*4.1. In Vitro Studies*

An in vitro study on Caco-2 cells demonstrated that adding AEA or cannabidiol (CBD) alongside IL-17A when incubating the cell monolayers for 48 h prevents mucosal damage and the changes in the epithelial permeability associated with inflammation [57]. In a similar study using the same cell types, delta-9-tetrahydrocannabinol (THC) and CBD were also able to reduce the intestinal permeability induced by IFN-γ and TNF-α, while AEA and 2-AG enhanced the cytokine-induced permeability [58]. This leads to the conclusion that the effects are CB1-related, and that the specific agonistic effects of various cannabinoids trigger specific effects [58,59]. These findings were validated ex vivo, in inflammatory and hypoxic states, on Caco-2 cells harvested from colorectal resections [60].

Improving on this experimental model, Couch et al. tested the effects of CBD and palmitoylethanolamide (PEA) on colon explants from patients with inflammatory conditions; these cannabinoids elicit anti-inflammatory properties by preventing the increased cytokine production and reducing the intracellular signaling phosphoprotein levels [61]. The anti-inflammatory properties of phytocannabinoids have been investigated using fresh and baked *C. sativa* flowers, and it was revealed that tetrahydrocannabinolic acid (THCA) is the main active compound and is able to reduce IL-8 levels in HCT116 colon cancer cells pretreated with TNF-α at concentrations of 114–207 µg/mL, partially via the GPR55 receptor [62]. The same study further confirmed the anti-inflammatory effects on patients suffering from IBD, through COX-2 and matrix metalloproteinase (MMP)-9 mediated mechanisms, and showed that THCA exerts superior effects and less cytotoxicity than CBD on these cell lines [62].

Another important anti-inflammatory property is the maintenance of the epithelial barrier's integrity. CBD is able to preserve the mucosal integrity in Caco-2 cells at concentrations of 10−<sup>7</sup> to 10−<sup>9</sup> M, opposing the effects of *Clostridium difficile* toxin A in an inflammation experimental model; this activity is CB1-mediated, as the use of AM251, a CB1 antagonist, inhibited the observed effects [63]. An in vitro study on HT29 cells showed that AEA, methanandamide (mAEA), and arachidonylcyclopropylamide (ACPA) stimulate CB1-dependent wound closure even in low nanomolar concentrations [64]. These findings consolidate the hypothesis of the intrinsic protective effect of endocannabinoids against intestinal barrier disruptions.

Conversely, CB2 receptors have also been implicated in mediating the anti-inflammatory effects of β-Caryophyllene on oral mucositis, which is characterized by the decrease of TNFα, IL-1β, IL-6 and IL-17A [65]. Furthermore, Matalon et al. showed, in their recent paper, that the synthetic CB2 agonist JWH-133 is able to reduce MMP-9 and IL-8 levels in inflamed colon biopsies from patients with IBD. These findings draw further attention to the specific involvement of the two cannabinoid receptors in the various facets of inflammation. There is extensive evidence that CB1 is normally expressed in the intestinal epithelium, while CB2 expression seems to be stimulated by inflammatory conditions, supporting its attributed role in the regulation of inflammation, cell growth and the immune response [64,66–68].

## *4.2. In Vivo Animal Studies*

The effectiveness of cannabinoids in inflammatory diseases has been demonstrated on various animal models. Couch et al. published a comprehensive meta-analysis presenting undeniable evidence that cannabinoids prove to be effective in intestinal inflammatory conditions [69]. The disease activity index (DAI) score and levels of myeloperoxidase (MPO) activity were the criteria of choice for the measurement of the effects of various synthetic and phyto-cannabinoids.

In an in vivo murine model of IBD induced by the intracolonic administration of dinitrobenzene sulphonic acid (DNBS), cannabigerol (CBG) reduced the expression of inducible nitric oxide synthase (iNOS), as well as the levels of IL-1β, IL-10 and IFN-γ, and the activity of myeloperoxidase, while increasing the activity of superoxide dismutase [70]. CBD appears to elicited analogous effects in a parallel study [71]. Using the same experimental model, Pagano et al. noted similar effects when using a combination of fish oil, CBD and CBG [72]. The association of fish oil and CBD enhances their anti-inflammatory effects, which occur at lower doses (20 mg and 0.3–10 mg/kg, respectively) than the per se administration of the two substances; these findings were reported in mice with induced colitis, in which the combination of fish oil and CBD decreased the myeloperoxidase (MPO) activity, DAI score, intestinal permeability, and levels of IL-1β and IL-6 [73].

An additional key factor in the evolution and management of intestinal inflammations is the gut microbiome. The interactions between various microbes and the intestinal mucosa seem to play an important role in the local immune response to various conditions including IBD and Crohn's disease [74]. CBD is able to induce changes in the gut microbiota, apparently independently from its effects on local inflammation [73]. This is of interest because there is evidence that the endocannabinoid system connects the intestinal microbiome with the physiological processes occurring in the adipose tissue, through regulatory pathways [75]. Furthermore, and more importantly, it appears that the alteration of the balance between the endocannabinoid system and the microbiota can negatively impact the integrity of the intestinal barrier [76].

In vivo studies on animal models confirmed that cannabinoids protect the intestinal barrier's integrity. In this regard, the role of CB1 has been validated using a knockout mice model which showed that CB1 is responsible for the intestinal physiological response to inflammation, leading to the secretion of IgA and the regulation of intestinal permeability, among other effects [77]. Synthetic cannabinoids can further enhance these properties. Cao et al. provoked acute lesions of the gastrointestinal mucosa in rats by inducing acute pancreatitis and then showed that synthetic cannabinoid HU210, a non-selective CB agonist, reversed the morphological and serum anomalies, demonstrating its anti-inflammatory properties [78]. A later study showed that HU210 protects the intestinal mucosa in a murine model of ulcerative colitis, most likely through the toll-like receptor 4 (TLR4) and mitogen-activated protein (MAP) signaling pathways [79]. Another synthetic cannabinoid, abnormal cannabidiol, is an isomer of cannabidiol that exerts its effects via non-CB1-non-CB2 signaling and elicits protective effects against chemically induced colitis in mice, stimulating wound healing while inhibiting neutrophil recruitment [80].

## *4.3. Human Clinical Trials*

Up to the moment of this review, limited information is available regarding the in vivo effects of cannabinoids on intestinal inflammatory conditions in humans. The outcome predictors in IBD trials usually include DAI, C-reactive protein (CRP), rapid fecal calprotectin (FC), the Mayo score and the Crohn's Disease Activity Index (CDAI), and the correct interpretation of the results relies on quality preparation for colonoscopy [81–83].

A recent paper by Kienzl et al. gathered retrospective data from patients suffering from IBD that used cannabis to alleviate symptoms [76]. Most studies included in the analysis showed that cannabis users benefited from symptom relief, improved DAI, and better quality of life. However, despite the overall good tolerance, the adverse reactions of cannabis inhalation are to be considered [76,84]. Furthermore, in one survey, patients with Crohn's disease presented a higher risk for surgery [85]. The legalization of medicinal marijuana did not affect its use in patients with IBD, despite an upward trend in the use of cannabis in the tested population throughout the previous 5 years [86].

A randomized clinical trial on healthy subjects performed by Couch et al. tested the effects of PEA and CBD on the regulation of the increased intestinal permeability induced by the pro-inflammatory effects of aspirin [87]. A dose of 600 mg of PEA and CBD, respectively, managed to prevent the increase in intestinal permeability caused by 600 mg of aspirin, evidenced by the measurement of lactulose and mannitol excretion in the urine.

Two randomized clinical trials were published by Naftali et al., in 2013 and 2017, which tested the effects of inhaled THC and oral CBD, respectively, in Crohn's disease [88,89]. While THC showed some clinical benefits, CBD failed to improve the DAI in active Crohn's disease. However, both trials received some criticism for methodological flaws, small patient groups and various other biases [69,90].

Two other randomized placebo-controlled studies studied the effects of cannabinoids in ulcerative colitis. Irving et al. tested a combination of THC and CBD capsules that were administered in increasing doses, and found that while the remission rates were similar to the placebo, the cannabinoid capsules offered subjective improvements to the patients' quality of life, despite mild to moderate side effects [91]. Naftali et al. tested the effects of inhaled THC and recorded significant improvements in the DAI, as well as decreases in the CRP and FC while registering no serious adverse effects [92].

These findings are encouraging, and the favorable in vitro results and animal studies observations warrant that further human clinical trials despite the low number of studies published to date. Furthermore, synthetic cannabinoids may demonstrate superior effectiveness and similar or fewer side effects, and could represent a major improvement in the treatment of intestinal inflammatory diseases.
