Endocannabinoid System as Therapeutic Target of PTSD: A Systematic Review

Post-Traumatic Stress Disorder (PTSD) is a complex disorder involving dysregulation of stress-related hormones and neurotransmitter systems. Research focused on the endocannabinoid system (eCBS) for anxiety and stress regulation, cognitive and emotional responses modulation and aversive memories extinction, leading to the hypothesis that it could represent a possible alternative treatment target for PTSD. In this systematic review, we summarize evidence about the efficacy and safety of medicinal cannabidiol (CBD), Δ9-tetrahydrocannabinol (Δ9-THC), and nabilone in PTSD treatment. The PRISMA statement guidelines were followed. A systematic literature search was conducted in MEDLINE/PubMed, Scopus and Web of Science by two independent researchers, who also performed data extraction and quality assessment. Among the initial 495 papers, 234 were screened for eligibility and 10 were included. Studies suggested that different medicinal cannabinoids at distinct doses and formulations could represent promising treatment strategies for the improvement of overall PTSD symptomatology as well as specific symptom domains (e.g., sleep disorders, arousal disturbances, suicidal thoughts), also influencing quality of life, pain and social impact. Although there is a robust rationale for treatment with drugs that target the eCBS and the results are promising, further studies are needed to investigate the safety and efficacy profile of their prolonged use.


Introduction
Post-Traumatic Stress Disorder (PTSD) is a psychiatric disorder that may occur in people after experiencing or witnessing a traumatic event. This nosographic entity was previously included in the Diagnostic and Statistical Manual of Mental Disorders, fourth edition (DSM-IV) as an anxiety disorder, whereas in the latest edition (DSM-5) PTSD was classified under a category named "Trauma and stress-related disorders" [1,2]. With a prevalence varying up to 5% in high-income countries, PTSD is often associated with significant comorbidity rates, relevant risk of chronicization, and substantial economic burden [3].
In the context of a maladaptive response to a traumatic stressor [4,5], individuals can develop symptoms such as persistent intrusive thoughts associated with the traumatic event, incessant avoidance of stimuli related to the traumatic event, negative changes in CB2, may present a beneficial effect in PTSD treatment, enhancing the consolidation of explicit fear extinction and attenuating aversive memories of the traumatic event [33][34][35][36]. Moreover, ∆ 9 -THC, a CB1 and CB2 partial agonist, showed a safe and well-tolerated profile in chronic PTSD with a significant improvement in global symptom severity, sleep quality, frequency of nightmares, and hyperarousal symptoms [37]. More recently, nabilone, a synthetic cannabinoid that activates CB1, was reported to reduce the frequency and intensity of nightmares in PTSD patients [38][39][40][41].
Despite promising preliminary evidence, no reviews to our best knowledge have systematically summarized the effectiveness of cannabinoids or cannabino-mimetic in the treatment of PTSD. Previous literature reviewed the evidence about the eCBS as a potential target for PTSD treatment and prevention, but without following a systematic approach and focusing also on pathophysiological correlates of the disorder [28,42,43]. In consideration of what is stated above, the present systematic review is aimed at summarizing the existent evidence on medicinal cannabinoids (e.g., THC, CBD, nabilone) in the treatment of PTSD in humans, critically analyzing and discussing both the efficacy and safety of these treatment approaches.

Materials and Methods
The present review was conducted following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement [44].

Literature Search
A systematic search of the electronic databases MEDLINE/PubMed, Scopus, and Web of Science was performed from inception to 23 November 2020 by entering the following search string: (((((((cannabidiol) OR cannabinoid) OR cannabinol) OR endocannabinoid) OR tetrahydrocannabinol) OR nabilone) OR palmitoyl-ethanolamide) AND ((posttraumatic stress disorder) OR PTSD) AND treatment.
Two investigators (EAC and GM) independently conducted the literature search, title/abstract screening, and full-text review. The reference list of selected articles was hand-screened to search for additional literature. Discrepancies were resolved through consensus and, whenever a final decision could not be achieved, a third investigator was consulted (LS jr).

Study Selection
Original studies investigating the effectiveness of medicinal cannabinoids in the treatment of PTSD diagnosed according to the DSM criteria were deemed eligible for inclusion, according to the PICO methodology. Literature considering specific symptoms of PTSD as treatment outcomes was also evaluated for the present review, with no language limits. Grey literature was included whenever sufficient information was provided. Articles presenting only an opinion or hypothesis without empirical investigation, reviews, letters to the editor, and commentaries were excluded. Case series were included if sufficient information was given. Studies reporting data on genetic and molecular aspects in animal models were not deemed eligible for the present review. As for studies with overlapping samples, more than one article was considered for inclusion if reporting on different aspects of the considered outcomes.

Quality Assessment
Considering the heterogeneity of included studies, the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) approach was used to rate the quality of the evidence [45]. This was done by two reviewers (EAC and GM) and disagreements were resolved via discussion with one further reviewer (LS jr). The quality of evidence was rated as "high", "moderate," "low," or "very low" based on GRADE rating standards. A "high-quality" rating indicates that future research is very unlikely to change existing evidence and that the true effect is similar to the estimated effect; a "moderate-quality" and a "low-quality" rating indicates that future research may change/is likely to change the evaluation results, respectively; a "very low-quality" rating indicates that it is highly uncertain about the existing evidence and that the true effect is likely to be substantially different from the estimated effect.

Data Extraction
Two blind researchers (EAC and GM) performed data extraction. In order to address the objectives of the review, the following information was extracted from the included papers: first author name, year and country of publication, analyzed period, study design, study sample (number, age, and gender), treatment provided, comparator group (e.g., placebo, active), study measure and outcomes. Reviewers extracted data independently from each relevant study and data was checked by all reviewers in case of discrepancies.

Risk of Bias Assessment
The quality of the evidence provided by the eligible studies was assessed by the two independent researchers (GM and EAC). The risk of bias for randomized controlled trials (RCTs) was evaluated with the Revised Cochrane Risk of Bias Tool (RoB 2.0) [46].

Search Results
The initial search returned 495 records (MEDLINE/PubMed = 182, Scopus = 182, Web of Science = 131). Among these, 261 were identified as duplicates and were subsequently excluded. Title and abstract screening were performed for the remaining 234 papers, 14 of which were included in the further evaluation. The full-text examination led to the selection of 8 papers. The hand-screening of references led to the inclusion of two additional records. Subsequently, 10 papers were deemed eligible for the present review (see flowchart in Figure 1).

Content Results
All studies were published over a period from 2009 to 2019 and mainly conducted in the US or Canada. Diagnosis of PTSD was made using DSM-IV or DSM-5 criteria. We included four retrospective studies [40,[47][48][49], two observational studies [50,51], two

Content Results
All studies were published over a period from 2009 to 2019 and mainly conducted in the US or Canada. Diagnosis of PTSD was made using DSM-IV or DSM-5 criteria. We included four retrospective studies [40,[47][48][49], two observational studies [50,51], two openlabel studies [37,38], and two randomized, double-blind, placebo-controlled studies [39,52]. Most of the studies included male subjects and two studies evaluated veterans or military personnel [39,48]. The mean age of participants ranged between 32 and 52 years [37,40,52]. Treatment with CBD only [47], THC only [37,52], medical cannabis (THC and CBD at variable percentages) [48][49][50][51], and nabilone [38][39][40] was evaluated in included studies considering different formulations (e.g., liquid oil spray, capsule), gradual titration and mean dose. The mean dose of CBD was 9 mg/d (range 1-16) for liquid oil spray formulation and 25 mg/d (range 25-100) for capsule [47]; THC was used at 7.5 mg in capsule formulation or oil 0.1 cc bid raising to 0.2 cc bid (0.1 cc = 2.5 mg) [37] while medical cannabis (THC and CBD at variable percentages) was started at 1 g/d and titrated based on clinical response to a maximum dose of 10 g/d [48,49] and a mean dose of 1.2-3 g/d [50,51]. The start dose of nabilone was 0.5-2 mg and titrated weekly to a maximum of 3-4 mg/d (range 0.5-6 mg) [38][39][40]. Only two studies used a placebo comparator group [39,52]. The treatment had variable duration with a minimum of three weeks [37] and a maximum of 43 weeks [40] of drug administration in the acute phase [52]. Regarding psychiatric measures, changes in PTSD Checklist for the DSM-5 (PCL-5) [47], Post-Traumatic Checklist-Civilian Version (PCL-C) [40], Clinician-Administered PTSD Scale (CAPS) [37,39,49,51], Clinical Global Impressions (CGI) [37,39], Pittsburgh Sleep Quality Index (PSQI), Nightmare Effects Survey (NES), Nightmare Effects Survey (NFQ) [37], Well Being Questionnaire (WBQ) [39], and Quality of Life Scale (QOLS) [51] scores were evident. Only one study evaluated the modulation of the amygdala and pre-frontal activation under an acute low dose of THC in patients with and without PTSD and healthy controls (HC) [52]. Considering outcomes, a reduction in PTSD symptoms (e.g., nightmares, intrusive thoughts, anxiety, arousal) as measured by psychometric scores was detected, as well as a decrease of suicidal thoughts. A decrease of PTSD social impact score from 6.6 to 2.7 as reported in clinical charts [48] and an improvement in the quality of life in QOLS score [51] and WBQ score [39] after treatment was also demonstrated. Therefore, results showed that cannabinoids (e.g., THC, CBD, nabilone) may be effective in treating PTSD symptoms as clinical response and psychometric scores demonstrated. Furthermore, acute administration of a low dose of cannabinoid acted on corticolimbic responses to threat-related processing. Data from included studies are reported in Table 1.

Quality and Risk of Bias Assessment
According to the GRADE approach, only two studies presented high quality, whilst most of the included research showed low-to-moderate quality. Detailed information concerning quality assessment is included in Table 1. According to the RoB 2.0 tool, there were some concerns about the possible risk of bias for included RCTs, mainly affecting the randomization process and data reporting (Table 2). Indeed, in the study with randomized, double-blind, placebo-controlled cross-over design [39] information about the sequence allocation was not available, whilst in the randomized, double-blind, placebo-controlled, between-subjects study [52] outcome data was not available for all randomized subjects.

Discussion
Studies included in the present review suggested that different medicinal cannabinoids at distinct doses and formulations could represent promising treatment strategies for PTSD-related symptoms, also presenting an impact on the quality of life in this population. Particularly, not only did treatment with cannabinoids determine an overall improvement of PTSD symptomatology [37,39,40,[47][48][49]51], but it also influenced specific symptom domains, namely sleep disorders [37][38][39], arousal disturbances [37], and suicidal thoughts [48]. Noteworthy, a significant reduction of pain [48,51] and social impact [48], as well as an improvement in quality of life and well-being [39,51], were demonstrated. A better understanding of the possible mechanisms through which such treatments could influence the clinical expression of PTSD is of particular interest since most medications used in clinical practice have the main aim of relieving symptom severity without specifically targeting underlying biological pathways [53].
To note, CB receptors are differently expressed in brain areas and nervous cells: CB1 receptors are highly expressed in the cortex, basal ganglia, hippocampus, hypothalamus, and cerebellum while CB2 receptors are mostly expressed in the immune and gastrointestinal cells and they may modulate central and peripheral regions [9]. CBD acts on both CB1 and CB2 receptors with lower affinity for ∆ 9 -THC, producing less adverse effects (e.g., tachycardia, sedation, hunger) but significantly decreasing fear-memory consolidation and anxiety symptoms of PTSD, possibly due to its action on limbic and paralimbic areas [54,55]. CBD can also exert an agonistic action on the 5-HT1a receptor that may explain its antiemetic and anxiolytic-like properties, on the GPR55 enhancing neuronal excitability, and on the TRPV1, highly localized in the hippocampus, reducing anxiety and aversive memories [32]. The inverse agonist action on CB2 may also explain the anti-inflammatory effects [56]. ∆ 9 -THC acts through neuronal presynaptic CB1 receptors as a partial agonist and inhibits directly or indirectly ongoing neurotransmitter release such as dopamine, glutamate, and acetylcholine [55] in the nucleus accumbens, prefrontal cortex, hippocampus, and amygdala with a consequential reduction in hypervigilance, anxiety, insomnia, nightmares, and extinction deficits related to PTSD [57,58]. Nabilone, a synthetic THC analog, is a CB1 receptor agonist and has been demonstrated to reduce recurrent nightmares, flashbacks, and improve sleep time even if the exact mechanism is not yet understood [38][39][40][41]59].
The efficacy of medicinal cannabinoids in reducing overall PTSD symptomatology might indeed find a possible explanation in their mechanism of action, given their modu-latory influence on different neurotransmitter systems, as demonstrated by the presence of CB receptors in pre-synaptic GABAergic and glutamatergic terminations, but also in noradrenergic, serotoninergic, and dopaminergic axon terminals [27,60].
Noteworthy, PTSD pathophysiology relies on abnormalities that cut across different neural circuits, involving distinct neurotransmitters at the same time [61]. Given the heterogeneity of PTSD symptomatology, belonging to several domains as stated in the diagnostic criteria, molecules that could target more than one system could represent adequate candidates for acting on different clinical features [28], thus reducing overall illness severity.
Besides, the eCBS plays a key role in the process of extinguishing aversive memories, which is mainly mediated by CB1 and could be thus facilitated by CBD administration, as already suggested by animal models, with further confirmation in human studies [34,62]. Similar mechanisms could also explain the improvement in arousal symptom clusters, that appear to be activated by emotional processing of fear-elicited stimuli and altered responses to trauma-related cues [63]. Interestingly, only one among the included studies considered the administration of CBD at variable doses [47], whilst in the remaining studies subjects were treated with different formulations of medicinal cannabis containing THC and CBD at different percentages, or synthetic compounds. Consequently, aversive memories extinction is not expected to be the only mechanism possibly explaining the efficacy of medicinal cannabis in reducing PTSD symptoms. The effect on global PTSD symptomatology might be mediated by the reduction of HPA axis hyperactivation following eCBS signaling enhancement. This could be related to the expression of CB receptors in brain areas that modulate stress, as well as emotional responses to fear and reward, such as the amygdala, pre-frontal cortex, and hippocampus [64]. Particularly, CB1 in the pre-frontal cortex plays a significant role in threat-related responses by modulating the amygdala region [65]. Noteworthy, one of the selected studies showed how the administration of medicinal THC in PTSD subjects could influence cortico-limbic circuitry, also reducing amygdala reactivity in response to potentially threatening stimuli [52]. This result should hopefully be replicated for CBD and synthetic Cannabis-derived compounds, to evaluate whether slightly different actions on eCBS receptors may underpin different effects on brain circuitry. Moreover, future studies should better clarify functional correlates of treatment with medications acting on the eCBS to provide further explanations about their mechanism of action in reducing PTSD symptoms. Furthermore, specific symptom clusters were targeted by treatment with medicinal cannabis in some of the included studies. Particularly, sleep problems such as PTSD-related nightmares were significantly improved by THC oil and nabilone at a maximum dose of 3-6 mg. This result may be of clinical relevance since nightmares are not typically targeted by medications that appear to be efficient in treating other PTSD symptoms [66]. The efficacy of Cannabis derivates in ameliorating sleep disorders was already elucidated by animal studies, with specific effects on sleep duration and depth varying based on means and site of administration (e.g., intraperitoneal, amygdala, brain ventriculus, etc.) [67,68]. Diverse effects of CBD were over time detected depending on medication doses, with higher doses up to 160 mg/day increasing sedative-hypnotic effects [69,70]. Such effects were hypothesized to be mediated by the monoaminergic system since CBD appeared to increase the expression of c-Fos in the dorsal raphe nuclei [71]. Furthermore, CBD can increase Anandamide (AEA) concentration by blocking the AEA membrane transporter (AMT) or the Fatty Acid Amide Hydrolase (FAAH) enzyme, which catalyzes AEA hydrolysis, increasing time of sleep and of slow-wave phase that is physiologically fostered by this endogenous CB1 ligand [72]. Nevertheless, most preclinical research focused mainly on the administration of CBD, which acts as a negative allosteric modulator of CB1 [73], while the included studies considered effects of TCH and its derivates, possibly operating as a partial agonist of the receptor [74]. In consideration of their different actions on endogenous receptors, further studies on PTSD populations should better clarify different actions of THC and CBD on sleep disorders. This could be of particular interest also due to the frequent association that specific arousal distur-bances may present with nightmares and sleep abnormalities, with evidence for nabilone improving such disturbances in non-PTSD populations [75].
Notably, pain indexes were significantly reduced after treatment with nabilone at a maximum dose of 6 mg/d and medicinal cannabis with 20-25% THC component at 2-3 g/d. The use of medicinal cannabis for chronic pain symptoms finds wide support in the medical literature given the modulatory effects of the eCBS on inflammation and pain-processing pathways in the central and peripheric nervous systems, mainly relying on CB2 expression in peripheral tissues and immune cells [76][77][78][79]. Pain is frequently associated with PTSD clinical features, with evidence about mutual influences that such symptoms may exert. Future research focused on this specific comorbidity may elucidate whether treatment with cannabinoids could represent a favorable treatment strategy for selected PTSD subjects with specific comorbidities.
Despite promising results on the improvement of quality of life, as well as reduction of the social impact of PTSD, the use of medicinal cannabinoids in this population could also present negative consequences on general well-being due to their abuse and dependence potential [59,80]. Indeed, subjects affected by PTSD demonstrated the tendency to use cannabis as a form of self-medication due to its anxiolytic and sedative properties [57,81,82], also displaying high rates of comorbid substance-related disorders [83]. Except for cannabidiol which is a phyto-derivative of cannabis devoid of any psychotropic effect, the recreational use of cannabis, particularly synthetic compounds with high THC content, may elicit the emergence of psychotic and anxiety symptoms, as well as cognitive dysfunctions [84][85][86]. Conversely, side effects of medicinal cannabinoids (e.g., dizziness, headache, fatigue, gastrointestinal problems) presented mild-to-moderate severity and were relatively well-tolerated, supporting the assumption that subjects with PTSD may consider them less burdensome than approved medications [87]. Future research should hopefully clarify the long-term effects of such treatments to provide further data concerning their safety profile.
Evidence reported by the included studies presents several limitations. First, most of the considered research is based on small sample sizes, with restricted generalizability of findings. Moreover, heterogeneous samples determine major limitations in the among-study comparability of results. Included studies show low-to-moderate quality and moderate-to-high risk of bias in most cases, which suggests that further studies are expected to address methodological issues to provide solid bases for the use of medicinal cannabinoids in clinical practice. In addition, the retrospective or observational design of most of the included studies precludes conclusions about causality. Only a few RCTs were conducted and comparators were used only in two cases, despite repeated calls for controlled studies that may elucidate the effectiveness of new medications in PTSD treatment [61,88]. Finally, medications were assumed for short periods in most cases and no follow-up data were thus provided. Other randomized, placebo-controlled studies designed or head-to-head comparator studies are therefore needed.

Conclusions
Post-Traumatic Stress Disorder is a particularly heterogeneous clinical entity since it does not invariably exhibit in all subjects the same variety of symptoms and neither do they have similar severity. Moreover, it frequently occurs in comorbidity with other physical or mental disorders. Although there is a robust rationale for the treatment with drugs active, directly or indirectly, on the cannabinoid system and the results to date are promising, further studies are needed to investigate the safety and efficacy profile of their prolonged use. Hopefully, continued research could provide clinicians with novel therapeutic options for a disease that currently is treated with drugs of limited efficacy.