**1. Introduction**

Autism spectrum disorder (ASD) is a heterogeneous neurodevelopmental disorder that is characterized by persistent deficits in social interaction and communication, restricted interests, and repetitive behaviors [1]. Phenotypes among individuals with ASD are highly diverse in terms of cognition, language abilities, irritability, sensory perception, anxiety, motor skills, executive functions, epilepsy, gastrointestinal problems, and more [1,2]. Up to 80% of children with ASD also have sleep disorders, including prolonged sleep onset latency, extended night awakenings, and early morning awakenings [3,4].

The etiology of sleep disorders in ASD is presumed to include multiple neuropsychological factors [5], among which alterations in the circadian sleep–wake cycle are the most well established [6,7]. Accordingly, the most common pharmacological treatment for sleep disorders in individuals with ASD is exogenous melatonin, given as an add on to behavioral interventions, and parental education [8].

One of the main regulators of the sleep–wake cycle is the endocannabinoid system [9]. The primary components of this cell-signaling system are the cannabinoid receptors and their endogenous ligands (endocannabinoids) [10]. The cannabis plant contains unique compounds (phytocannabinoids) that can interact with the endocannabinoid system either directly, using Δ9-tetrahydrocannabinol (THC), or indirectly, using cannabidiol (CBD). THC is the major psychoactive component of the cannabis plant. It activates the type 1 cannabinoid receptor (CB1R) in the brain and might lead to anxiety and psychosis [11]. CBD is the major non-psychoactive phytocannabinoid. While it is an allosteric modulator of the CB1R, which may decrease the effects of CB1R agonists, it concomitantly increases the levels of the endocannabinoids that activate the CB1R (Figure 1) [11]. As opposed to THC, CBD has a relatively high toxicity threshold and it also appears to have anxiolytic, antipsychotic, antiepileptic, and neuroprotective properties that may be mediated through receptors, such as serotonin 5-HT1A, TRPV1, GPR55, GABAA, and PPARγ, and through the inhibition of adenosine reuptake (Figure 1) [12–16].

**Figure 1.** Multiple molecular targets for cannabidiol (CBD) in neurons.

Alterations in the endocannabinoid system have been found in several animal models of ASD [17,18]. Recent human studies have demonstrated lower circulating endocannabinoid levels in children with ASD [19–21] and evidence of successful CBD-rich cannabinoid treatment for the core symptoms and comorbidities in children with ASD is accumulating [22–25]. However, the effect of phytocannabinoids on the sleep of ASD children is still unclear.

In general, cannabinoid therapy with various THC to CBD ratios is being increasingly used to alleviate sleep disorders, regardless of the cause. Among patients with chronic pain, treatment with medical cannabis seems to result in a small improvement in sleep quality [26]. Preliminary evidence of successful treatment is also available for sleep apnea, and posttraumatic stress disorder-related nightmares [27]. However, currently there is insufficient evidence to support this line of treatment for any individual sleep disorder [27–29].

In the current study, we aimed to evaluate the impact of a CBD-rich cannabinoid treatment on sleep, as part of a placebo-controlled trial, which assessed the effects of cannabinoids on the behavior of children and adolescents with ASD. We used two CBD-rich preparations. The first contained only purified CBD and purified THC isolates (pure cannabinoids) and the second contained a full-spectrum (whole-plant) extract, which, in addition to the same amounts of CBD and THC, also contained minor cannabinoids, terpenes, and flavonoids that might enhance the efficacy and tolerability.

We found that an improvement in sleep (after receiving either cannabinoids or the placebo) was associated with an improvement in the autistic core symptoms and disruptive behavior. However, CBD-rich cannabinoid treatment did not improve sleep disturbances more than the placebo treatment.

The main cannabinoid receptor in neurons is cannabinoid receptor type 1 (CB1R). The primary neuronal effect of the CB1R is a decrease in the synaptic transmission during increased synaptic activity, which can also promote sleep and reduce seizures and excitotoxicity. CBD is a negative allosteric modulator of the CB1R. However, CBD can activate the endocannabinoid system through the CB1R by inhibiting the endocannabinoid membrane transporter (EMT) and the degradation of anandamide (AEA) through fatty acid amide hydrolase (FAAH). This, in turn, increases the levels of the endocannabinoids AEA (main agonist of CB1R) and 2-Arachidonoylglycerol (2-AG). Other neuronal effects of CBD are mediated through agonism at the 5-HT1A serotonin receptors and at the TRPV1 channel, reducing anxiety and pain, and through agonism of the nuclear PPARγ receptors, increasing the expression of the cytoprotective enzymes. CBD also has direct antioxidative effects.

ENT—equilibrative nucleotide transporter; 5-HT1A—5-hydroxytriptamine 1A receptor; GPR55—G protein coupled receptor 55; PPARγ—peroxisome proliferator-activated receptor gamma; ROS—reactive oxygen species; TRPV1—transient receptor potential vanilloid 1.

#### **2. Materials and Methods**
