Randomized controlled trial of sulforaphane and metabolite discovery in children with Autism Spectrum Disorder

Abstract

Background: Sulforaphane (SF), an isothiocyanate in broccoli, has potential benefits relevant to autism spectrum disorder (ASD) through its effects on several metabolic and immunologic pathways. Previous clinical trials of oral SF demonstrated positive clinical effects on behavior in young men and changes in urinary metabolomics in children with ASD.

Methods: We conducted a 15-week randomized parallel double-blind placebo-controlled clinical trial with 15-week open-label treatment and 6-week no-treatment extensions in 57 children, ages 3-12 years, with ASD over 36 weeks. Twenty-eight were assigned SF and 29 received placebo (PL). Clinical effects, safety and tolerability of SF were measured as were biomarkers to elucidate mechanisms of action of SF in ASD.

Results: Data from 22 children taking SF and 23 on PL were analyzed. Treatment effects on the primary outcome measure, the Ohio Autism Clinical Impressions Scale (OACIS), in the general level of autism were not significant between SF and PL groups at 7 and 15 weeks. The effect sizes on the OACIS were non-statistically significant but positive, suggesting a possible trend toward greater improvement in those on treatment with SF (Cohen's d 0.21; 95% CI - 0.46, 0.88 and 0.10; 95% CI - 0.52, 0.72, respectively). Both groups improved in all subscales when on SF during the open-label phase. Caregiver ratings on secondary outcome measures improved significantly on the Aberrant Behavior Checklist (ABC) at 15 weeks (Cohen's d - 0.96; 95% CI - 1.73, - 0.15), but not on the Social Responsiveness Scale-2 (SRS-2). Ratings on the ABC and SRS-2 improved with a non-randomized analysis of the length of exposure to SF, compared to the pre-treatment baseline (p < 0.001). There were significant changes with SF compared to PL in biomarkers of glutathione redox status, mitochondrial respiration, inflammatory markers and heat shock proteins. Clinical laboratory studies confirmed product safety. SF was very well tolerated and side effects of treatment, none serious, included rare insomnia, irritability and intolerance of the taste and smell.

Limitations: The sample size was limited to 45 children with ASD and we did not impute missing data. We were unable to document significant changes in clinical assessments during clinical visits in those taking SF compared to PL. The clinical results were confounded by placebo effects during the open-label phase.

Conclusions: SF led to small yet non-statistically significant changes in the total and all subscale scores of the primary outcome measure, while for secondary outcome measures, caregivers' assessments of children taking SF showed statistically significant improvements compared to those taking PL on the ABC but not the SRS-2. Clinical effects of SF were less notable in children compared to our previous trial of a SF-rich preparation in young men with ASD. Several of the effects of SF on biomarkers correlated to clinical improvements. SF was very well tolerated and safe and effective based on our secondary clinical measures.

Trial registration: This study was prospectively registered at clinicaltrials.gov (NCT02561481) on September 28, 2015. Funding was provided by the U.S. Department of Defense.

Keywords: Autism spectrum disorder (ASD); Biomarkers; Clinical trial; Placebo effects; Sulforaphane.

Conflict of interest statement

AWZ reports giving testimony in legal proceedings on behalf of plaintiffs and defendants in matters related to pediatric neurology and Autism Spectrum Disorder. JWF retired from the full-time faculty at Johns Hopkins in mid-2020, and now serves as a scientific advisor to Brassica Protection Products LLC (Baltimore, MD, USA), which produces a glucoraphanin-rich broccoli seed extract that it supplies to the supplement industry. AWZ is named on a patent on the use of sulforaphane for the treatment of autism that has been assigned to Johns Hopkins University.

Figures

Fig. 1

Study timeline (6 total visits); in Phase 1 (1–15 weeks) participants were randomly assigned to either SF or placebo with visits at baseline, 7 and 15 weeks. In Phase 2 (16–30 weeks), all participants received SF and returned for follow-up at 22 and 30 weeks. In Phase 3 (31–36 weeks), there was no treatment, with a final visit at 36 weeks

Fig. 2

Randomization and follow-up. SF sulforaphane and PL placebo

Fig. 3

Change in mean (95% CI) total SRS-2 raw scores from baseline using sex- and fever response-matched pairs. Scores between sulforaphane (SF) and placebo (PL) groups did not differ significantly. Both groups’ scores improved during the open label phase after 15 weeks (shaded box). Note: 95% CI not shown for 36 weeks due to small sample size; see Additional file 1: Table S5 for mean (SD)

Fig. 4

Change in mean (95% CI) total ABC raw scores from baseline using sex- and fever response-matched pairs. Change in mean score was significantly different between the sulforaphane (SF) and placebo (PL) groups at 15 weeks. Both groups’ scores improved during the open label phase after 15 weeks (shaded box). Note: 95% CI not shown for week 36 due to small sample size; see Additional file 1: Table S8 for mean (SD). *p = 0.02

Fig. 5

Plasma cyclocondensation of SF metabolite (DTC) levels. “PL” directly under the X-axis indicates Placebo arm and “SF” indicates Sulforaphane arm. Further annotation indicates actual treatment delivery at times indicated

Fig. 6

Natural log of relative gene expression for biomarkers, SF versus PL, from baseline to 15 weeks. N = 42. Small circles outside of the boxes denote outliers. Large circles inside the boxes denote means; center horizontal lines inside the boxes denote medians

Fig. 7

Natural log of relative gene expression of biomarkers for participants with more than 20% improvement on ABC total scores from baseline to 15 weeks. N = 42. Small circles outside of the boxes denote outliers. Large circles inside the boxes denote means; center horizontal lines inside the boxes denote medians

Fig. 8

Relation between respiratory parameters and both treatment with Sulforaphane (SF) and change in Aberrant Behavior Checklist (ABC) scores. a ATP-Linked respiration significantly increased (p < 0.05) with SF treatment (N = 13) as compared to PL (N = 14); b Individuals with developmental regression demonstrated a trend for decreased ATP-Linked Respiration and Maximal Respiratory Capacity over the DBPC treatment period while those without developmental regression demonstrated the opposite trend. c Greater improvement in ABC scores was associated with a greater increase in ATP-Linked Respiration and d decrease in Proton Leak Respiration, across the entire study period