An interventional study of Sodium Butyrate and Placebo (cornstarch) in Autism Spectrum Disorder (ASD), sponsored by Istituto Superiore di Sanità. Active, not recruiting at 3 sites in Italy. Open to participants aged 3 Years to 6 Years. Per ClinicalTrials.gov, last updated 2026-11-09.
Sponsored by Istituto Superiore di Sanità · Not applicable, Interventional, and Treatment
Autism spectrum disorder (ASD) is a neurodevelopmental condition occurring in 1:77 Italian children. Several comorbidities are reported, including functional gastrointestinal disorders (FGIDs) present in up to 70% of patients. FGIDs are disorders resulting from a combination of symptoms affecting motility, hypersensitivity, and other functions, which are not caused by anatomic or organic origin and that impact the severity of ASD core symptoms and complicate the clinical management of ASD children, especially those who are non-verbal. Evidence reports gut microbiome (GM) remodelling in ASD children, and postbiotic butyrate, a GM-derived metabolite, attenuates FGIDs in children and restores social behavior in ASD mouse models. Clinical data on butyrate effects in ASD are still scanty. The present study investigates the therapeutic effects of a 16-week oral postbiotic supplementation on clinical/behavioral profiles, gastrointestinal disturbances, gut microbiome, and immune and inflammatory biomarkers in peripheral blood and fecal samples in children with ASD and FGIDs. Using the Machine Learning (ML) approach, a subset of artificial intelligence, this study also aims to identify predictive factors implicated in the effect of the postbiotic supplementation on FGIDs, important for prevention through modulation of the microbiota. The investigators expect that treating FGIDs will have an impact on the behavioral and core symptoms of ASD and the quality of life of children and their families.
ASD is a heterogeneous group of neurodevelopmental disorders with complex multifactorial etiologies requiring personalized, timely, and evidence-based interventions to improve the lives of children and their families. Medical comorbidities are common in ASD and include FGIDs, which are disorders resulting from a combination of symptoms affecting motility, hypersensitivity, and other functions, which are not caused by anatomic or organic origin. FGIDs are often associated with sleep and behavioral problems with a negative impact on clinical functioning of ASD children. Pharmacological agents that could target FGIDs in ASD children are advocated. Immune system, epigenetic, gut microbiome, mitochondrial metabolism, and gut-brain axis alterations have been implicated in ASD and FGIDs etiology and may represent potential intervention targets. The term postbiotics can be regarded as an umbrella term for all synonyms and related terms of microbial fermentation components, including many different constituents such as short-chain fatty acids, microbial cell fractions, functional proteins, extracellular polysaccharides, cell lysates, teichoic acid, peptidoglycan-derived muropeptides, and pili-type structures. Emerging data indicate that postbiotics can have direct immunomodulatory and clinically relevant effects. Evidence can be found for using postbiotics in healthy individuals to improve overall health and relieve symptoms of various diseases. In particular, the postbiotic butyrate, a Gut Microbiota-derived short-chain fatty acid, exerts protective action against FGIDs and ASD through a wide range of activities involving Gut Microbiota, immune system, epigenetic mechanisms, and mitochondrial function. Taken together, these data strongly support the hypothesis that butyrate oral supplementation can exert a therapeutic action against FGIDs and behavioral symptoms in ASD children. Butyrate has been proposed for FGIDs treatment, but its promising effect on ASD is limited to preclinical data. The present multicenter double blinded randomized controlled trial has been rigorously designed to investigate the therapeutic effects of oral butyrate supplementation in children with ASD (either idiopathic or syndromic) and FGIDs. In line with precision medicine and designing and improving diagnosis, therapeutics, and prognosis using large complex datasets, this study will implement high-performance computing and artificial intelligence in multidimensional clinical and biological datasets. The great heterogeneity of ASD, in terms of etiology and clinical presentation, requires a better understanding of who is most likely to respond to which interventions, at what intensity, and for what duration so they could be assigned to those most likely to benefit while reducing the cost of treatment to the National Health Service. Thus, there is a need to identify mediators of treatment response and moderators (factors that predict greater responsiveness to treatment, including factors that influence individuals' responses to various treatments), allowing clinicians to tailor early interventions more carefully. To this aim, mediators and moderators implicated in the effect of postbiotic supplementation on FGIDs will be explored through ML performed on clinical and biological variables collected in the digital platform Research Electronic Data Capture (REDCap). Identifying patterns/profiles of the child responding to treatment may be important in assessing risk during the early stage and planning for individualized treatment and prevention of ASD through modulation of the microbiota.
Increasing evidence suggests that the gut microbiome is one of the key modulators of gut-brain communication in ASD. The brain-gut-microbiome axis has become a compelling area of investigation in ASD, specifically in children. Previous studies suggested the presence of alteration in gut microbiome structure and function in ASD children as well as in FGIDs: decreased Bacteroidetes/Firmicutes (well-known butyrate producers) ratio in fecal samples of ASD children and significantly reduced butyrate level production by the gut microbiome of ASD children.
Butyrate is a widely known histone deacetylase inhibitor due to its ability to cross the blood brain barrier. Previous research has shown that treatment with oral sodium butyrate improved behavioral deficits in BTBR mice, one of the most robust animal models of ASD. The gene expression analyses of the frontal cortex revealed that the effects of butyrate on behavior were attributable to a positive modulation of genes involved in excitatory/inhibitory balance and neuronal activation. Specifically, sodium butyrate downregulated the neuronal activation marker genes and upregulated the inhibitory neurotransmitter genes. In addition, a double-blind trial in 3-8 year old children with ASD was recently conducted to assess the effect of L. Reuteri supplementation (6 months) on social deficits, gastrointestinal (GI) symptoms, and immune response. Unpublished findings showed that L. Reuteri significantly increased scores of Adaptive Behavior Assessment System social subdomains and decreased the total score of the Social Responsiveness Scale and gastrointestinal (GI) symptoms at 6 months. Behavioral results based on intelligent quotient (IQ) stratification also revealed the L. Reuteri efficacy on social and GI domains in ASD children with normal IQ range. These studies highlight the promising role of probiotics and postbiotics in ASD children.
Aims. A multicenter, double-blind, sequential Randomized Controlled Trial of butyrate vs placebo will be carried out in 128 ASD children (estimated sample size) to evaluate the efficacy of the postbiotic supplementation on functional gastrointestinal disorders in children with ASD. A secondary aim is to define whether and to what extent the butyrate supplementation could influence ASD behavioral and core symptoms. The third aim is to identify clinical and/or biological determinants of ASD children's response to postbiotic supplementation.
Materials and Methods. Azienda Ospedaliera Universitaria Federico II of Naples and Policlinico Tor Vergata Hospital will invite parents of 3-6 years old ASD children to participate in the study. If they agree, full information about the protocol will be provided and written signed informed consent will be collected. ASD diagnosis, severity, and clinical functioning will be defined according to Diagnostic and Statistical Manual of Mental Disorders (DSM-5) criteria and gold-standard tests; the FGIDs diagnosis will be performed using the Rome III Criteria questionnaire, and the 6-GSI. Children's dietary habits will be assessed by the 3-day food diary. Clinical centers will send age, gender, and IQ data to Istituto Superiore di Sanità for the centralized randomization procedure in the REDCap platform. At baseline (Timepoint 0), parents will be invited to Azienda Ospedaliera Universitaria Federico II of Naples and Policlinico Tor Vergata Hospital for a comprehensive clinical evaluation including anamnestic interview and the administration of standardized tests for autism and gastrointestinal symptoms, global development and adaptive and behavioral functioning. Blood and faecal samples for genetic and metabolic analysis will be collected, and electroencephalography will also be performed.
Parents will receive butyrate or placebo supplementation for 16 weeks (4 months), according to the allocation process. Treatment will be numbered according to the randomization scheme with no reference to group assignment, only known by the Istituto Superiore di Sanità statistician who generates the list. The Central Pharmacy Service of the Academic Hospital Federico II of Naples will prepare the sachets and indistinguishable (same color, smell, taste) treatments' content. Clinical centers will repeat a full evaluation at the end of the supplementation period (Timepoint 1). At Timepoint 2 (after 4 months by the end of Timepoint 1), parents will be asked to fill Timepoint 1 questionnaires to evaluate the long-term effect - consolidation - of supplementation. To avoid further discomfort to children and families, blood and fecal sampling will not be collected at Timepoint 2.
Determinants of response to the butyrate supplementation and biomarkers that differentiate responders from non-responders will be identified by implementing high-performance computing and artificial intelligence to the multidimensional clinical and biological datasets collected in the digital platform developed and managed by Istituto Superiore di Sanità. Using the ML approach, a subset of artificial intelligence, the University Federico II of Naples will analyze the dataset to establish relationships without preassigned modeling. ML is an innovative approach to provide robust predictions, overcoming descriptive statistical models, and proving a digital twin for the considered phenomena. Clinical, behavioral, inflammatory, and immunological factors are altered in ASD. However, the great variability among individuals with ASD of these problems makes it difficult to predict who is most likely to respond to a specific intervention, at what intensity, and for what duration. To this aim, the investigators will develop a probabilistic multi-domain data integration model consisting of the gut microbiome, immune, and inflammatory biomarkers in peripheral blood and fecal samples, and clinical/behavioral biomarkers using ML to improve therapeutics and predict the prognosis of ASD. Clinical and behavioral variables to be included in the dataset will be collected through the tests/questionnaires described in the experimental design. Gut microbiome, immune and inflammatory variables will be gathered on blood and fecal samples at baseline (Timepoint 0) and at the end of supplementation (Timepoint 1). The composition of the gut microbiome will be characterized by shotgun metagenomics. Genomes of dominant strains will also be reconstructed with a recently validated standard pipeline and compared to define if a strain-level variation exists upon treatment with butyrate. Fecal Short-Chain Fatty Acids (SCFAs: acetate, propionate, and butyrate) levels will be determined by gas chromatograph-mass spectrometry, as previously described. Altered composition of the gut microbiome may alter the gut barrier, potentially allowing the translocation of bacteria and their antigens, toxins, and metabolites and result in multiple alterations of cytokines' production, immune cell populations, as well as cellular activation markers.
Randomization and allocation concealment will be performed by the Istituto Superiore di Sanità. Randomization will be generated using the procedure Ralloc in STATA, managed through the RECap platform after enrollment and provided to clinicians by the Istituto Superiore di Sanità. Specific standard operative procedures (SOPs) were defined to garantee study and trial integrity. A blinded statistician will analyze data using Stata 16.1.
1,724 studies on the registry are indexed under Autism Spectrum Disorder; 500 are open to participants now.
This study's planned enrollment of 128 is above the median of 52 across 1,368 interventional studies indexed under Autism Spectrum Disorder.
Browse Autism Spectrum Disorder studies →Istituto Superiore di Sanità is the lead sponsor of 17 studies on the registry; 2 are open to participants now.
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Go through the criteria one by one — yes, no or not sure — and take the list to your doctor or the study team. Kept in this browser only.
Exclusion Criteria:
Underlined terms in the criteria open these too. Explained by clinicaltrials.gg, not part of the registry record. The study team decides who can take part.
A condition in which the immune system attacks the body’s own tissues, such as rheumatoid arthritis, lupus, psoriasis, type 1 diabetes or inflammatory bowel disease.
Immunotherapy studies often leave out active autoimmune disease, and some name exceptions, such as thyroid disease on replacement hormone. Check the exceptions this study lists.
Autoimmune disease
A condition in which the immune system attacks the body’s own tissues, such as rheumatoid arthritis, lupus, psoriasis, type 1 diabetes or inflammatory bowel disease.
Immunotherapy studies often leave out active autoimmune disease, and some name exceptions, such as thyroid disease on replacement hormone. Check the exceptions this study lists.
Explained by clinicaltrials.gg, not part of the registry record. The study team decides who can take part.
The butyrate groups (butyrate \<=20 kg, butyrate \>20 kg) will receive oral sodium butyrate (dose of 20 mg/kg body weight/daily in sachets, up to 800 mg/day maximum dose) plus ASD standard care.
Dietary Supplement: Sodium Butyrate
The placebo groups (placebo \<=20 kg, placebo \>20 kg) will receive placebo (cornstarch) at the same dose and time plus ASD standard care.
Dietary Supplement: Placebo (cornstarch)
Daily supplementation with 1 sachet per day for 16 weeks
Daily supplementation with 1 sachet per day for 16 weeks
Gastrointestinal symptom severity.
Rate of subjects presenting a clinically relevant improvement in Functional Gastrointestinal Disorders (FGIDs) severity, measured by 6-item Gastrointestinal Severity Index (6-GSI) score decrease \>4 points. The Gastrointestinal Severity Index (6-GSI) score is the sum of the individual item scores. The higher it is, the worse is the outcome. Score range for each item: 0, 1, 2 (0 no symptoms, 1 mild symptoms, 2 severe symptoms).
Time frame: At baseline (Timepoint 0) and at 4 months (Timepoint 1)
Incidence of Treatment-Emergent Adverse Events [Safety and Tolerability]
Parental questionnaires on the presence of nausea, skin irritation, itching, patches on the skin, stomach ache, flatulence, meteorism, vomiting, constipation, increased/decreased appetite, heartburn, reflux, burping. Measured in hours or days of symptoms' duration.
Time frame: At 4 months (Timepoint 1) and at 8 months (Timepoint 2)
Unexplained daytime irritability
Parental questionnaires on the presence of angry outbursts, bouts of crying, easy frustration, mood swings, aggression, restlessness. Measured by symptom intensity during daytime: light, mild, severe.
Time frame: At 4 months (Timepoint 1) and at 8 months (Timepoint 2)
Nocturnal awakening
Parental questionnaires on the presence of night waking. Measured as Yes (number of episodes) or No.
Time frame: At 4 months (Timepoint 1) and at 8 months (Timepoint 2)
Persistency of Functional Gastrointestinal Disorders severity improvement
Measured by 6-item Gastrointestinal Severity Index (6-GSI) score: the sum of the individual item scores. The higher it is, the worse is the outcome. Score range for each item: 0, 1, 2 (0 no symptoms, 1 mild symptoms, 2 severe symptoms).
Time frame: At 4 months (Timepoint 1) and at 8 months (Timepoint 2)
Gut Microbiota structure and function (fecal Short-Chain Fatty Acids levels, SCFAs)
Bacterial DNA extraction from one fecal sample will be carried out by shotgun metagenomics. Fecal SCFAs (acetate, propionate and butyrate) levels will be measured using the second fecal sample by gas chromatograph-mass spectrometry and expressed in mM (millimolar) with a range of 0-∞.
Time frame: At baseline (Timepoint 0) and at 4 months (Timepoint 1)
Changes in Autism Spectrum Disorder severity
Measured by Social Responsiveness Scale-2 (SRS-2). Score range for each item: 0-100. The higher the score, the worse the outcome.
Time frame: At baseline (Timepoint 0), at 4 months (Timepoint 1) and 8 months (Timepoint 2)
Changes in Autism Spectrum Disorder behavior
Measured by Aberrant Behavior Checklist (ABC). Score range for each item: 0-999. The higher the score, the worse the outcome.
Time frame: At baseline (Timepoint 0), at 4 months (Timepoint 1) and 8 months (Timepoint 2)
Changes in Autism Spectrum Disorder symptomatology: repetitive behaviors
Delta of scores at Repetitive Behavior Scale-Revised (RBS-R), a 43-item scale. Score range for each item: 0-100. Higher scores are related to greater severity of symptoms.
Time frame: At baseline (Timepoint 0), at 4 months (Timepoint 1) and 8 months (Timepoint 2)
Changes in Sensory input reaction
Measured by Sensory Profile 2 (SP-2) scores. Score range for raw score: 86-430. Score range for percentiles: 0-100. The further away from the mean score, the more pathological. Score categories for sensitivity level: 1,2,3,4,5. 1. Severe hyposensitivity (\< -2 Standard Deviation) 2. Mild hyposensitivity (between -1 e -2 Standard Deviation) 3. Typical sensitivity (between -1 and 1 Standard Deviation) 4. Mild hypersensitivity (between 1 and 2 Standard Deviation) 5. Severe hypersensitivity (\> 2 Standard Deviation)
Time frame: At baseline (Timepoint 0), at 4 months (Timepoint 1) and 8 months (Timepoint 2)
Changes in Adaptive Functioning
Measured by Vineland Adaptive Behavior Scales-II (VABS-II). Score range for each item: 0-160. The higher the score, the better the outcome.
Time frame: At baseline (Timepoint 0), at 4 months (Timepoint 1) and 8 months (Timepoint 2)
Changes in emotional/behavioral problems
Measured by Child Behavior Checklist (CBCL syndrome subscales). Score range for each item: 0-999. The higher the score, the worse the outcome.
Time frame: At baseline (Timepoint 0), at 4 months (Timepoint 1) and 8 months (Timepoint 2)
Changes in sleep disorders
Measured by Children's Sleep Habits Questionnaire Abbreviated (CSHQ). Score ranges for items: Bedtime (0-63), Sleep behavior (0-49), Waking during the night (0-14), Morning wake up (0-28). Total sleep disturbance score: the sum of the individual item scores (0-154). The higher the score, the worse the outcome.
Time frame: At baseline (Timepoint 0), at 4 months (Timepoint 1) and 8 months (Timepoint 2)
Sleep disturbance assessment
Measured by Sleep Disturbance Scale for Children (SDSC). Score ranges for items: Sleep onset and maintenance disorders (7-35), Sleep breathing disorders (3-15), Arousal disorders (3-15), Wake-Sleep Transition Disorder (6-30), Excessive sleepiness disorder (5-25), Nocturnal hyperhidrosis (2-10). Total score: the sum of the individual item scores (26-130). Higher scores indicate more acute sleep disturbances.
Time frame: At baseline (Timepoint 0), at 4 months (Timepoint 1) and 8 months (Timepoint 2)
Impact on parental quality of life
Evaluated through the Quality of life questionnaire (EUROHIS QOL-8). Score range for each item: 1-5. Higher score indicates a better condition. Overall index score: the mean of the individual scores, ranging from 1 to 5 with a higher score indicating a better quality of life.
Time frame: At baseline (Timepoint 0), at 4 months (Timepoint 1) and 8 months (Timepoint 2)
Serum proinflammatory and regulatory cytokines
Intracellular expression of cytokines (IL-1beta, IL-5, IL-6, IL-10, IL-15, IL-17, TGF-beta, TNF-alpha, IFN-alpha, IFN-gamma) measured by Enzyme-Linked Immuno Assay (ELISA). All reported as pg/ml.
Time frame: At baseline (Timepoint 0) and at 4 months (Timepoint 1)
Plasmatic levels of chemochines involved in the migration of inflammatory cells within the CNS (Part 1)
Expression of ICAM-1, PECAM-1, and P-selectin measured by Enzyme-Linked Immuno Assay (ELISA). All reported as ng/ml.
Time frame: At baseline (Timepoint 0) and at 4 months (Timepoint 1)
Plasmatic levels of chemochines involved in the migration of inflammatory cells within the CNS (Part 2)
Expression of L-selectin measured by Enzyme-Linked Immuno Assay (ELISA). Reported as pg/ml.
Time frame: At baseline (Timepoint 0) and at 4 months (Timepoint 1)
Peripheral blood mononuclear cells features (Part 1)
Characterization of peripheral blood mononuclear cells immunophenotype by flow cytometry (Cytoflex, Beckman Coulter). All expressed in % (range 0,000-100,000): Lymphocytes, Monocytes, Granulocytes,CD3, CD4, CD8, CD19, CD16, CD56, HLA-DR Monocytes, HLA-DR Lymphocytes, HLADRMONOLYMPH, CD169Leukocytes, CD169Lymphocytes, CD169Monocytes, CD169Granulocytes, HLADRLLeukocytes, HLADRLymphocytes, HLADRMonocytes, HLADRGranulocytes, HLADRCD169Leukocytes, HLADRCD169Lymphocytes, HLADRCD169Monocytes, HLADRCD169Granulocytes, CD3\_tube2, CD4\_tube2, CD4CM, CD4NAIVE, CD4EM, CD4TEM, CD8\_tube2, CD8CM, CD8NAIVE, CD8EM, CD8TEM, CD4PD1CD57, CD4CD57, CD4PD1, CD8PD1CD57, CD8CD57, CD8PD1, CD19\_tube2, Marginal, NaiveB, IgDmCD27m, IgDmCD27p, CD21lowCD38low, IgMpIgDp, IgMmIgDp, IgDmIgMm, IgMpIgDm, IgMpCd27pCD38p, Unswitchedmemory, IgMpCD27mCD38dim, IgMpCD27mCD38high, IgDmIgMmCD27pCD38high, Switchedmemory, IgMmIgDmCD27mCD38low, IgMmIgDmCD27mCD38high, Plasmablasts, IgMpCD27m, TransitionalBcells
Time frame: At baseline (Timepoint 0) and at 4 months (Timepoint 1)
Peripheral blood mononuclear cells features (Part 2)
Characterization of peripheral blood mononuclear cells immunophenotype by flow cytometry (Cytoflex, Beckman Coulter). All expressed as absolute number/ml: CD169MEDLYMPH, CD169MEDMONO, CD64MEDLYMPH, CD64MEDNEUTRO
Time frame: At baseline (Timepoint 0) and at 4 months (Timepoint 1)
Peripheral blood mononuclear cells features (Part 3)
Characterization of peripheral blood mononuclear cells immunophenotype by flow cytometry (Cytoflex, Beckman Coulter). All expressed as Ratio, absolute number: CD169 Monocytes/Lymphocytes RMFI, CD64 Neutrophils/Lymphocytes RMFI
Time frame: At baseline (Timepoint 0) and at 4 months (Timepoint 1)
Plan to share: No
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Studies of the drug tested here: Butyric Acid
Istituto Superiore di Sanità