A Placebo-controlled Trial of Folinic Acid in Children With ASD

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).