Sulforaphane Treatment of Children With Autism Spectrum Disorder (ASD)

• Sponsor: University of Massachusetts, Worcester (Other)
• Overall Status: Completed
• CT.gov ID: NCT02561481
• Collaborator: Congressionally Directed Medical Research Programs (U.S. Fed), Johns Hopkins University (Other)
• Enrollment: 60
• Location: 1
• Arms: 2
• Actual Duration (Months): 49
• Patients Per Site Per Month: 1.2

Study Details

Study Description

Brief Summary

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.

Condition or Disease	Intervention/Treatment	Phase
Autism Spectrum Disorder	Drug: Sulforaphane; Drug: Placebo	Phase 1/Phase 2

Detailed Description

Background: 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.

Hypothesis: Based on the observation that fever is frequently associated with behavioral improvements in children with ASD, it is hypothesized that sulforaphane might lead to functional improvements in ASD because it can up-regulate cell-protective responses, such as heat shock proteins and related mechanisms that are also up-regulated during fever. These mechanisms are central to multiple cellular processes in the central nervous system, including synaptic transmission, and may improve long-range cerebral cortical connectivity, which is depressed in ASD.

Specific aims: The 3 specific aims in this study are: 1) To determine if there are measurable effects on social responsiveness and problem behaviors during treatment of children with sulforaphane; 2) To determine if treatment with sulforaphane in children is safe and well tolerated; and 3) To elucidate cellular biomarkers that respond to treatment with sulforaphane.

Design: This is a randomized, double blind, single-arm crossover phase 1/2 clinical trial of orally administered 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.

The children will be monitored with physical examinations and for toxicity with clinical laboratory studies and examine possible biomarkers: Nrf2, oxidative stress and mitochondrial function, mTOR pathway and cytokine expression.

In addition, prior to the main clinical trial, a pilot study will be performed 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.

Study Design

• Study Type: Interventional
• Actual Enrollment: 60 participants
• Allocation: Randomized
• Intervention Model: Parallel Assignment
• Masking: Quadruple (Participant, Care Provider, Investigator, Outcomes Assessor)
• Primary Purpose: Treatment
• Official Title: Sulforaphane Treatment of Children With Autism Spectrum Disorder (ASD)
• Actual Study Start Date: Dec 1, 2015
• Actual Primary Completion Date: Dec 1, 2018
• Actual Study Completion Date: Jan 1, 2020

Arms and Interventions

Arm	Intervention/Treatment
Active Comparator: Sulforaphane Sulforaphane (SF) will be administered once a day orally in an approximate dose of 1 µmol/lb (2.2 kg µmol/kg) body weight. Each SF tablet will contain 125 mg broccoli seed powder (equivalent to ~ 15 µmol SF). The total dose per day will depend on participants' body weight: 30-50 lb: 3 tablets (45 µmol/day) 50-70 lb: 4 tablets (60 µmol/day) 70-90 lb: 6 tablets (90 µmol/day) 90-110 lb: 7 tablets (105 µmol/day) 110-130 lb: 8 tablets (120 µmol/day) For pilot study, all participants (n=10) will receive SF for 14 days. For main clinical trial, 25 participants will randomly receive SF for 15 weeks (phase 1). During phase 2, a single-arm crossover from placebo to sulforaphane arm will take place, and all participants will receive SF from 15-30 weeks.	Drug: Sulforaphane; See under active arm description
Placebo Comparator: Placebo Placebo tablets identical in size and similar in appearance to the active tablets will be used. Number of placebo tablets will be equivalent to the active tablets depending on participants' body weight. For the main clinical trial, 25 participants will be randomly allocated to receive placebo for 15 weeks (phase 1). During phase 2, a single-arm crossover from placebo arm to sulforaphane arm will take place, and all participants will receive sulforaphane from 15-30 weeks.	Drug: Placebo; See under placebo arm description

Outcome Measures

Primary Outcome Measures

1. Change in Ohio Autism Clinical Impressions Scale - Improvement (OACIS-I) Average Score From Baseline [7 weeks, 15 weeks, 22 weeks, 30 weeks, 36 weeks]

   The Ohio Autism Clinical Impressions Scale-Severity (OACIS-S) rates severity of symptoms in 10 categories: general level of autism, social interaction, aberrant and repetitive behavior, verbal and nonverbal communication, hyperactivity, anxiety, sensory sensitivities and restricted and narrow interests. Each category is rated from 1 (normal) to 7 (most severe). The OACIS-S was a reference at follow up visits when the OACIS-Improvement was compared to the OACIS-S, from 1 to 7: 4 was no change; 3 to 1 minimal to marked improvement and 5 to 7 minimal to marked worsening. The numerical score of the OACIS-S is independent and unrelated quantitatively to the OACIS-I. For analysis, the OACIS-I general score and subscale values were recoded, in which 4 (no change) was recoded as 0; 3 to 1 were recoded as +1 to +3 to denote improvement, and 5 to 7 were recoded as -1 to -3 for worsening.

Secondary Outcome Measures

1. OACIS-I Response Rate on Aberrant Behaviors Subscale [7 weeks, 15 weeks, 22 weeks, 30 weeks, 36 weeks]

   See above in the primary outcome measure for a description of OACIS-I scale. This section describes the change from baseline of the OACIS-I subdomain of aberrant behaviors. This subdomain has a range of 1 to 7 - 1 is extremely improved from baseline, 7 is extremely worse from baseline, and 4 is no change. For analysis, the OACIS-I general score and subscale values were recoded, in which 4 (no change) was recoded as 0; 3 to 1 were recoded as +1 to +3 to denote improvement, and 5 to 7 were recoded as -1 to -3 for worsening.

2. OACIS-I Response Rate on Social Communication Subscale [7 weeks, 15 weeks, 22 weeks, 30 weeks, 36 weeks]

   See above in the primary outcome measure for a description of OACIS-I scale. This section describes the change from baseline of the OACIS-I subdomain of social communication. This subdomain has a range of 1 to 7 - 1 is extremely improved from baseline, 7 is extremely worse from baseline, and 4 is no change. For analysis, the OACIS-I general score and subscale values were recoded, in which 4 (no change) was recoded as 0; 3 to 1 were recoded as +1 to +3 to denote improvement, and 5 to 7 were recoded as -1 to -3 for worsening.

3. Change in Total Aberrant Behavior Checklist Score From Baseline [7 weeks, 15 weeks, 22 weeks, 30 weeks, 36 weeks]

   Aberrant Behavior Checklist (ABC) is a 58 item scale that primarily evaluates how aberrant or abnormal a patient's daily behaviors are. The items evaluate behaviors as they pertain to irritability, lethargy/social withdrawal, stereotypic behavior, hyperactivity/noncompliance, and inappropriate speech. Each item is scored on a scale of 0 to 3, with 0 being better outcome and 3 being worse outcome. The score from each item is added up to calculate a total score. This outcome describes change in total ABC score from baseline at each follow up visit. The scores from all items are added to calculate a total score (0 to 174). This outcome describes change in total ABC score from baseline at each follow up visit.

4. Change in Total SRS-2 Score From Baseline [7 weeks, 15 weeks, 22 weeks, 30 weeks, 36 weeks]

   The Social Responsiveness Scale-2 (SRS-2) is a 65-item scale that measures total scores as well as subscales: four social behaviors (awareness, cognition, communication and motivation) and autistic mannerisms. Each item is rated from 1 to 4 (not true to almost always true) on worksheets that are blinded to the rater with respect to values. Total and subscale scores are calculated as raw scores and can be converted to T-scores. Raw scores (range 0-180) are reported here (unadjusted for general population, since all children had ASD). Higher or lower values at follow up visits compared to baseline indicated worsening or improvement, respectively.

5. Dithiocarbamate Plasma Concentration Detected by Cyclocondensation at Each Visit [Week 0, Week 7, Week 15, Week 22, Week 30, Week 36]

   Sulforaphane (and other isothiocyanates, ITC) are conjugated by glutathione (GSH) which then undergoes further enzymatic modifications to give rise sequentially to the cysteinylglycine-, cysteine- and N-acetylcysteine-ITC conjugates, all of which are dithiocarbamates (DTC) and are detected in the cyclocondensation reaction-HPLC assay.

6. Comparison of Free Reduced Glutathione (GSH), Total GSH, Oxidized Glutathione (GSSG) at Week 0 and 15 [Week 0 and Week 15]

   F-statistic calculated by comparison of Free Reduced GSH, Total GSH and oxidized Glutathione (GSSG) of Week 15 to Week 0.

7. Free GSH:GSSG and Total GSH:GSSG Ratios at Week 15 [Week 15]

   Ratios of free GSH:GSSG and total GSH:GSSG were calculated by obtaining ratios of f-statistic scores from baseline to week 15 between free reduced GSH and GSSG and between total GSH and GSSG.

Other Outcome Measures

1. NQO1: NAD(P)H:Quinone Oxidoreductase-1 [Week 0, Week 15, Week 30]

   Cytoprotective enzyme regulated by nuclear factor erythroid 2-related factor 2 (Nrf2), the master regulator of cellular redox homeostasis and an inhibitor of a key pro-inflammatory pathway, of which both functions are critical factors in the neuropathology of ASD. Total cellular RNA was isolated from peripheral blood mononuclear cells (PBMCs) and complementary DNAs (cDNA) were synthesized. Quantitative real-time PCR analysis was performed using the Applied Biosystems QuantStudio™ 3 Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA). Relative mRNA expression was normalized to GAPDH. Gene expression was calculated using the comparative 2-ΔΔCT method.

2. xCT [Week 0, Week 15, Week 30]

   xCT (SLC7A11): Cystine/glutamate antiporter encoded by the SLC7A11 gene.Total cellular RNA was isolated from peripheral blood mononuclear cells (PBMCs) and complementary DNAs (cDNA) were synthesized. Quantitative real-time PCR analysis was performed using the Applied Biosystems QuantStudio™ 3 Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA). Relative mRNA expression was normalized to GAPDH. Gene expression was calculated using the comparative 2-ΔΔCT method.

3. HO-1 (Heme Oxygenase 1) [Week 0, Week 15, Week 30]

   HO-1 (heme oxygenase 1): an essential and Nrf2-dependent enzyme in heme catabolism. Total cellular RNA was isolated from peripheral blood mononuclear cells (PBMCs) and complementary DNAs (cDNA) were synthesized. Quantitative real-time PCR analysis was performed using the Applied Biosystems QuantStudio™ 3 Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA). Relative mRNA expression was normalized to GAPDH. Gene expression was calculated using the comparative 2-ΔΔCT method.

4. HSP70 [Week 0, Week 15, Week 30]

   HSP70 (Heat shock protein 70) was examined because it is upregulated by SF in vitro. Total cellular RNA was isolated from peripheral blood mononuclear cells (PBMCs) and complementary DNAs (cDNA) were synthesized. Quantitative real-time PCR analysis was performed using the Applied Biosystems QuantStudio™ 3 Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA). Relative mRNA expression was normalized to GAPDH. Gene expression was calculated using the comparative 2-ΔΔCT method.

5. HSP27 [Week 0, Week 15, Week 30.]

   HSP27 (Heat shock protein 27) was examined because it is upregulated by SF in vitro. Total cellular RNA was isolated from peripheral blood mononuclear cells (PBMCs) and complementary DNAs (cDNA) were synthesized. Quantitative real-time PCR analysis was performed using the Applied Biosystems QuantStudio™ 3 Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA). Relative mRNA expression was normalized to GAPDH. Gene expression was calculated using the comparative 2-ΔΔCT method.

6. IL-6 [Week 0, Week 15, Week 30]

   IL-6 (interleukin 6) cytokine gene expression, a nuclear factor-kappa B - regulated inflammatory biomarker. Total cellular RNA was isolated from peripheral blood mononuclear cells (PBMCs) and complementary DNAs (cDNA) were synthesized. Quantitative real-time PCR analysis was performed using the Applied Biosystems QuantStudio™ 3 Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA). Relative mRNA expression was normalized to GAPDH. Gene expression was calculated using the comparative 2-ΔΔCT method.

7. IL-1β [Week 0, Week 15, Week 30]

   IL-1β: Interleukin-1 beta cytokine gene expression, a nuclear factor-kappa B - regulated inflammatory biomarker. Total cellular RNA was isolated from peripheral blood mononuclear cells (PBMCs) and complementary DNAs (cDNA) were synthesized. Quantitative real-time PCR analysis was performed using the Applied Biosystems QuantStudio™ 3 Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA). Relative mRNA expression was normalized to GAPDH. Gene expression was calculated using the comparative 2-ΔΔCT method.

8. Cox-2 [Week 0, Week 15, Week 30]

   Cox-2 (cyclooxygenase-2): a nuclear factor-kappa B - regulated inflammatory biomarker. Total cellular RNA was isolated from peripheral blood mononuclear cells (PBMCs) and complementary DNAs (cDNA) were synthesized. Quantitative real-time PCR analysis was performed using the Applied Biosystems QuantStudio™ 3 Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA). Relative mRNA expression was normalized to GAPDH. Gene expression was calculated using the comparative 2-ΔΔCT method.

9. TNF-α [Week 0, Week 15, Week 30]

   TNF-α (Tumor necrosis factor alpha), a cytokine as inflammatory biomarker. Total cellular RNA was isolated from peripheral blood mononuclear cells (PBMCs) and complementary DNAs (cDNA) were synthesized. Quantitative real-time PCR analysis was performed using the Applied Biosystems QuantStudio™ 3 Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA). Relative mRNA expression was normalized to GAPDH. Gene expression was calculated using the comparative 2-ΔΔCT method.

Eligibility Criteria

Criteria

• Ages Eligible for Study: 3 Years to 12 Years
• Sexes Eligible for Study: All
• Accepts Healthy Volunteers: No

Inclusion Criteria:

• Autism Spectrum Disorder (ASD) diagnosis of moderate or greater severity

• Age 3 through 12 years inclusive

Exclusion Criteria:

• Absence of a parent or legal guardian and consent

• Inability to speak/understand English language

• Seizure within 1 year of screening: This exclusion is based on the theoretical concern that cellular activation by sulforaphane might exacerbate seizures in patients with known seizure disorders. As noted in our previous trial of sulforaphane in young adult males, a seizure occurred in each of 2 participants: one during treatment (in a participant with a previously undisclosed seizure), the other 3 weeks after discontinuing sulforaphane.

• Impaired renal function (serum creatinine > 1.2 mg/dl), impaired hepatic function (SGOT/SGPT> 2x upper limit of normal), impaired thyroid function (Thyroid Stimulating Hormone (TSH) outside normal limits): This exclusion is based on a theoretical possibility of activation of underlying cellular metabolic abnormalities by sulforaphane. Current infection or treatment with antibiotics: this exclusion is to avoid complications of inter-current illness that may occur due to the clinical trial or obscure possible effects of sulforaphane.

• Medications that may modify the course or testing of ASD parameters (e.g., prednisone): This exclusion is necessary in order not to interfere with or complicate effects of sulforaphane.

• Chronic medical disorder (e.g., cardiovascular disease, stroke or diabetes) or major surgery within 3 months prior to enrollment: Serious medical illness in the child may be complicated by the clinical trial and make it difficult to discern a change in ASD associated with treatment.

• Less than 3 years or more than 13 years of age: this age range was selected to cover the ages from usual diagnosis of ASD up to adolescence.

• A diagnosis of autism spectrum disorder of mild severity (for example, earlier categories of Asperger disorder, Pervasive Developmental Disorder - Not Otherwise Specified (PDD-NOS)), according to Autism Diagnostic Observation Schedule (ADOS) criteria.

• Prisoners

• Pregnant women

Contacts and Locations

Locations

	Site	City	State	Country	Postal Code
1	University of Massachusetts Medical School	Worcester	Massachusetts	United States	01655

Sponsors and Collaborators

• University of Massachusetts, Worcester
• Congressionally Directed Medical Research Programs
• Johns Hopkins University

Investigators

• Principal Investigator: Andrew W Zimmerman, MD, University of Massachusetts, Worcester

Study Documents (Full-Text)

• Study Protocol and Statistical Analysis Plan - Jul 3, 2019
• Informed Consent Form - Jul 3, 2019

More Information

Publications

None provided.

• Responsible Party: Andrew Zimmerman, Clinical Professor of Pediatrics and Neurology, University of Massachusetts, Worcester
• ClinicalTrials.gov Identifier: NCT02561481
• Other Study ID Numbers: H00007832; AR140087
• First Posted: Sep 28, 2015
• Last Update Posted: Dec 17, 2020
• Last Verified: Dec 1, 2020

• Individual Participant Data (IPD) Sharing Statement: No
• Plan to Share IPD: No
• Additional relevant MeSH terms: Autistic Disorder; Autism Spectrum Disorder; Child Development Disorders, Pervasive; Sulforaphane

Study Results

Participant Flow

Arm/Group Title	Sulforaphane	Placebo
Arm/Group Description	Sulforaphane (SF) will be administered once a day orally in an approximate dose of 1 µmol/lb (2.2 kg µmol/kg) body weight. Each SF tablet will contain 125 mg broccoli seed powder (equivalent to ~ 15 µmol SF). The total dose per day will depend on participants' body weight: 30-50 lb: 3 tablets (45 µmol/day) 50-70 lb: 4 tablets (60 µmol/day) 70-90 lb: 6 tablets (90 µmol/day) 90-110 lb: 7 tablets (105 µmol/day) 110-130 lb: 8 tablets (120 µmol/day) For pilot study, all participants (n=10) will receive SF for 14 days. For main clinical trial, 25 participants will randomly receive SF for 15 weeks (phase 1). During phase 2, a single-arm crossover from placebo to sulforaphane arm will take place, and all participants will receive SF from 15-30 weeks. Sulforaphane: See under active arm description	Placebo tablets identical in size and similar in appearance to the active tablets will be used. Number of placebo tablets will be equivalent to the active tablets depending on participants' body weight. For the main clinical trial, 25 participants will be randomly allocated to receive placebo for 15 weeks (phase 1). During phase 2, a single-arm crossover from placebo arm to sulforaphane arm will take place, and all participants will receive sulforaphane from 15-30 weeks. Placebo: See under placebo arm description
Period Title: Pilot Trial
STARTED	10	0
COMPLETED	10	0
NOT COMPLETED	0	0
Period Title: Pilot Trial
STARTED	25	25
COMPLETED	22	23
NOT COMPLETED	3	2
Period Title: Pilot Trial
STARTED	45	0
COMPLETED	32	0
NOT COMPLETED	13	0

Baseline Characteristics

Arm/Group Title	Sulforaphane	Placebo	Total
Arm/Group Description	Sulforaphane (SF) will be administered once a day orally in an approximate dose of 1 µmol/lb (2.2 kg µmol/kg) body weight. Each SF tablet will contain 125 mg broccoli seed powder (equivalent to ~ 15 µmol SF). The total dose per day will depend on participants' body weight: 30-50 lb: 3 tablets (45 µmol/day) 50-70 lb: 4 tablets (60 µmol/day) 70-90 lb: 6 tablets (90 µmol/day) 90-110 lb: 7 tablets (105 µmol/day) 110-130 lb: 8 tablets (120 µmol/day) For pilot study, all participants (n=10) will receive SF for 14 days. For main clinical trial, 25 participants will randomly receive SF for 15 weeks (phase 1). During phase 2, a single-arm crossover from placebo to sulforaphane arm will take place, and all participants will receive SF from 15-30 weeks. Sulforaphane: See under active arm description	Placebo tablets identical in size and similar in appearance to the active tablets will be used. Number of placebo tablets will be equivalent to the active tablets depending on participants' body weight. For the main clinical trial, 25 participants will be randomly allocated to receive placebo for 15 weeks (phase 1). During phase 2, a single-arm crossover from placebo arm to sulforaphane arm will take place, and all participants will receive sulforaphane from 15-30 weeks. Placebo: See under placebo arm description	Total of all reporting groups
Overall Participants	22	23	45
Age (years) [Mean (Standard Deviation) ]
Mean (Standard Deviation) [years]	7.4 (3.0)	7.0 (2.5)	7.2 (2.8)
Sex: Female, Male (Count of Participants)
Female	2 9.1%	3 13%	5 11.1%
Male	20 90.9%	20 87%	40 88.9%
Race/Ethnicity, Customized (Count of Participants)
White	17 77.3%	15 65.2%	32 71.1%
Other (asian, mixed, and unknown)	5 22.7%	8 34.8%	13 28.9%
Region of Enrollment (participants) [Number]
United States	22 100%	23 100%	45 100%

Outcome Measures

1. Primary Outcome

Title	Change in Ohio Autism Clinical Impressions Scale - Improvement (OACIS-I) Average Score From Baseline
Description	The Ohio Autism Clinical Impressions Scale-Severity (OACIS-S) rates severity of symptoms in 10 categories: general level of autism, social interaction, aberrant and repetitive behavior, verbal and nonverbal communication, hyperactivity, anxiety, sensory sensitivities and restricted and narrow interests. Each category is rated from 1 (normal) to 7 (most severe). The OACIS-S was a reference at follow up visits when the OACIS-Improvement was compared to the OACIS-S, from 1 to 7: 4 was no change; 3 to 1 minimal to marked improvement and 5 to 7 minimal to marked worsening. The numerical score of the OACIS-S is independent and unrelated quantitatively to the OACIS-I. For analysis, the OACIS-I general score and subscale values were recoded, in which 4 (no change) was recoded as 0; 3 to 1 were recoded as +1 to +3 to denote improvement, and 5 to 7 were recoded as -1 to -3 for worsening.
Time Frame	7 weeks, 15 weeks, 22 weeks, 30 weeks, 36 weeks

Outcome Measure Data

Analysis Population Description

Patients started dropping out at subsequent visits so the number analyzed at latter visits is less

Arm/Group Title	Sulforaphane	Placebo
Arm/Group Description	Sulforaphane (SF) will be administered once a day orally in an approximate dose of 1 µmol/lb (2.2 kg µmol/kg) body weight. Each SF tablet will contain 125 mg broccoli seed powder (equivalent to ~ 15 µmol SF). The total dose per day will depend on participants' body weight: 30-50 lb: 3 tablets (45 µmol/day) 50-70 lb: 4 tablets (60 µmol/day) 70-90 lb: 6 tablets (90 µmol/day) 90-110 lb: 7 tablets (105 µmol/day) 110-130 lb: 8 tablets (120 µmol/day) For pilot study, all participants (n=10) will receive SF for 14 days. For main clinical trial, 25 participants will randomly receive SF for 15 weeks (phase 1). During phase 2, a single-arm crossover from placebo to sulforaphane arm will take place, and all participants will receive SF from 15-30 weeks. Sulforaphane: See under active arm description	Placebo tablets identical in size and similar in appearance to the active tablets will be used. Number of placebo tablets will be equivalent to the active tablets depending on participants' body weight. For the main clinical trial, 25 participants will be randomly allocated to receive placebo for 15 weeks (phase 1). During phase 2, a single-arm crossover from placebo arm to sulforaphane arm will take place, and all participants will receive sulforaphane from 15-30 weeks. Placebo: See under placebo arm description
Measure Participants	22	23
7 weeks	0.28 (0.67)	0.28 (0.57)
15 weeks	0.28 (0.57)	0.33 (0.69)
22 weeks	0.47 (0.72)	0.59 (0.71)
30 weeks	0.47 (0.67)	0.69 (0.77)
36 weeks	0.29 (0.73)	0.29 (0.61)

2. Secondary Outcome

Title	OACIS-I Response Rate on Aberrant Behaviors Subscale
Description	See above in the primary outcome measure for a description of OACIS-I scale. This section describes the change from baseline of the OACIS-I subdomain of aberrant behaviors. This subdomain has a range of 1 to 7 - 1 is extremely improved from baseline, 7 is extremely worse from baseline, and 4 is no change. For analysis, the OACIS-I general score and subscale values were recoded, in which 4 (no change) was recoded as 0; 3 to 1 were recoded as +1 to +3 to denote improvement, and 5 to 7 were recoded as -1 to -3 for worsening.
Time Frame	7 weeks, 15 weeks, 22 weeks, 30 weeks, 36 weeks

Outcome Measure Data

Analysis Population Description

Patients started dropping out at subsequent visits so the number analyzed at latter visits is less

Arm/Group Title	Sulforaphane	Placebo
Arm/Group Description	Sulforaphane (SF) will be administered once a day orally in an approximate dose of 1 µmol/lb (2.2 kg µmol/kg) body weight. Each SF tablet will contain 125 mg broccoli seed powder (equivalent to ~ 15 µmol SF). The total dose per day will depend on participants' body weight: 30-50 lb: 3 tablets (45 µmol/day) 50-70 lb: 4 tablets (60 µmol/day) 70-90 lb: 6 tablets (90 µmol/day) 90-110 lb: 7 tablets (105 µmol/day) 110-130 lb: 8 tablets (120 µmol/day) For pilot study, all participants (n=10) will receive SF for 14 days. For main clinical trial, 25 participants will randomly receive SF for 15 weeks (phase 1). During phase 2, a single-arm crossover from placebo to sulforaphane arm will take place, and all participants will receive SF from 15-30 weeks. Sulforaphane: See under active arm description	Placebo tablets identical in size and similar in appearance to the active tablets will be used. Number of placebo tablets will be equivalent to the active tablets depending on participants' body weight. For the main clinical trial, 25 participants will be randomly allocated to receive placebo for 15 weeks (phase 1). During phase 2, a single-arm crossover from placebo arm to sulforaphane arm will take place, and all participants will receive sulforaphane from 15-30 weeks. Placebo: See under placebo arm description
Measure Participants	22	23
7 weeks	0.06 (0.42)	0.28 (0.57)
15 weeks	0.22 (0.43)	0.33 (0.69)
22 weeks	0.59 (0.80)	0.53 (0.87)
30 weeks	0.63 (0.89)	0.59 (0.84)
36 weeks	0.14 (0.77)	0.14 (0.53)

3. Secondary Outcome

Title	OACIS-I Response Rate on Social Communication Subscale
Description	See above in the primary outcome measure for a description of OACIS-I scale. This section describes the change from baseline of the OACIS-I subdomain of social communication. This subdomain has a range of 1 to 7 - 1 is extremely improved from baseline, 7 is extremely worse from baseline, and 4 is no change. For analysis, the OACIS-I general score and subscale values were recoded, in which 4 (no change) was recoded as 0; 3 to 1 were recoded as +1 to +3 to denote improvement, and 5 to 7 were recoded as -1 to -3 for worsening.
Time Frame	7 weeks, 15 weeks, 22 weeks, 30 weeks, 36 weeks

Outcome Measure Data

Analysis Population Description

Patients started dropping out at subsequent visits so the number analyzed at latter visits is less

Arm/Group Title	Sulforaphane	Placebo
Arm/Group Description	Sulforaphane (SF) will be administered once a day orally in an approximate dose of 1 µmol/lb (2.2 kg µmol/kg) body weight. Each SF tablet will contain 125 mg broccoli seed powder (equivalent to ~ 15 µmol SF). The total dose per day will depend on participants' body weight: 30-50 lb: 3 tablets (45 µmol/day) 50-70 lb: 4 tablets (60 µmol/day) 70-90 lb: 6 tablets (90 µmol/day) 90-110 lb: 7 tablets (105 µmol/day) 110-130 lb: 8 tablets (120 µmol/day) For pilot study, all participants (n=10) will receive SF for 14 days. For main clinical trial, 25 participants will randomly receive SF for 15 weeks (phase 1). During phase 2, a single-arm crossover from placebo to sulforaphane arm will take place, and all participants will receive SF from 15-30 weeks. Sulforaphane: See under active arm description	Placebo tablets identical in size and similar in appearance to the active tablets will be used. Number of placebo tablets will be equivalent to the active tablets depending on participants' body weight. For the main clinical trial, 25 participants will be randomly allocated to receive placebo for 15 weeks (phase 1). During phase 2, a single-arm crossover from placebo arm to sulforaphane arm will take place, and all participants will receive sulforaphane from 15-30 weeks. Placebo: See under placebo arm description
Measure Participants	22	23
7 weeks	0.33 (0.69)	0.56 (0.70)
15 weeks	0.44 (0.70)	0.56 (0.62)
22 weeks	0.65 (0.79)	1.35 (0.61)
30 weeks	0.94 (0.83)	1.25 (0.61)
36 weeks	0.32 (0.61)	0.61 (0.66)

4. Secondary Outcome

Title	Change in Total Aberrant Behavior Checklist Score From Baseline
Description	Aberrant Behavior Checklist (ABC) is a 58 item scale that primarily evaluates how aberrant or abnormal a patient's daily behaviors are. The items evaluate behaviors as they pertain to irritability, lethargy/social withdrawal, stereotypic behavior, hyperactivity/noncompliance, and inappropriate speech. Each item is scored on a scale of 0 to 3, with 0 being better outcome and 3 being worse outcome. The score from each item is added up to calculate a total score. This outcome describes change in total ABC score from baseline at each follow up visit. The scores from all items are added to calculate a total score (0 to 174). This outcome describes change in total ABC score from baseline at each follow up visit.
Time Frame	7 weeks, 15 weeks, 22 weeks, 30 weeks, 36 weeks

Outcome Measure Data

Analysis Population Description

Patients started dropping out at subsequent visits so the number analyzed at later visits is less

Arm/Group Title	Sulforaphane	Placebo
Arm/Group Description	Sulforaphane (SF) will be administered once a day orally in an approximate dose of 1 µmol/lb (2.2 kg µmol/kg) body weight. Each SF tablet will contain 125 mg broccoli seed powder (equivalent to ~ 15 µmol SF). The total dose per day will depend on participants' body weight: 30-50 lb: 3 tablets (45 µmol/day) 50-70 lb: 4 tablets (60 µmol/day) 70-90 lb: 6 tablets (90 µmol/day) 90-110 lb: 7 tablets (105 µmol/day) 110-130 lb: 8 tablets (120 µmol/day) For pilot study, all participants (n=10) will receive SF for 14 days. For main clinical trial, 25 participants will randomly receive SF for 15 weeks (phase 1). During phase 2, a single-arm crossover from placebo to sulforaphane arm will take place, and all participants will receive SF from 15-30 weeks. Sulforaphane: See under active arm description	Placebo tablets identical in size and similar in appearance to the active tablets will be used. Number of placebo tablets will be equivalent to the active tablets depending on participants' body weight. For the main clinical trial, 25 participants will be randomly allocated to receive placebo for 15 weeks (phase 1). During phase 2, a single-arm crossover from placebo arm to sulforaphane arm will take place, and all participants will receive sulforaphane from 15-30 weeks. Placebo: See under placebo arm description
Measure Participants	22	23
7 weeks	-6 (3.4)	-16.23 (14.02)
15 weeks	-22.6 (10.34)	-11.68 (5.40)
22 weeks	-36.33 (14.55)	-22.4 (15.45)
30 weeks	-22.8 (13.5)	-10.29 (9.97)
36 weeks	1.14 (3.53)	-7.8 (5.56)

5. Secondary Outcome

Title	Change in Total SRS-2 Score From Baseline
Description	The Social Responsiveness Scale-2 (SRS-2) is a 65-item scale that measures total scores as well as subscales: four social behaviors (awareness, cognition, communication and motivation) and autistic mannerisms. Each item is rated from 1 to 4 (not true to almost always true) on worksheets that are blinded to the rater with respect to values. Total and subscale scores are calculated as raw scores and can be converted to T-scores. Raw scores (range 0-180) are reported here (unadjusted for general population, since all children had ASD). Higher or lower values at follow up visits compared to baseline indicated worsening or improvement, respectively.
Time Frame	7 weeks, 15 weeks, 22 weeks, 30 weeks, 36 weeks

Outcome Measure Data

Analysis Population Description

Patients dropped out (or families did not return forms) at subsequent visits so the number analyzed at later visits is less.

Arm/Group Title	Sulforaphane	Placebo
Arm/Group Description	Sulforaphane (SF) will be administered once a day orally in an approximate dose of 1 µmol/lb (2.2 kg µmol/kg) body weight. Each SF tablet will contain 125 mg broccoli seed powder (equivalent to ~ 15 µmol SF). The total dose per day will depend on participants' body weight: 30-50 lb: 3 tablets (45 µmol/day) 50-70 lb: 4 tablets (60 µmol/day) 70-90 lb: 6 tablets (90 µmol/day) 90-110 lb: 7 tablets (105 µmol/day) 110-130 lb: 8 tablets (120 µmol/day) For pilot study, all participants (n=10) will receive SF for 14 days. For main clinical trial, 25 participants will randomly receive SF for 15 weeks (phase 1). During phase 2, a single-arm crossover from placebo to sulforaphane arm will take place, and all participants will receive SF from 15-30 weeks. Sulforaphane: See under active arm description	Placebo tablets identical in size and similar in appearance to the active tablets will be used. Number of placebo tablets will be equivalent to the active tablets depending on participants' body weight. For the main clinical trial, 25 participants will be randomly allocated to receive placebo for 15 weeks (phase 1). During phase 2, a single-arm crossover from placebo arm to sulforaphane arm will take place, and all participants will receive sulforaphane from 15-30 weeks. Placebo: See under placebo arm description
Measure Participants	22	23
7 weeks	1.14 (5.33)	-8.06 (7.89)
15 weeks	-16.86 (10.56)	-7.92 (4.45)
22 weeks	-14.61 (9.90)	-13.67 (11.23)
30 weeks	-19.83 (15.0)	-18.59 (11.57)
36 weeks	0.80 (3.30)	-19.59 (10.17)

6. Secondary Outcome

Title	Dithiocarbamate Plasma Concentration Detected by Cyclocondensation at Each Visit
Description	Sulforaphane (and other isothiocyanates, ITC) are conjugated by glutathione (GSH) which then undergoes further enzymatic modifications to give rise sequentially to the cysteinylglycine-, cysteine- and N-acetylcysteine-ITC conjugates, all of which are dithiocarbamates (DTC) and are detected in the cyclocondensation reaction-HPLC assay.
Time Frame	Week 0, Week 7, Week 15, Week 22, Week 30, Week 36

Outcome Measure Data

Analysis Population Description

Number analyzed varied due to participants who dropped out, their blood samples could not be obtained, or data could not be obtained from the assay.

Arm/Group Title	Sulforaphane	Placebo
Arm/Group Description	Sulforaphane (SF) will be administered once a day orally in an approximate dose of 1 µmol/lb (2.2 kg µmol/kg) body weight. Each SF tablet will contain 125 mg broccoli seed powder (equivalent to ~ 15 µmol SF). The total dose per day will depend on participants' body weight: 30-50 lb: 3 tablets (45 µmol/day) 50-70 lb: 4 tablets (60 µmol/day) 70-90 lb: 6 tablets (90 µmol/day) 90-110 lb: 7 tablets (105 µmol/day) 110-130 lb: 8 tablets (120 µmol/day) For pilot study, all participants (n=10) will receive SF for 14 days. For main clinical trial, 25 participants will randomly receive SF for 15 weeks (phase 1). During phase 2, a single-arm crossover from placebo to sulforaphane arm will take place, and all participants will receive SF from 15-30 weeks. Sulforaphane: See under active arm description	Placebo tablets identical in size and similar in appearance to the active tablets will be used. Number of placebo tablets will be equivalent to the active tablets depending on participants' body weight. For the main clinical trial, 25 participants will be randomly allocated to receive placebo for 15 weeks (phase 1). During phase 2, a single-arm crossover from placebo arm to sulforaphane arm will take place, and all participants will receive sulforaphane from 15-30 weeks. Placebo: See under placebo arm description
Measure Participants	22	24
Week 0	0.007 (0.008)	0.006 (0.008)
Week 7	0.299 (0.297)	0.003 (0.005)
Week 15	0.329 (0.350)	0.005 (0.008)
Week 22	0.248 (0,232)	0.205 (0.253)
Week 30	0.165 (0.183)	0.214 (0.228)
Week 36	0.015 (0.024)	0.008 (0.012)

7. Secondary Outcome

Title	Comparison of Free Reduced Glutathione (GSH), Total GSH, Oxidized Glutathione (GSSG) at Week 0 and 15
Description	F-statistic calculated by comparison of Free Reduced GSH, Total GSH and oxidized Glutathione (GSSG) of Week 15 to Week 0.
Time Frame	Week 0 and Week 15

Outcome Measure Data

Analysis Population Description

[Not Specified]

Arm/Group Title	Sulforaphane	Placebo
Arm/Group Description	Sulforaphane (SF) will be administered once a day orally in an approximate dose of 1 µmol/lb (2.2 kg µmol/kg) body weight. Each SF tablet will contain 125 mg broccoli seed powder (equivalent to ~ 15 µmol SF). The total dose per day will depend on participants' body weight: 30-50 lb: 3 tablets (45 µmol/day) 50-70 lb: 4 tablets (60 µmol/day) 70-90 lb: 6 tablets (90 µmol/day) 90-110 lb: 7 tablets (105 µmol/day) 110-130 lb: 8 tablets (120 µmol/day) For pilot study, all participants (n=10) will receive SF for 14 days. For main clinical trial, 25 participants will randomly receive SF for 15 weeks (phase 1). During phase 2, a single-arm crossover from placebo to sulforaphane arm will take place, and all participants will receive SF from 15-30 weeks. Sulforaphane: See under active arm description	Placebo tablets identical in size and similar in appearance to the active tablets will be used. Number of placebo tablets will be equivalent to the active tablets depending on participants' body weight. For the main clinical trial, 25 participants will be randomly allocated to receive placebo for 15 weeks (phase 1). During phase 2, a single-arm crossover from placebo arm to sulforaphane arm will take place, and all participants will receive sulforaphane from 15-30 weeks. Placebo: See under placebo arm description
Measure Participants	22	23
Free Reduced GSH	1.51	0.11
Total GSH	0.00	0.08
GSSG	1.97	0.46

8. Secondary Outcome

Title	Free GSH:GSSG and Total GSH:GSSG Ratios at Week 15
Description	Ratios of free GSH:GSSG and total GSH:GSSG were calculated by obtaining ratios of f-statistic scores from baseline to week 15 between free reduced GSH and GSSG and between total GSH and GSSG.
Time Frame	Week 15

Outcome Measure Data

Analysis Population Description

[Not Specified]

Arm/Group Title	Sulforaphane	Placebo
Arm/Group Description	Sulforaphane (SF) will be administered once a day orally in an approximate dose of 1 µmol/lb (2.2 kg µmol/kg) body weight. Each SF tablet will contain 125 mg broccoli seed powder (equivalent to ~ 15 µmol SF). The total dose per day will depend on participants' body weight: 30-50 lb: 3 tablets (45 µmol/day) 50-70 lb: 4 tablets (60 µmol/day) 70-90 lb: 6 tablets (90 µmol/day) 90-110 lb: 7 tablets (105 µmol/day) 110-130 lb: 8 tablets (120 µmol/day) For pilot study, all participants (n=10) will receive SF for 14 days. For main clinical trial, 25 participants will randomly receive SF for 15 weeks (phase 1). During phase 2, a single-arm crossover from placebo to sulforaphane arm will take place, and all participants will receive SF from 15-30 weeks. Sulforaphane: See under active arm description	Placebo tablets identical in size and similar in appearance to the active tablets will be used. Number of placebo tablets will be equivalent to the active tablets depending on participants' body weight. For the main clinical trial, 25 participants will be randomly allocated to receive placebo for 15 weeks (phase 1). During phase 2, a single-arm crossover from placebo arm to sulforaphane arm will take place, and all participants will receive sulforaphane from 15-30 weeks. Placebo: See under placebo arm description
Measure Participants	22	23
Free GSH:GSSG	12.72	0.87
Total GSH:GSSG	5.16	0.03

9. Other Pre-specified Outcome

Title	NQO1: NAD(P)H:Quinone Oxidoreductase-1
Description	Cytoprotective enzyme regulated by nuclear factor erythroid 2-related factor 2 (Nrf2), the master regulator of cellular redox homeostasis and an inhibitor of a key pro-inflammatory pathway, of which both functions are critical factors in the neuropathology of ASD. Total cellular RNA was isolated from peripheral blood mononuclear cells (PBMCs) and complementary DNAs (cDNA) were synthesized. Quantitative real-time PCR analysis was performed using the Applied Biosystems QuantStudio™ 3 Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA). Relative mRNA expression was normalized to GAPDH. Gene expression was calculated using the comparative 2-ΔΔCT method.
Time Frame	Week 0, Week 15, Week 30

Outcome Measure Data

Analysis Population Description

N varies due to missing data, variable clinic visits or assay failure.

Arm/Group Title	Sulforaphane	Placebo
Arm/Group Description	Children receiving sulforaphane 0 - 30 weeks	Children receiving placebo from 0 - 15 weeks; received sulforaphane (SF) from 16 - 30 weeks.
Measure Participants	22	23
Week 0 (baseline)	2.23 (0.18)	2.25 (0.22)
Week 15	2.19 (0.16)	2.14 (0.20)
Week 30	2.15 (0.15)	2.13 (0.11)

10. Other Pre-specified Outcome

Title	xCT
Description	xCT (SLC7A11): Cystine/glutamate antiporter encoded by the SLC7A11 gene.Total cellular RNA was isolated from peripheral blood mononuclear cells (PBMCs) and complementary DNAs (cDNA) were synthesized. Quantitative real-time PCR analysis was performed using the Applied Biosystems QuantStudio™ 3 Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA). Relative mRNA expression was normalized to GAPDH. Gene expression was calculated using the comparative 2-ΔΔCT method.
Time Frame	Week 0, Week 15, Week 30

Outcome Measure Data

Analysis Population Description

N varies due to missing data, variable clinic visits or assay failure.

Arm/Group Title	Sulforaphane	Placebo
Arm/Group Description	Children receiving sulforaphane 0 - 30 weeks	Children receiving placebo 0 - 15 weeks; sulforaphane from 16 - 30 weeks
Measure Participants	22	23
Week 0	0.24 (0.94)	0.40 (0.71)
Week 15	0.25 (0.65)	0.30 (0.95)
Week 30	0.24 (1.02)	0.27 (1.00)

11. Other Pre-specified Outcome

Title	HO-1 (Heme Oxygenase 1)
Description	HO-1 (heme oxygenase 1): an essential and Nrf2-dependent enzyme in heme catabolism. Total cellular RNA was isolated from peripheral blood mononuclear cells (PBMCs) and complementary DNAs (cDNA) were synthesized. Quantitative real-time PCR analysis was performed using the Applied Biosystems QuantStudio™ 3 Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA). Relative mRNA expression was normalized to GAPDH. Gene expression was calculated using the comparative 2-ΔΔCT method.
Time Frame	Week 0, Week 15, Week 30

Outcome Measure Data

Analysis Population Description

N varies due to missing data, variable clinic visits or assay failure

Arm/Group Title	Sulforaphane	Placebo
Arm/Group Description	Children receiving sulforaphane 0 - 30 weeks	Children receiving placebo 0 - 15 weeks; sulforaphane from 16 - 30 weeks
Measure Participants	22	23
Week 0	0.10 (1.21)	0.27 (0.98)
Week 15	0.74 (0.69)	0.55 (0.48)
Week 30	0.74 (0.56)	0.30 (1.40)

12. Other Pre-specified Outcome

Title	HSP70
Description	HSP70 (Heat shock protein 70) was examined because it is upregulated by SF in vitro. Total cellular RNA was isolated from peripheral blood mononuclear cells (PBMCs) and complementary DNAs (cDNA) were synthesized. Quantitative real-time PCR analysis was performed using the Applied Biosystems QuantStudio™ 3 Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA). Relative mRNA expression was normalized to GAPDH. Gene expression was calculated using the comparative 2-ΔΔCT method.
Time Frame	Week 0, Week 15, Week 30

Outcome Measure Data

Analysis Population Description

N varies due to missing data, variable clinic visits or assay failure.

Arm/Group Title	Sulforaphane	Placebo
Arm/Group Description	Children receiving sulforaphane 0 - 30 weeks	Children receiving placebo 0 - 15 weeks; sulforaphane from 16 - 30 weeks
Measure Participants	22	23
Week 0	1.11 (0.29)	0.99 (0.43)
Week 15	1.33 (0.43)	1.14 (0.30)
Week 30	1.23 (0.34)	1.20 (0.40)

13. Other Pre-specified Outcome

Title	HSP27
Description	HSP27 (Heat shock protein 27) was examined because it is upregulated by SF in vitro. Total cellular RNA was isolated from peripheral blood mononuclear cells (PBMCs) and complementary DNAs (cDNA) were synthesized. Quantitative real-time PCR analysis was performed using the Applied Biosystems QuantStudio™ 3 Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA). Relative mRNA expression was normalized to GAPDH. Gene expression was calculated using the comparative 2-ΔΔCT method.
Time Frame	Week 0, Week 15, Week 30.

Outcome Measure Data

Analysis Population Description

N varies due to missing data, variable clinic visits or assay failure

Arm/Group Title	Sulforaphane	Placebo
Arm/Group Description	Children receiving sulforaphane 0 - 30 weeks	Children receiving placebo 0 - 15 weeks; sulforaphane from 16 - 30 weeks
Measure Participants	22	23
Week 0	0.21 (0.46)	-0.13 (0.98)
Week 15	0.21 (0.63)	-0.83 (1.21)
Week 30	-0.47 (1.21)	-0.58 (1.46)

14. Other Pre-specified Outcome

Title	IL-6
Description	IL-6 (interleukin 6) cytokine gene expression, a nuclear factor-kappa B - regulated inflammatory biomarker. Total cellular RNA was isolated from peripheral blood mononuclear cells (PBMCs) and complementary DNAs (cDNA) were synthesized. Quantitative real-time PCR analysis was performed using the Applied Biosystems QuantStudio™ 3 Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA). Relative mRNA expression was normalized to GAPDH. Gene expression was calculated using the comparative 2-ΔΔCT method.
Time Frame	Week 0, Week 15, Week 30

Outcome Measure Data

Analysis Population Description

N varies due to missing data, variable clinic visits or assay failure.

Arm/Group Title	Sulforaphane	Placebo
Arm/Group Description	Children receiving sulforaphane 0 - 30 weeks	Children receiving placebo 0 - 15 weeks; sulforaphane from 16 - 30 weeks
Measure Participants	22	23
Week 0	1.76 (0.39)	1.82 (0.30)
Week 15	2.05 (0.28)	1.87 (0.42)
Week 30	1.99 (0.23)	2.08 (0.22)

15. Other Pre-specified Outcome

Title	IL-1β
Description	IL-1β: Interleukin-1 beta cytokine gene expression, a nuclear factor-kappa B - regulated inflammatory biomarker. Total cellular RNA was isolated from peripheral blood mononuclear cells (PBMCs) and complementary DNAs (cDNA) were synthesized. Quantitative real-time PCR analysis was performed using the Applied Biosystems QuantStudio™ 3 Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA). Relative mRNA expression was normalized to GAPDH. Gene expression was calculated using the comparative 2-ΔΔCT method.
Time Frame	Week 0, Week 15, Week 30

Outcome Measure Data

Analysis Population Description

N varies due to missing data, variable clinic visits or assay failure.

Arm/Group Title	Sulforaphane	Placebo
Arm/Group Description	Children receiving sulforaphane 0 - 30 weeks	Children receiving placebo 0 - 15 weeks; sulforaphane from 16 - 30 weeks
Measure Participants	22	23
Week 0	0.90 (1.16)	1.14 (1.11)
Week 15	1.45 (0.59)	1.47 (0.50)
Week 30	1.50 (0.49)	1.59 (0.43)

16. Other Pre-specified Outcome

Title	Cox-2
Description	Cox-2 (cyclooxygenase-2): a nuclear factor-kappa B - regulated inflammatory biomarker. Total cellular RNA was isolated from peripheral blood mononuclear cells (PBMCs) and complementary DNAs (cDNA) were synthesized. Quantitative real-time PCR analysis was performed using the Applied Biosystems QuantStudio™ 3 Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA). Relative mRNA expression was normalized to GAPDH. Gene expression was calculated using the comparative 2-ΔΔCT method.
Time Frame	Week 0, Week 15, Week 30

Outcome Measure Data

Analysis Population Description

N varies due to missing data, variable clinic visits or assay failure.

Arm/Group Title	Sulforaphane	Placebo
Arm/Group Description	Children receiving sulforaphane 0 - 30 weeks	Children receiving placebo 0 - 15 weeks; sulforaphane from 16 - 30 weeks
Measure Participants	22	23
Week 0	0.14 (0.86)	-0.20 (1.06)
Week 15	-0.07 (1.59)	0.16 (0.66)
Week 30	-0.46 (1.57)	-0.17 (1.17)

17. Other Pre-specified Outcome

Title	TNF-α
Description	TNF-α (Tumor necrosis factor alpha), a cytokine as inflammatory biomarker. Total cellular RNA was isolated from peripheral blood mononuclear cells (PBMCs) and complementary DNAs (cDNA) were synthesized. Quantitative real-time PCR analysis was performed using the Applied Biosystems QuantStudio™ 3 Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA). Relative mRNA expression was normalized to GAPDH. Gene expression was calculated using the comparative 2-ΔΔCT method.
Time Frame	Week 0, Week 15, Week 30

Outcome Measure Data

Analysis Population Description

N varies due to missing data, variable clinic visits or assay failure

Arm/Group Title	Sulforaphane	Placebo
Arm/Group Description	Children receiving sulforaphane 0 - 30 weeks	Children receiving placebo 0 - 15 weeks; sulforaphane from 16 - 30 weeks
Measure Participants	22	23
Week 0	0.70 (1.00)	0.91 (0.37)
Week 15	0.98 (1.03)	1.08 (0.49)
Week 30	1.17 (0.79)	1.10 (0.73)

Adverse Events

Time Frame	The pilot study took place over 2 weeks for each of 10 participants. The main study took place over 2 years, 6 months; 1/1/16 - 6/30/18 for 45 participants, each of whom was assessed from screening (time 0), 7, 15, 22 and up to 36 weeks for the final visit. Participants in the pilot study were followed for adverse events during regular clinical visits for at least 12 months after the study and none were reported.
Adverse Event Reporting Description	Adverse event reporting followed clinicaltrials.gov Definitions.
Arm/Group Title	Sulforaphane		Placebo
Arm/Group Description	Sulforaphane (SF) will be administered once a day orally in an approximate dose of 1 µmol/lb (2.2 kg µmol/kg) body weight. Each SF tablet will contain 125 mg broccoli seed powder (equivalent to ~ 15 µmol SF). The total dose per day will depend on participants' body weight: 30-50 lb: 3 tablets (45 µmol/day) 50-70 lb: 4 tablets (60 µmol/day) 70-90 lb: 6 tablets (90 µmol/day) 90-110 lb: 7 tablets (105 µmol/day) 110-130 lb: 8 tablets (120 µmol/day) For pilot study, all participants (n=10) will receive SF for 14 days. For main clinical trial, 25 participants will randomly receive SF for 15 weeks (phase 1). During phase 2, a single-arm crossover from placebo to sulforaphane arm will take place, and all participants will receive SF from 15-30 weeks. Sulforaphane: See under active arm description		Placebo tablets identical in size and similar in appearance to the active tablets will be used. Number of placebo tablets will be equivalent to the active tablets depending on participants' body weight. For the main clinical trial, 25 participants will be randomly allocated to receive placebo for 15 weeks (phase 1). During phase 2, a single-arm crossover from placebo arm to sulforaphane arm will take place, and all participants will receive sulforaphane from 15-30 weeks. Placebo: See under placebo arm description
All Cause Mortality
	Sulforaphane		Placebo
	Affected / at Risk (%)	# Events	Affected / at Risk (%)	# Events
Total	0/32 (0%)		0/23 (0%)
Serious Adverse Events
	Sulforaphane		Placebo
	Affected / at Risk (%)	# Events	Affected / at Risk (%)	# Events
Total	0/32 (0%)		1/23 (4.3%)
Endocrine disorders
Hashimoto thyroiditis	0/32 (0%)	0	1/23 (4.3%)	1
Other (Not Including Serious) Adverse Events
	Sulforaphane		Placebo
	Affected / at Risk (%)	# Events	Affected / at Risk (%)	# Events
Total	8/32 (25%)		0/23 (0%)
Gastrointestinal disorders
gastrointestinal upset	2/32 (6.3%)		2/23 (8.7%)
General disorders
taste/smell	2/32 (6.3%)		2/23 (8.7%)
Nervous system disorders
insomnia	2/32 (6.3%)		2/23 (8.7%)
irritability	2/32 (6.3%)		2/23 (8.7%)

Limitations/Caveats

The sample size was limited to 45 children with ASD and we could not impute missing data for those participants who were lost to follow up.

More Information

Certain Agreements

All Principal Investigators ARE employed by the organization sponsoring the study.

There is NOT an agreement between Principal Investigators and the Sponsor (or its agents) that restricts the PI's rights to discuss or publish trial results after the trial is completed.