Efficacy of Sulforaphane in Treatment of Children with Autism Spectrum Disorder: A Randomized Double-Blind Placebo-Controlled Multi-center Trial.

Sulforaphane has been reported to possibly improve core symptoms associated with autism spectrum disorders from mostly small size studies. Here we present results of a larger randomized clinical trial (N = 108) in China. There were no significant changes in caregiver rated scales between sulforaphane and placebo groups. However, clinician rated scales showed a significant improvement in the sulforaphane group, and one third of participants showed at least a 30% decrease in score by 12 weeks treatment. The effects of sulforaphane were seen across the full range of intelligence and greater in participants over 10 years. Sulforaphane was safe and well-tolerated even for young children. The inconsistent results between caregiver and clinician rated scales suggest more clinical trials are needed to confirm our findings.

A Presurgical-Window Intervention Trial of Isothiocyanate-Rich Broccoli Sprout Extract in Patients with Breast Cancer.

SCOPE: Dietary isothiocyanates (ITCs) from cruciferous vegetables have shown potent anti-breast cancer activities in preclinical models, but their anticancer effects in vivo in breast cancer patients remain elusive. A proof-of-principle, presurgical window of opportunity trial is conducted to assess the anticancer effects of dietary ITCs in breast cancer patients. METHODS AND RESULTS: Thirty postmenopausal breast cancer patients are randomly assigned to receive ITC-rich broccoli sprout extract (BSE) (200 µmol ITC per day) or a placebo for 2 weeks. Expression of biomarkers related to ITCs functions are measured in breast cancer tissue specimens at pre- and post-interventions using immunohistochemistry staining. First morning urine samples are collected at both timepoints for proteomic analysis. Overall, the study shows high compliance (100%) and low toxicity (no grade 4 adverse event). Trends of increase in cleaved caspase 3 and tumor-infiltrating lymphocytes (TILs) and trends of decrease in Ki-67 and nuclear to cytoplasm ratio of estrogen receptor (ER)-α are observed in the BSE arm only, consistent with the significantly altered signaling pathways identified in urinary proteomic analysis. CONCLUSIONS: Anticancer activities of ITCs are observed in breast cancer patients, supporting the potential beneficial roles of ITC-containing cruciferous vegetables in breast cancer prognosis.

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

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.

The Challenges of Designing and Implementing Clinical Trials With Broccoli Sprouts and Turning Evidence Into Public Health Action.

Broccoli sprouts are a convenient and rich source of the glucosinolate glucoraphanin, which can generate the chemopreventive agent sulforaphane through the catalytic actions of plant myrosinase or ß-thioglucosidases in the gut microflora. Sulforaphane, in turn, is an inducer of cytoprotective enzymes through activation of Nrf2 signaling, and a potent inhibitor of carcinogenesis in multiple murine models. Sulforaphane is also protective in models of diabetes, neurodegenerative disease, and other inflammatory processes, likely reflecting additional actions of Nrf2 and interactions with other signaling pathways. Translating this efficacy into the design and implementation of clinical chemoprevention trials, especially food-based trials, faces numerous challenges including the selection of the source, placebo, and dose as well as standardization of the formulation of the intervention material. Unlike in animals, purified sulforaphane has had very limited use in clinical studies. We have conducted a series of clinical studies and randomized clinical trials to evaluate the effects of composition (glucoraphanin-rich [± myrosinase] vs. sulforaphane-rich or mixture beverages), formulation (beverage vs. tablet) and dose, on the efficacy of these broccoli sprout-based preparations to evaluate safety, pharmacokinetics, pharmacodynamic action, and clinical benefit. While the challenges for the evaluation of broccoli sprouts in clinical trials are themselves formidable, further hurdles must be overcome to bring this science to public health action.

Mitigating potential public health problems associated with edible cannabis products through adequate regulation: A landscape analysis.

An edible cannabis product (ECP) manufactured with food ingredients is subject to the same types of contamination as any conventional food product. Physical, microbial, and chemical hazards are a potential threat to anyone consuming cannabinoid-containing products by mouth. Preventing the unintentional ingestion of ECPs is also a concern for public health professionals. An analysis of the regulatory landscape in the United States (US) was conducted to identify best practices specific to ECPs and to pinpoint preventative safety measures that had not been extensively implemented. Widespread adoption of some of the more useful precedents set by US jurisdictions, as examined in this work, could be of great value in protecting public health.

Biomarker Exploration in Human Peripheral Blood Mononuclear Cells for Monitoring Sulforaphane Treatment Responses in Autism Spectrum Disorder.

Autism Spectrum Disorder (ASD) is one of the most common neurodevelopmental disorders with no drugs treating the core symptoms and no validated biomarkers for clinical use. The multi-functional phytochemical sulforaphane affects many of the biochemical abnormalities associated with ASD. We investigated potential molecular markers from three ASD-associated physiological pathways that can be affected by sulforaphane: redox metabolism/oxidative stress; heat shock response; and immune dysregulation/inflammation, in peripheral blood mononuclear cells (PBMCs) from healthy donors and patients with ASD. We first analyzed the mRNA levels of selected molecular markers in response to sulforaphane ex vivo treatment in PBMCs from healthy donors by real-time quantitative PCR. All of the tested markers showed quantifiability, accuracy and reproducibility. We then compared the expression levels of those markers in PBMCs taken from ASD patients in response to orally-delivered sulforaphane. The mRNA levels of cytoprotective enzymes (NQO1, HO-1, AKR1C1), and heat shock proteins (HSP27 and HSP70), increased. Conversely, mRNA levels of pro-inflammatory markers (IL-6, IL-1ß, COX-2 and TNF-α) decreased. Individually none is sufficiently specific or sensitive, but when grouped by function as two panels, these biomarkers show promise for monitoring pharmacodynamic responses to sulforaphane in both healthy and autistic humans, and providing guidance for biomedical interventions.

Maternal Dyslipidemia, Plasma Branched-Chain Amino Acids, and the Risk of Child Autism Spectrum Disorder: Evidence of Sex Difference.

In contrast to the well-observed associations between obesity, diabetes, and autism spectrum disorder (ASD), the roles of maternal dyslipidemia and sex disparity in ASD have not been well-studied. We examined the joint associations of maternal plasma cholesterols, branched-chain amino acids (BCAAs) and child sex on child ASD risk. We analyzed data from 756 mother-infant pairs (86 ASD) from the Boston Birth Cohort. Maternal plasma cholesterols and BCAAs were measured in samples collected 24-72 h postpartum. We found that in this urban, low-income prospective birth cohort, low maternal high-density lipoprotein cholesterol (HDL-C), above-median maternal plasma BCAA concentrations, and male sex additively or synergistically increased risk of ASD. Additional studies are necessary to confirm our findings.

Broccoli or Sulforaphane: Is It the Source or Dose That Matters?

There is robust epidemiological evidence for the beneficial effects of broccoli consumption on health, many of them clearly mediated by the isothiocyanate sulforaphane. Present in the plant as its precursor, glucoraphanin, sulforaphane is formed through the actions of myrosinase, a ß-thioglucosidase present in either the plant tissue or the mammalian microbiome. Since first isolated from broccoli and demonstrated to have cancer chemoprotective properties in rats in the early 1990s, over 3000 publications have described its efficacy in rodent disease models, underlying mechanisms of action or, to date, over 50 clinical trials examining pharmacokinetics, pharmacodynamics and disease mitigation. This review evaluates the current state of knowledge regarding the relationships between formulation (e.g., plants, sprouts, beverages, supplements), bioavailability and efficacy, and the doses of glucoraphanin and/or sulforaphane that have been used in pre-clinical and clinical studies. We pay special attention to the challenges for better integration of animal model and clinical studies, particularly with regard to selection of dose and route of administration. More effort is required to elucidate underlying mechanisms of action and to develop and validate biomarkers of pharmacodynamic action in humans. A sobering lesson is that changes in approach will be required to implement a public health paradigm for dispensing benefit across all spectrums of the global population.

Compartmentalization of anti-oxidant and anti-inflammatory gene expression in current and former smokers with COPD.

BACKGROUND: Patients with chronic obstructive pulmonary disease (COPD) have high oxidative stress associated with the severity of the disease. Nuclear factor erythroid-2 related factor 2 (Nrf2)-directed stress response plays a critical role in the protection of lung cells to oxidative stress by upregulating antioxidant genes in response to tobacco smoke. There is a critical gap in our knowledge about Nrf-2 regulated genes in active smokers and former-smokers with COPD in different cell types from of lungs and surrogate peripheral tissues. METHODS: We compared the expression of Nrf2 and six of its target genes in alveolar macrophages, nasal, and bronchial epithelium and peripheral blood mononuclear cells (PBMCs) in current and former smokers with COPD. We compared cell-type specific of Nrf2 and its target genes as well as markers of oxidative and inflammatory stress. RESULTS: We enrolled 89 patients; expression all Nrf2 target gene measured were significantly higher in the bronchial epithelium from smokers compared to non-smokers. None were elevated in alveolar macrophages and only one was elevated in each of the other compartments. CONCLUSION: Bronchial epithelium is the most responsive tissue for transcriptional activation of Nrf2 target genes in active smokers compared to former-smokers with COPD that correlated with oxidative stress and inflammatory markers. There were no consistent trends in gene expression in other cell types tested. TRIAL REGISTRATION: Clinicaltrials.gov : NCT01335971.

Maternal Obesity/Diabetes, Plasma Branched-Chain Amino Acids, and Autism Spectrum Disorder Risk in Urban Low-Income Children: Evidence of Sex Difference.

Maternal metabolic conditions are known risk factors for child autism spectrum disorder (ASD). Branched-chain amino acids (BCAAs) are also associated with ASD. We examined the joint associations of maternal metabolic conditions and BCAAs on the risk of child ASD and whether the associations differed by child's sex. We analyzed 789 mother-infant pairs, a subset of the Boston Birth Cohort, from a predominantly urban, low-income, minority population. Maternal plasma BCAAs were measured by liquid chromatography-tandem mass spectrometry in samples collected 24-72 hr postpartum. A composite BCAA score was created using factor analysis, and prepregnancy obesity and diabetes (ob/DM) were combined into one variable. Logistic regression was used to explore the role of BCAAs as mediators or cofactors with ob/DM and child's sex on ASD risk. BCAA-ob/DM and BCAA-sex interactions were also examined. Maternal BCAAs alone were not associated with ASD and did not mediate the path between ob/DM and ASD. In the presence of maternal ob/DM, BCAA score was significantly associated with ASD (adjusted OR 2.33, 95% CI 1.18, 4.60). Interactions were present for valine with ob/DM and for valine and isoleucine with male sex on ASD risk. The odds ratio (OR) for risk of ASD was the greatest with all three risk factors combined-male sex, above median BCAA score, and ob/DM (OR 10.79, 95% CI 4.40, 26.42). Similar patterns were found for other developmental disorders, though not as strong as for ASD. Additional studies are warranted to clarify the role of maternal BCAAs, ob/DM, and child's sex in ASD. Autism Res 2019, 12: 1562-1573. © 2019 International Society for Autism Research, Wiley Periodicals, Inc. LAY SUMMARY: This study investigated whether maternal obesity/diabetes and maternal circulating branched-chain amino acids (BCAAs) can jointly affect child ASD risk and whether the associations differ by child's sex. We found that the risk of ASD was greater among mothers with obesity/diabetes who also had elevated concentrations of BCAAs and that this risk was even greater for male children. These findings provide new evidence on fetal origins of ASD and sex difference and warrant additional investigation.

A Strategy to Deliver Precise Oral Doses of the Glucosinolates or Isothiocyanates from Moringa oleifera Leaves for Use in Clinical Studies.

The tropical tree Moringa oleifera produces high yields of protein-rich leaf biomass, is widely used as a food source, contains an abundance of phytochemicals, and thus has great potential for chronic disease prevention and perhaps, treatment. We have developed and characterized standardized ways of preparing aqueous "teas" from moringa leaves to deliver precisely calibrated levels of phytochemicals for use in clinical trials. These phytochemicals, especially the glucosinolate glucomoringin and the isothiocyanate moringin, produced from it following hydrolysis by the enzyme myrosinase, provide potent anti-inflammatory and cytoprotective indirect antioxidant activity. The taste of both hot and cold teas is palatable without the need for flavor masking. These teas can be easily and reproducibly prepared in underserved tropical regions of the world where moringa is cultivated. Isothiocyanate yield from a cold extraction was rapid and essentially complete after 30 min and its anti-inflammatory potential is comparable to that of equimolar purified moringin. A preparation similar to this may be safe to consume with respect to its bacterial titer even after 48 h without refrigeration. Thus, facile delivery of moringa tea to both adults and children for clinical evaluation of their effects on such conditions as autism, diabetes, and hypertension, is now possible.

Bioavailability of Sulforaphane Following Ingestion of Glucoraphanin-Rich Broccoli Sprout and Seed Extracts with Active Myrosinase: A Pilot Study of the Effects of Proton Pump Inhibitor Administration.

We examined whether gastric acidity would affect the activity of myrosinase, co-delivered with glucoraphanin (GR), to convert GR to sulforaphane (SF). A broccoli seed and sprout extract (BSE) rich in GR and active myrosinase was delivered before and after participants began taking the anti-acid omeprazole, a potent proton pump inhibitor. Gastric acidity appears to attenuate GR bioavailability, as evidenced by more SF and its metabolites being excreted after participants started taking omeprazole. Enteric coating enhanced conversion of GR to SF, perhaps by sparing myrosinase from the acidity of the stomach. There were negligible effects of age, sex, ethnicity, BMI, vegetable consumption, and bowel movement frequency and quality. Greater body mass correlated with reduced conversion efficiency. Changes in the expression of 20 genes in peripheral blood mononuclear cells were evaluated as possible pharmacodynamic indicators. When grouped by their primary functions based on a priori knowledge, expression of genes associated with inflammation decreased non-significantly, and those genes associated with cytoprotection, detoxification and antioxidant functions increased significantly with bioavailability. Using principal components analysis, component loadings of the changes in gene expression confirmed these groupings in a sensitivity analysis.

Investigation into the use of histone deacetylase inhibitor MS-275 as a topical agent for the prevention and treatment of cutaneous squamous cell carcinoma in an SKH-1 hairless mouse model.

Cutaneous squamous cell carcinomas are a common form of highly mutated keratinocyte skin cancers that are of particular concern in immunocompromised patients. Here we report on the efficacy of topically applied MS-275, a clinically used histone deacetylase inhibitor, for the treatment and management of this disease. At 2 mg/kg, MS-275 significantly decreased tumor burden in an SKH-1 hairless mouse model of UVB radiation-induced skin carcinogenesis. MS-275 was cell permeable as a topical formulation and induced histone acetylation changes in mouse tumor tissue. MS-275 was also effective at inhibiting the proliferation of patient derived cutaneous squamous cell carcinoma lines and was particularly potent toward cells isolated from a regional metastasis on an immunocompromised individual. Our findings support the use of alternative routes of administration for histone deacetylase inhibitors in the treatment of high-risk squamous cell carcinoma which may ultimately lead to more precise delivery and reduced systemic toxicity.

Crucifers and related vegetables and supplements for neurologic disorders: what is the evidence?

PURPOSE OF REVIEW: Neurologic disorders have varied pathophysiology, yet many of them appear to have core molecular pathways that are aberrant. We review the evidence that a dietary component may have utility in ameliorating or preventing at least some of them. RECENT FINDINGS: The weight of evidence supporting prescriptive dietary recommendations to promote or enhance healthspan has been building for decades. Cruciferous vegetables are a key part of the arsenal of nutrition-based approaches for reducing the burden of chronic disease. Much new evidence suggests that neurological disorders are among the potential targets for this approach. This evidence includes at least nine clinical studies of neurodevelopmental conditions like autism spectrum disorder and schizophrenia, and there are a great many studies in animal model systems, of neurodegenerative disorders like Alzheimer's and Parkinson's diseases. This review highlights the most bioactive and most well-studied compounds from crucifers - the isothiocyanates, in particular sulforaphane. SUMMARY: There is great promise for the regular use of cruciferous vegetables or supplements containing standardized levels of bioactives in the treatment and prevention of neurologic disorders. Many clinical and animal studies are underway, and the evidence is building to support this strategy.

Sulforaphane Augments Glutathione and Influences Brain Metabolites in Human Subjects: A Clinical Pilot Study.

Schizophrenia and other neuropsychiatric disorders await mechanism-associated interventions. Excess oxidative stress is increasingly appreciated to participate in the pathophysiology of brain disorders, and decreases in the major antioxidant, glutathione (GSH), have been reported in multiple studies. Technical cautions regarding the estimation of oxidative stress-related changes in the brain via imaging techniques have led investigators to explore peripheral GSH as a possible pathological signature of oxidative stress-associated brain changes. In a preclinical model of GSH deficiency, we found a correlation between whole brain and peripheral GSH levels. We found that the naturally occurring isothiocyanate sulforaphane increased blood GSH levels in healthy human subjects following 7 days of daily oral administration. In parallel, we explored the potential influence of sulforaphane on brain GSH levels in the anterior cingulate cortex, hippocampus, and thalamus via 7-T magnetic resonance spectroscopy. A significant positive correlation between blood and thalamic GSH post- and pre-sulforaphane treatment ratios was observed, in addition to a consistent increase in brain GSH levels in response to treatment. This clinical pilot study suggests the value of exploring relationships between peripheral GSH and clinical/neuropsychological measures, as well as the influences sulforaphane has on functional measures that are altered in neuropsychiatric disorders.

Identification of urinary metabolites that correlate with clinical improvements in children with autism treated with sulforaphane from broccoli.

Background: Children with autism spectrum disorder (ASD) have urinary metabolites suggesting impairments in several pathways, including oxidative stress, inflammation, mitochondrial dysfunction, and gut microbiome alterations. Sulforaphane, a supplement with indirect antioxidant effects that are derived from broccoli sprouts and seeds, was recently shown to lead to improvements in behavior and social responsiveness in children with ASD. We conducted the current open-label study to determine if we could identify changes in urinary metabolites that were associated with clinical improvements with the goal of identifying a potential mechanism of action. Methods: Children and young adults enrolled in a school for children with ASD and related neurodevelopmental disorders were recruited to participate in a 12-week, open-label study of sulforaphane. Fasting urinary metabolites and measures of behavior (Aberrant Behavior Checklist-ABC) and social responsiveness (Social Responsiveness Scale-SRS) were measured at baseline and at the end of the study. Pearson's correlation coefficient was calculated for the pre- to post-intervention change in each of the two clinical scales (ABS and SRS) versus the change in each metabolite. Results: Fifteen children completed the 12-week study. Mean scores on both symptom measures showed improvements (decreases) over the study period, but only the change in the SRS was significant. The ABC improved - 7.1 points (95% CI - 17.4 to 3.2), and the SRS improved - 9.7 points (95% CI - 18.7 to - 0.8). We identified 77 urinary metabolites that were correlated with changes in symptoms, and they clustered into pathways of oxidative stress, amino acid/gut microbiome, neurotransmitters, hormones, and sphingomyelin metabolism. Conclusions: Urinary metabolomics analysis is a useful tool to identify pathways that may be involved in the mechanism of action of treatments targeting abnormal physiology in ASD. Trial registration: This study was prospectively registered at clinicaltrials.gov (NCT02654743) on January 11, 2016.

The Diversity of Chemoprotective Glucosinolates in Moringaceae (Moringa spp.).

Glucosinolates (GS) are metabolized to isothiocyanates that may enhance human healthspan by protecting against a variety of chronic diseases. Moringa oleifera, the drumstick tree, produces unique GS but little is known about GS variation within M. oleifera, and even less in the 12 other Moringa species, some of which are very rare. We assess leaf, seed, stem, and leaf gland exudate GS content of 12 of the 13 known Moringa species. We describe 2 previously unidentified GS as major components of 6 species, reporting on the presence of simple alkyl GS in 4 species, which are dominant in M. longituba. We document potent chemoprotective potential in 11 of 12 species, and measure the cytoprotective activity of 6 purified GS in several cell lines. Some of the unique GS rank with the most powerful known inducers of the phase 2 cytoprotective response. Although extracts of most species induced a robust phase 2 cytoprotective response in cultured cells, one was very low (M. longituba), and by far the highest was M. arborea, a very rare and poorly known species. Our results underscore the importance of Moringa as a chemoprotective resource and the need to survey and conserve its interspecific diversity.

Wild and domesticated Moringa oleifera differ in taste, glucosinolate composition, and antioxidant potential, but not myrosinase activity or protein content.

Taste drives consumption of foods. The tropical tree Moringa oleifera is grown worldwide as a protein-rich leafy vegetable and for the medicinal value of its phytochemicals, in particular its glucosinolates, which can lead to a pronounced harsh taste. All studies to date have examined only cultivated, domestic variants, meaning that potentially useful variation in wild type plants has been overlooked. We examine whether domesticated and wild type M. oleifera differ in myrosinase or glucosinolate levels, and whether these different levels impact taste in ways that could affect consumption. We assessed taste and measured levels of protein, glucosinolate, myrosinase content, and direct antioxidant activity of the leaves of 36 M. oleifera accessions grown in a common garden. Taste tests readily highlighted differences between wild type and domesticated M. oleifera. There were differences in direct antioxidant potential, but not in myrosinase activity or protein quantity. However, these two populations were readily separated based solely upon their proportions of the two predominant glucosinolates (glucomoringin and glucosoonjnain). This study demonstrates substantial variation in glucosinolate composition within M. oleifera. The domestication of M. oleifera appears to have involved increases in levels of glucomoringin and substantial reduction of glucosoonjnain, with marked changes in taste.