of Sulforaphane in Children With Autism. [R. 
Hendren, University of California, San Francisco]. This is an open-label, 4-month study examining 
the effects of Sulforaphane (SF) on behavior in children with ASD and the correlation between 
behavior change and urinary metabolites. The goal is to determine a potential mechanism of action 
of SF in this population.  
Status: Enrolling by Invitation 
 

Clinical Protocol: Sulforaphane for ASD 
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50. NCT02656420. Broccoli Sprout Dose Response. [T. Kensler, Johns Hopkins Bloomberg School 
of Public Health]. This study will examine the extent to which lower doses of a broccoli-derived 
beverage enhance the detoxication of air pollutants excreted in urine as compared to an maximal 
dose shown to be effective previously. 
Status: Enrolling by Invitation 
 
51. NCT02677051. Sulforaphane in a New Jersey (NJ) Population of Individuals With Autism. [W. 
G. Johnson, Rutgers, The State University of New Jersey]. This study is a double blind treatment trial 
that will test if sulforaphane improves core symptoms in autism. The investigators expect to see 
clinical improvement in some of these areas. Sulforaphanes come from eating certain vegetables such 
as broccoli. The investigators will be using a preparation that gives specific and reproducible 
amounts. The investigators will also test specific chemicals and genes needed for sulforaphane usage 
to try to understand differences in response. 
Status: Not Yet Recruiting 

Clinical Protocol: Sulforaphane for ASD 
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Clinical Protocol: Sulforaphane for ASD 
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