Romano, Barbara (2014) NON-PSYCHOTROPIC PHYTOCANNABINOIDS IN INTESTINAL INFLAMMATION AND COLON CANCER. [Tesi di dottorato]

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Background and aim. Preparations from Cannabis sativa and/or its main active ingredient Δ9-tetrahydrocannabinoid (Δ9-THC) have been traditionally used for the relief of inflammatory bowel disease (IBD) symptoms and as antiemetics in cancer patients. The plant Cannabis contains, in addition to Δ9-THC, non-psychotropic cannabinoids with potential therapeutic interest. These include cannabidiol (CBD), cannabigerol (CBG), cannabichromene (CBC) and Δ9-tetrahydrocannabivarin (THCV). Pharmacodynamic studies have shown that such phytocannabinoids may interact with specific targets (e.g. components of the so-called “endogenous cannabinoid system”, TRP channels) which play a key role in intestinal inflammation and colon carcinogenesis. In this thesis, the effect and the mode of action of CBD, CBC, CBG and THCV as well as CBD BDS (i.e. a standardized Cannabis extract with high content in CBD) in experimental models of IBD and colon cancer were evaluated. Main Methods. Experimental colitis was induced in mice by intracolonic administration of dinitrobenzene sulfonic acid (DNBS). Inflammation was assessed by evaluating inflammatory markers/parameters (e.g. colon weight/colon length ratio, myeloperoxidase and superoxide dismutase activity) as well as by evaluating intestinal permeability, by histological and immunohistochemistry analysis; interleukin-1β (IL-1β), interleukin-10 (IL-10), interferon-γ (IFN- γ) levels were measured by ELISA; inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) by western blot and RT-PCR. Murine macrophages were used to evaluate the in vitro anti-inflammatory action of phytocannabinoids; Radioligand binding assay and endocannabinoids levels (by isotope dilution chromatography-atmospheric pressure chemical ionisation mass spectrometry) were also performed. Carcinogenesis in vivo was evaluated in the azoxymentane(AOM) and xenograft model of colon cancer. Cell growth was evaluated by the MTT assay both in colorectal cancer as well as intestinal healthy epithelial cells. Key Results Inflammation: CBG, CBC and THCV exerted anti-inflammatory effects in the DNBS model of colitis. More in depth ex vivo investigations on CBG showed that its anti-inflammatory action was associated to modulation of cytokine (IL-1β, IL-10 and IFN- γ) levels and down-regulation of iNOS expression. Studies on peritoneal macrophages suggest that the three phytocannabinoids inhibited NO production, an effect associated to inhibition of iNOS expression (for CBG and THCV, but not for CBC). The effect of THCV, but not CBG or CBC, was mediated by CB2 receptor activation. By contrast, an endogenous cannabinoid ‘‘tone’’ at CB1 and CB2 receptors is likely coupled negatively to CBC and CBG anti-inflammatory actions, respectively. Interestingly, CBC increased the oleylethanolamide, an endogenous anti-inflammatory lipid. Cancer: CBD, CBD BDS and CBG exert chemopreventive and curative effects in experimental models of colon cancer and inhibited the growth of tumoural - but not healthy - intestinal cells. CBD BDS and CBD exerted antiproliferative effects via cannabinoid-mediated mechanisms, with TRPV1 and PPAR-γ possibly involved in the antiproliferative action of CBD. By contrast, CBG inhibited the growth of colorectal cancer cells, but with a mechanism not involving activation of cannabinoid receptors. Notably, the inhibitory effect of CBG on cell growth was mimicked by other TRPM8 antagonists, thus suggesting that such receptor might be, at least in part, involved in its actions Conclusions and implications The results reported in this thesis supports the notion that the Cannabis plant is a treasure trove of potentially novel therapeutic agents for the prevention and/or the treatment of IBD and colon cancer.

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