Autism Spectrum Disorder (ASD) is a group of pervasive neurodevelopmental disorders that appear in early childhood. These disorders are characterised by social functioning deficits, restricted or repeated patterns of behaviour across the lifespan [1], and other comorbidities such as intellectual disabilities, epilepsy, anxiety, hyperactivity, irritability, and sleep disturbances. [2, 3] These symptoms can significantly impact a person’s daily functioning. [4, 5] Globally, the prevalence of ASD is estimated to range from 0.4% to 1.7% [6]. In the last decade, the prevalence rate of young children (under five years) diagnosed with ASD has increased by 6.7%, from 2.7 million in 1990 to 2.9 million in 2019 [7].

The families of children with ASD face a substantial financial burden, with recent global estimates indicating 2834 EUR total costs for health-related services combined with indirect societal costs over two months [8]. The estimated lifetime social cost in the US alone was $7 trillion for the last three decades (1990-2019) and is forecasted to increase to $11-15 trillion in the following decade [9]. The continuing rise in ASD prevalence is further projected to generate an enormous economic burden on US healthcare for decades and to reach an annual cost estimate of $5.54 trillion by 2060, with substantial potential yearly savings of $1.9 trillion through ASD prevention strategies [10]. Despite thorough investigation, no evidence supports the effectiveness of pharmacological treatments in relieving some of the symptoms that cause people with ASD distress. [11, 12]. However, these pharmacological treatments often result in adverse side effects in children, with a notable variation in effectiveness between individuals [13] and a lack of adherence [14]. The complexity of the ASD behavioural phenotype and its aetiology resulting from the interaction of multiple genetic and environmental risk factors further limits targeted interventions and the development of effective therapies [15, 16]. Therefore, to address core behaviours and co-morbid symptoms associated with ASD, targeted therapeutic interventions are urgently needed.

The endocannabinoid system (ECS), consisting of cannabinoid receptors (CB1 and CB2), endocannabinoids (endogenous bioactive ligands) and metabolic enzymes [17] presents as a potential pharmacological target for developing therapeutics in the management of ASD symptoms since it has a crucial role in modulation of neuronal plasticity during development [18] and associated with social-emotional processes [19] which are impaired in ASD-related phenotypes [20, 21]. The central endocannabinoid receptors CB1 and CB2 are expressed mainly in sensory neurons and immune cells, including microglia [17]. The endogenous cannabinoids are produced as required, activating presynaptic receptors to reduce calcium uptake and inhibit the release of neurotransmitters, consequently influencing a broad range of biological responses [22]. Alterations in ECS functionality have been reported in several preclinical ASD models [23–25] and clinical studies [26–28], suggesting that ASD pathogenesis likely involves dysregulation of the endocannabinoid signalling pathway. These findings suggest that endocannabinoid anandamide (AEA) and its associated metabolic enzymes may be a potential therapeutic target for ASD-related behavioural impairment. In animal studies, treatment with inhibitors of the enzyme fatty acid amide hydrolase that degrades anandamide resulted in increased AEA levels, restoring cognitive and social deficits in the ASD-related knockout models [29–31].

The increased understanding of the ECS signalling concurrent with growing evidence for the therapeutic effects of cannabis (Cannabis sativa L.) in neurodevelopment disorders has been the catalyst for extensive research on the two most abundant active constituents Δ9-Tetrahydrocannabinol (THC) and Cannabidiol (CBD). During the last two decades, there has been a surge in clinical trials of CBD owing to the absence of psychoactive effects and its confirmed favourable safety profile in humans for the treatment of several neurological and psychiatric disorders [32–34]. In addition, accumulating evidence from experimental and animal model studies on the effect of CBD has demonstrated modulation of a broad range of biological responses, including antidepressant [35], anti-inflammatory [36], antinociceptive [37], anti-seizure [38], immunomodulatory [39] and neuroprotective activities [40]. Cannabinoids are generally well-tolerated, but evidence suggests that the risk of hepatotoxicity may be increased when combined with other medications. This risk is usually low, dose-dependent, and more likely to occur in individuals with a history of liver disease.[41] Rigorous controlled clinical studies are needed to determine the efficacy and safety profile of cannabinoids in children.

There is growing evidence that using CBD in combination with other plant compounds found in cannabis as a full-spectrum botanical medicine could provide unique therapeutic benefits, as suggested by anecdotal reports and emerging research in the field. [42] This effect, known as the entourage effect, was initially proposed as a regulatory mechanism for the endocannabinoid system, whereby multiple compounds enhance the activity of endogenous cannabinoids. A recent meta-analysis of observational studies showed that twice as many patients with treatment-resistant epilepsy improved when treated with an herbal formulation that offered a better safety profile than isolated CBD product [46]. Furthermore, promising evidence from preclinical cancer models supports the potential for the entourage effect of botanical preparations to produce enhanced therapeutic responses than individual cannabinoids [47–49]. Given that the current single molecule approach of individual phytocannabinoids remains the predominant pharmaceutical development strategy [50], for complex diseases such as ASD, multi-target strategies and botanical drug development may be an effective treatment strategy.

Evidence from animal studies suggests that CBD positively affects various physiological responses. However, there is limited evidence from clinical trials to support the potential therapeutic benefits of this non-psychoactive phytocannabinoid for pediatric neurodevelopmental disorders [51]. Only three randomised controlled studies have been published on medicinal cannabis use in children with ASD; two were short-term [58, 59], and the other only reported sleep outcomes [60]. Additionally, most studies on this topic are open-label observational or retrospective design [52–57].

This single centre, phase I/II open-label study aimed to evaluate the efficacy and safety of a novel oil-based full-spectrum medicinal cannabis plant extract with 0.08% THC (NTI-164) in children with ASD over four weeks of daily treatment.