Autism spectrum disorder (ASD) is a neurodevelopmental condition characterized by difficulties in social communication and the presence of restricted or repetitive behaviors. Although behavioral and educational interventions can be helpful, there is currently no established medication for the core symptoms of ASD. Medications approved for associated irritability may be effective in some children but are often associated with significant adverse effects.

Folinic acid (also known as leucovorin) is a reduced form of folate that plays an important role in brain development, neurotransmitter production, DNA methylation, and cellular metabolism. Previous clinical studies have suggested that folinic acid may improve communication, social functioning, and behavioral symptoms in some children with ASD. However, existing studies have generally been small and have used different outcome measures, and the current evidence is insufficient to establish the efficacy and optimal dosing of folinic acid in ASD.

This multicenter, randomized, double-blind, placebo-controlled clinical trial is designed to evaluate the safety, tolerability, and efficacy of folinic acid in children with ASD. A total of 150 children aged 3 to 6 years with ASD and clinically significant behavioral symptoms will be enrolled at multiple sites in Israel. Participants will be randomly assigned in a 1:1 ratio to receive either folinic acid or matching placebo for 9 weeks in addition to their existing treatments.

The primary objective of the study is to determine whether folinic acid improves behavioral symptoms compared with placebo, as measured by the Aberrant Behavior Checklist Irritability Subscale (ABC-I). Secondary objectives include evaluating the effects of folinic acid on communication, socialization, adaptive functioning, autism symptoms, emotional regulation, disruptive behavior, sleep, gastrointestinal symptoms, caregiver quality of life, and overall clinical improvement.

Following completion of the initial 9-week placebo-controlled phase, participants will enter a second 8-week double-blind treatment phase in which they will be randomly assigned to receive one of two folinic acid dose regimens. This phase is intended to explore whether different maintenance doses are associated with differences in clinical outcomes.

The study will also investigate potential biological markers associated with treatment response. Blood and stool samples will be collected to assess folate-related biomarkers, folate receptor alpha autoantibodies, oxidative stress markers, transcriptomic profiles, proteomic signatures, and gut microbiota composition. The study will also examine whether these biological measures are associated with symptom severity or response to treatment.

The results of this study are expected to provide important information regarding the efficacy, safety, and optimal use of folinic acid in children with ASD and may help identify biological factors associated with treatment response.

Autism spectrum disorder (ASD) is a heterogeneous neurodevelopmental condition characterized by persistent deficits in social communication and social interaction, together with restricted, repetitive patterns of behavior, interests, or activities. In addition to these core features, many children with ASD experience associated behavioral difficulties including irritability, emotional dysregulation, hyperactivity, sleep disturbances, and gastrointestinal symptoms. ASD affects approximately 1-3% of children worldwide and is associated with substantial long-term impact on affected individuals, families, educational systems, and healthcare resources.

At present, there is no established pharmacological treatment for the core symptoms of ASD. While behavioral and educational interventions remain the foundation of treatment, access, intensity, and effectiveness vary considerably among individuals. Pharmacological treatments currently approved for ASD-related irritability may reduce disruptive behaviors in some children but are often associated with adverse effects including weight gain, metabolic abnormalities, sedation, and extrapyramidal symptoms. Consequently, there remains a significant unmet need for safe and effective interventions that address both core and associated symptoms of ASD.

Folate plays a central role in neurodevelopment and brain function. Folate-dependent pathways are involved in DNA synthesis and repair, epigenetic regulation through methylation, neurotransmitter synthesis, mitochondrial function, and cellular redox balance. Disturbances in folate transport and metabolism have been implicated in a subset of individuals with ASD.

One mechanism that has received increasing attention is dysfunction of folate transport into the central nervous system. The folate receptor alpha (FRα) is responsible for transporting 5-methyltetrahydrofolate into the brain. Autoantibodies directed against this receptor may interfere with folate transport and contribute to cerebral folate deficiency. Multiple studies have reported an increased prevalence of folate receptor alpha autoantibodies in children with ASD compared with the general population. In addition, genetic variants affecting folate metabolism and transport pathways may influence neurodevelopment and treatment response.

Folinic acid (leucovorin calcium) is a reduced form of folate that bypasses several metabolic steps required for folic acid utilization and can utilize transport mechanisms that are less dependent on folate receptor alpha function. Folinic acid has been used safely for many years in pediatric and adult medicine for a variety of indications and has a well-characterized safety profile.

Over the past decade, several randomized placebo-controlled studies have suggested that high-dose folinic acid may improve language abilities, social communication, adaptive functioning, and behavioral symptoms in some children with ASD. These findings have generated considerable interest among clinicians and families and have led to increasing off-label use of folinic acid in clinical practice. However, the currently available evidence remains limited. Previous studies were generally small, used different outcome measures, enrolled relatively heterogeneous populations, and were not designed to determine the optimal duration or dose of treatment. Furthermore, uncertainty remains regarding which biological characteristics may predict treatment response.

As a result, major clinical guidelines do not currently recommend routine treatment with folinic acid for children with ASD, and there is a need for adequately powered confirmatory trials using standardized outcome measures and rigorous methodology.

The current study was designed to address these knowledge gaps. The trial will evaluate whether folinic acid is superior to placebo in improving behavioral symptoms and adaptive functioning in young children with ASD. The study will use a randomized, double-blind, placebo-controlled design to minimize bias and provide high-quality evidence regarding efficacy, safety, and tolerability.

In addition to evaluating clinical outcomes, the study incorporates an extensive translational research program intended to improve understanding of the biological mechanisms associated with treatment response. Biological samples will be collected to assess folate-related biomarkers, folate receptor alpha autoantibodies, markers of oxidative stress, transcriptomic signatures, proteomic profiles, and gut microbiota composition. These analyses may help identify biological subgroups that are more likely to benefit from treatment and may contribute to future precision medicine approaches in ASD.

Maternal folate receptor alpha autoantibody status will also be evaluated because emerging evidence suggests that maternal folate-related factors may influence neurodevelopmental outcomes. Exploratory analyses will investigate associations among biological markers, clinical characteristics, and treatment outcomes.

The study includes an initial placebo-controlled phase followed by an active-treatment dose-comparison phase. This design allows rigorous evaluation of efficacy against placebo while also generating information regarding potential dose-related differences in response and tolerability. The study therefore aims not only to determine whether folinic acid is effective, but also to provide data that may guide future treatment strategies and trial design.

By combining a large multicenter randomized clinical trial with detailed biological characterization, this study seeks to provide definitive evidence regarding the role of folinic acid in ASD and to advance understanding of folate-related mechanisms in neurodevelopmental disorders. The findings may help identify children most likely to benefit from treatment, improve evidence-based clinical decision-making, and support the development of more individualized therapeutic approaches for ASD.

Inclusion Criteria:

• Children aged 3 to 6 years.
• Diagnosis of autism spectrum disorder (ASD) according to DSM-5 criteria.
• ASD diagnosis confirmed by Autism Diagnostic Observation Schedule, Second Edition (ADOS-2).
• ASD diagnosis made at least 6 months before screening.
• Non-syndromic ASD, defined as ASD occurring in the absence of a known genetic syndrome, chromosomal abnormality, major congenital anomaly, or identifiable metabolic or neurological disorder.
• CGI-S score ≥ 4.
• ABC-I score ≥ 12.
• SRS-2 total T-score ≥ 66.
• Body weight between 11.45 kg and <27 kg.

Exclusion Criteria:

• A seizure or a change in antiepileptic medication within 8 weeks prior to randomization.
• Clinically significant abnormalities on physical examination or laboratory testing, including significant impairment of cardiac, hepatic, or renal function.
• Treatment with folinic acid within 3 months prior to randomization.
• Any change in pharmacological treatment, behavioral treatment, home environment, or school setting (other than school holidays) within 4 weeks prior to randomization, or planned changes during study participation.
• Predicted inability or unwillingness to comply with study procedures.
• Use of antifolate medications or other agents known to interfere with folate metabolism (e.g., methotrexate, trimethoprim).

De-identified individual participant data that underlie the results reported in publications arising from this study, including demographic and baseline characteristics, clinical outcome measures (ABC-I, VABS-3, CGI-I, MB-CDI, SRS-2 and other study assessments), adverse event data, laboratory safety data, and selected biomarker datasets generated during the study

The study protocol, Statistical Analysis Plan (SAP), and Informed Consent Form (ICF) will be made publicly available prior to study initiation. De-identified individual participant data (IPD) underlying the published results will become available 12 months after publication of the primary study results and will remain available for 5 years thereafter

Participants, parents/caregivers, investigators, study coordinators, treating physicians, outcome assessors, and study personnel involved in study conduct and data collection will remain blinded to treatment assignment. Folinic acid and placebo will be supplied in identical bottles and packaging and will be matched in appearance, taste, and smell. Randomization lists and treatment codes will be generated and maintained by an independent unblinded biostatistician and will not be accessible to blinded study personnel until study completion and database lock. During the second treatment phase, participants and study personnel will also remain blinded to the assigned folinic acid dose.