Clinical research on Nutraceutical Supplements for Autism Spectrum Disorder (ASD)

INTERVENTION: This is a two‐arm, randomized, double‐blind, controlled clinical trial aimed at evaluating the efficacy and safety of a combined nutraceutical approach for treating autism spectrum disorder (ASD) in children. Autism spectrum disorder ASD is a neurodevelopmental disorder with a multifactorial etiology, strongly involving immunometabolic mechanisms such as neuroinflammation, oxidative stress, mitochondrial dysfunction, epigenetic alterations, autoimmunity, increased intestinal permeability, and dysbiosis. Recent evidence suggests that certain nutraceutical substances—including vitamins, minerals, and phytocompounds—can modulate these mechanisms, offering clinical benefits that complement conventional behavioral and therapeutic approaches. The study will include children aged 3 to 7 years with a confirmed diagnosis of ASD, recruited through extensive media and social media outreach. Following screening and the signing of the informed consent form, eligible participants will undergo an assessment using the Autism Treatment Evaluation Checklist (ATEC). A washout period of 2 to 4 weeks will be observed to allow for the discontinuation of other supplements and clinical stabilization. Randomization will be performed by the compounding pharmacy itself, without the knowledge of the participants or members of the assessment team. Participants (estimated total of 88; 44 per group, with 75% statistical power) will be randomized into one of two arms: the intervention group will receive an optimized nutraceutical formulation for 60 days—at safe, literature‐based dosages—comprising vitamins A, D3, K2 (MK‐7), folinic acid (active B9), and methylcobalamin (B12); magnesium (bisglycinate) and P5P (active B6); zinc (bisglycinate); sulforaphane; phytosomal quercetin and bromelain; coenzyme Q10 (ubiquinone); and N‐acetylcysteine. Inulin (prebiotic), betaine, and choline bitartrate. The formulations were developed with a sweet taste to mask the flavor of the components. Control group: will receive a placebo identical in appearance, taste, and dosage form. Clinical and outcome assessments will take place at two time points: baseline (T0) and after 60 days of intervention (T1). The primary outcome will be the change in the total score on the Autism Treatment Evaluation Checklist (ATEC) between baseline and T1. The ATEC was chosen because it is an internationally validated instrument for assessing interventions in autism spectrum disorder, is easy to administer, and is sensitive to clinical changes. Secondary outcomes include: changes in ATEC subdomains (language, sociability, cognition/sensory, physical/behavioral health). Adverse events, adherence, and intervention acceptability will be monitored. Statistical analysis: repeated‐measures analysis of variance (ANOVA) and/or linear mixed models will be used, comparing groups and time points (baseline vs. T1), with significance set at p less than 0.05. The sample size calculation was based on a minimum clinical difference of 15 points on the ATEC score. Ethical aspects: the project adheres to the principles of the Declarat CONDITION: Childhood autism ; Autism Spectrum Disorder F03.625.164.113 SECONDARY OUTCOME: Expected outcome 2: A greater mean reduction in the autism treatment evaluation checklist speech/language/communication domain score is expected in the intervention group compared to the control/placebo group after 60 days, assessed by comparing scores obtained at baseline (T0) and post‐intervention (T1), considering statistical significance at p less than 0.05. Expected outcome 3: A greater mean reduction in the autism treatment evaluation checklist sociability domain score is expected in the intervention group compared to the control/placebo group after 60 days, assessed by comparing scores obtained at baseline (T0) and after the intervention (T1), considering statistical significance at p less than 0.05. Expected outcome 4: a greater mean reduction in the autism treatment evaluation checklist Sensory/Cognitive domain score is expected in the intervention group compared to the control/placebo group after 60 days, assessed by comparing scores obtained at baseline (T0) and after the intervention (T1), considering statistical significance at p less than 0.05. Expected outcome 5: a greater mean reduction in the autism treatment evaluation checklist physical health/behavior domain score is expected in the intervention group compared to the control/placebo group after 60 days, assessed by comparing scores obtained at baseline (T0) and after the intervention (T1), considering statistical significance at p less than 0.05. Expected outcome 6: a higher proportion of clinical responders is expected in the intervention group compared to the control/placebo group after 60 days, assessed via the autism treatment evaluation checklist administered at baseline (T0) and after the intervention (T1), with clinical response defined as a reduction of at least 15 points in the total autism treatment evaluation checklist ATEC score. Expected outcome 7: satisfactory adherence to and good acceptability of the nutraceutical intervention are expected over the 60‐day period, verified by the caregivers' records of correctly administered doses and reports on palatability and ease of use; adherence exceeding 80 per cent of the scheduled doses is considered satisfactory. Expected outcome 8: a low incidence of adverse events related to the nutraceutical intervention is expected—predominantly mild and transient—verified through weekly contact with families, a structured form completed by caregivers, and clinical monitoring, with events classified as mild, moderate, or severe and their frequencies compared between groups. Outcome 3: A reduction in the sociability domain score was observed in both groups after 60 days. In the intervention group, the mean decreased from 14.7 to 9.9 points, with a mean improvement of 4.8 points; in the control/placebo group, it decreased from 17.3 to 11.8 points, with a mean improvement of 5.4 points. The difference in improvement between the groups was ‐0.7 points, without statistical significance (p equal to 0.762). Outcome 4: a reduction in the sensory/cognitive domain score was observed in both groups after 60 days. In the intervention group, the mean decreased from 12.7 to 8.4 points, with a mean improvement of 4.3 points; in the control/placebo group, it decreased from 14.2 to 9.5 points, with a mean improvement of 4.7 points. The difference in improvement between the groups was ‐0.5 points, without statistical significance (p equal to 0.812). Outcome 5: a reduction in the physical health/behavior domain score was observed in both groups after 60 days. In the intervention group, the mean decreased from 27.7 to 21.0 points, with a mean improvement of 6.8 points; in the control/placebo group, it decreased from 29.4 to 22.2 points, with a mean improvement of 7.2 points. The difference in improvement between the groups was ‐0.5 points, without statistical significance (p equal to 0.898). Outcome 6: A reduction of at least 15 points in the total Autism Treatment Evaluation Checklist (ATEC) score was observed in 50.0% of participants in the intervention group and in 66.7% of participants in the control/placebo group, as determined by comparing assessments conducted at baseline and after 60 days. There was no statistically significant difference between the groups according to Fisher's exact test (p equal to 0.353). Outcome 7: completion of the 60‐day paired assessments was observed for 42 of the 74 allocated participants, corresponding to a completion rate of 56.8 per cent and a loss to follow‐up of 43.2 per cent, as shown in the participant flow. The loss rate was 36.8 per cent in the intervention group and 50.0 per cent in the control/placebo group, with no statistically significant difference between the groups. Withdrawals were primarily related to the palatability of the formulations, indicating lower‐than‐expected acceptability. Outcome 8: Adverse events observed were predominantly mild and transient, occurring with similar frequency in both groups. In the intervention group and the control/placebo group, respectively, the following were recorded: gastrointestinal symptoms in 7 and 4 participants; irritability or agitation in 5 and 5; sleep disturbances in 2 and 1; headache in 2 and 1; increased appetite or weight in 2 and 0; and cutaneous allergic reaction in 0 and 1 participant. No serious adverse event attributed to the nutraceutical formula occurred, and the allergic reaction that led to discontinuation occurred in the control/placebo group. Outcome found 2: a reduction in the speech/language/communication domain score was observed in both groups after 60 days. In the intervention group, the average decreased from 10.4 to 7.7 points, with an average improvement of 2.7 points; in the control/placebo group, it decreased from 14.2 to 9.4 points, with an average improvement of 4.8 points. The difference in improvement between the groups was ‐2.1 points, without statistical significance (p equal to 0.222). INCLUSION CRITERIA: Children aged 3 to 7 years. Confirmed diagnosis of Autism Spectrum Disorder, established by a qualified professional based on the criteria of the Diagnostic and Statistical Manual of Mental Disorders, 5th Edition, Text Revision. Baseline score on the Autism Treatment Evaluation Checklist consistent with mild, moderate, or severe Autism Spectrum Disorder. Clinical stability (no hospital admission or acute medical condition within the last 4 weeks). Parents or legal guardians consenting to participation by signing the Informed Consent Form. Commitment by parents/guardians to follow protocol guidelines and attend scheduled assessments PRIMARY OUTCOME: Expected outcome 1: A greater mean reduction in the total Autism Treatment Evaluation Checklist (ATEC) score is expected in the intervention group compared to the control/placebo group after 60 days, as assessed by caregivers administering the Autism Treatment Evaluation Checklist (ATEC) at the start of the intervention (T0) and after 60 days (T1), with a 15‐point reduction considered the minimum clinically relevant difference between the groups. Outcome 1: A statistically significant reduction in total autism treatment evaluation checklist scores was observed in both groups after 60 days. In the intervention group, the mean score decreased from 65.5 more or less 31.0 to 47.0 more or less 22.7 points, corresponding to a mean improvement of 18.5 points (p=0.001). In the control/placebo group, the mean score decreased from 75.1 more or less 27.9 to 52.9 more or less 24.4 points, corresponding to a mean improvement of 22.2 points (p=0.001). The difference in improvement between the groups was ‐3.7 points (intervention minus control), which was not statistically significant (p=0.595). The analysis of variance ANCOVA adjusted for baseline autism treatment evaluation checklist and age also showed no independent effect of the intervention (ß=‐0.29; 95% CI ‐11.7 to 11.1; p=0.959). Therefore, the superiority of the nutraceutical formula over the placebo was not demonstrated.