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Compounds extracted from the cannabis plant, including the psychoactive Δ9-tetrahydrocannabinol (THC) and related phytocannabinoids, evoke multiple diverse biological actions as ligands of the G protein-coupled cannabinoid receptors CB1 and CB2. In addition, there is increasing evidence that phytocannabinoids also have non-CB targets, including several ion channels of the transient receptor potential superfamily. We investigated the effects of six non-THC phytocannabinoids on the epithelial calcium channels TRPV5 and TRPV6, and found that one of them, Δ9-tetrahydrocannabivarin (THCV), exerted a strong and concentration-dependent inhibitory effect on mammalian TRPV5 and TRPV6 and on the single zebrafish orthologue drTRPV5/6. Moreover, THCV attenuated the drTRPV5/6-dependent ossification in zebrafish embryos in vivo. Oppositely, 11-hydroxy-THCV (THCV-OH), a product of THCV metabolism in mammals, stimulated drTRPV5/6-mediated Ca2+ uptake and ossification. These results identify the epithelial calcium channels TRPV5 and TRPV6 as novel targets of phytocannabinoids, and suggest that THCV-containing products may modulate TRPV5- and TRPV6-dependent epithelial calcium transport. ispartof: PHARMACOLOGICAL RESEARCH vol:136 pages:83-89 ispartof: location:Netherlands status: published

Historical and scientific evidence suggests that Cannabis use has immunomodulatory and anti-inflammatory effects. We have here investigated the effect of the non-psychotropic phytocannabinoid Δ9-tetrahydrocannabivarin (THCV) and of a Cannabis sativa extract with high (64.8%) content in THCV (THCV-BDS) on nitric oxide (NO) production, and on cannabinoid and transient receptor potential (TRP) channel expression in lipopolysaccharide (LPS)-stimulated murine peritoneal macrophages. THCV-BDS and THCV exhibited similar affinity in radioligand binding assays for CB1 and CB2 receptors, and inhibited, via CB2 but not CB1 cannabinoid receptors, nitrite production evoked by LPS in peritoneal macrophages. THCV down-regulated the over-expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and interleukin 1β (IL-1β) proteins induced by LPS. Furthermore, THCV counteracted LPS-induced up-regulation of CB1 receptors, without affecting the changes in CB2, TRPV2 or TRPV4 mRNA expression caused by LPS. Other TRP channels, namely, TRPA1, TRPV1, TRPV3 and TRPM8 were poorly expressed or undetectable in both unstimulated and LPS-challenged macrophages. It is concluded that THCV - via CB2 receptor activation - inhibits nitrite production in macrophages. The effect of this phytocannabinoid was associated with a down-regulation of CB1, but not CB2 or TRP channel mRNA expression.

Compounds extracted from the cannabis plant, including the psychoactive Δ

Colorectal cancer (CRC) is a major health problem in Western countries. The endocannabinoid 2-arachidonoyl-glycerol (2-AG) exerts antiproliferative actions in a number of tumoral cell lines, including CRC cells. Monoacylglycerol lipase (MAGL), a serine hydrolase that inactivates 2-AG, is highly expressed in aggressive human cancer cells. Here, we investigated the role of MAGL in experimental colon carcinogenesis. The role of MAGL was assessed in vivo by using the xenograft and the azoxymethane models of colon carcinogenesis; MAGL expression was evaluated by RT-PCR and immunohistochemistry; 2-AG levels were measured by liquid chromatography mass spectrometry; angiogenesis was evaluated in tumor tissues [by microvessel counting and by investigating the expression of vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2) proteins] as well as in human umbilical vein endothelial cells (HUVEC); cyclin D1 was evaluated by RT-PCR. MAGL and 2-AG were strongly expressed in tumor tissues. The MAGL inhibitor URB602 reduced xenograft tumor volume, this effect being associated to down-regulation of VEGF and FGF-2, reduction in the number of vessels and down-regulation of cyclin D1. In HUVEC, URB602 exerted a direct antiangiogenic effect by inhibiting FGF-2 induced proliferation and migration, and by modulating pro/anti-angiogenic agents. In experiments aiming at investigating the role of MAGL in chemoprevention, URB602 attenuated azoxymethane-induced preneoplastic lesions, polyps and tumors. MAGL, possibly through modulation of angiogenesis, plays a pivotal role in experimental colon carcinogenesis. Pharmacological inhibition of MAGL could represent an innovative therapeutic approach to reduce colorectal tumor progression. (C) 2017 Elsevier Ltd. All rights reserved.

Given that the pharmacological or genetic inactivation of fatty acid amide hydrolase (FAAH) counteracts pain and inflammation, and on the basis of the established involvement of transient receptor potential vanilloid type-1 (TRPV1) channels in inflammatory pain, we tested the capability of a dual FAAH/TRPV1 blocker, N-arachidonoyl-serotonin (AA-5-HT), to relieve oedema and pain in a model of acute inflammation, and compared its efficacy with that of a single FAAH inhibitor (URB597) or TRPV1 antagonist (capsazepine). Acute inflammation was induced by intraplantar injection of λ-carrageenan into mice and the anti-inflammatory and anti-nociceptive actions of AA-5-HT were assessed at different doses, time points and treatment schedule. In addition, endocannabinoid levels were measured in paw skin and spinal cord. Systemic administration of AA-5-HT elicited dose-dependent anti-oedemigen and anti-nociceptive effects, whereas it was devoid of efficacy when given locally. When tested in a therapeutic regimen, the compound retained comparable anti-inflammatory effects. TRPV1 receptor mediated the anti-inflammatory property of AA-5-HT, whereas both CB1 and TRPV1 receptors were involved in its anti-hyperalgesic activity. These effects were accompanied by an increase of the levels of the endocannabinoid anandamide (AEA) in both inflamed paw and spinal cord. AA-5-HT was more potent than capsazepine as anti-oedemigen and anti-hyperalgesic drug, whereas it shows an anti-oedemigen property similar to URB597, which was, however, devoid of the anti-nociceptive effect. AA-5-HT did not induce unwanted effects on locomotion and body temperature. In conclusion AA-5-HT has both anti-inflammatory and anti-hyperalgesic properties and its employment offers advantages, in terms of efficacy and lack of adverse effects, deriving from its dual activity. © 2010 Elsevier Ltd.

The endocannabinoids anandamide and 2-arachidonoylglycerol, and the anandamide-congener, palmitoylethanolamide, are all substrates for the enzyme fatty acid amide hydrolase, and are endowed with anti-inflammatory actions exerted via cannabinoid receptors or, in the case of palmitoylethanolamide, also via other targets. We investigated the role of the endocannabinoid system during granuloma formation, a model of chronic inflammation sustained by neoangiogenesis, in rats. Granuloma was induced by subcutaneous λ-carrageenin-soaked sponge implants on the back of male Wistar rats. After 96 h, granulomas were detached and tissue formation was evaluated as wet weight; the endocannabinoid system was evaluated by the measurement of endocannabinoid levels, by LC–MS, and of cannabinoid receptor expression, by western blot analysis. Moreover, angiogenesis was evaluated by the measurement of both hemoglobin content and CD31 protein expression. Arachidonoylserotonin (AA-5-HT, 12.5–50 μg/ml), an inhibitor of FAAH, and palmitoylethanolamide (PEA, 200–800 μg/ml) were given locally only once at the time of implantation. Granuloma formation was accompanied by a significant decrease in endocannabinoid and palmitoylethanolamide levels paralleled by increased levels of the fatty acid amide hydrolase, responsible for their breakdown. Moreover, an increase of cannabinoid receptor expression was also observed. Pharmacological elevation of endocannabinoids and palmitoylethanolamide, obtained separately by arachidonoylserotonin and exogenous palmitoylethanolamide treatment, dose-dependently reduced inflammatory hallmarks including tumor necrosis factor-α as well as granuloma-dependent angiogenesis. The effect of arachidonoylserotonin was accompanied by near-normalization of 2-arachidonoylglycerol and palmitoylethanolamide levels in the tissue. These findings suggest that chronic inflammation might develop also because of endocannabinoid and palmitoylethanolamide tissue concentration impairment, the correction of which might be exploited to develop new anti-inflammatory drugs.

There is considerable evidence to support the role of anandamide (AEA), an endogenous ligand of cannabinoid receptors, in neuropathic pain modulation. AEA also produces effects mediated by other biological targets, of which the transient receptor potential vanilloid type 1 (TRPVI) has been the most investigated. Both, inhibition of AEA breakdown by fatty acid amide hydrolase (FAAH) and blockage of TRPVI have been shown to produce anti-nociceptive effects. Recent research suggests the usefulness of dual-action compounds, which may afford greater anti-allodynic efficacy. Therefore, in the present study, we examined the effect of N-arachidonoyl-serotonin (AA-5-HT), a blocker of FAAH and TRPV1, in a rat model of neuropathic pain after intrathecal administration. We found that treatment with AA-5-HT increased the pain threshold to mechanical and thermal stimuli, with highest effect at the dose of 500 nM, which was most strongly attenuated by AM-630, CB2 antagonist, administration. The single action blockers PF-3845 (1000 nM, for FAAH) and I-RTX (1 nM, for TRPV1) showed lower efficacy than AA-5-HT. Moreover AA-5-HT (500 nM) elevated AEA and palmitoylethanolamide (PEA) levels. Among the possible targets of these mediators, only the mRNA levels of CB2, GPR18 and GPR55, which are believed to be novel cannabinoid receptors, were upregulated in the spinal cord and/or DRG of CCI rats. It was previously reported that AA-5-HT acts in CB1 and TRPV1-dependent manner after systemic administration, but here for the first time we show that AA-5-HT action at the spinal level involves CB2, with potential contributions from GRP18 and/or GPR55 receptors. (C) 2016 Elsevier Ltd. All rights reserved.

To investigate the possible interactions between kavalactone-based molecules and proteins of the endocannabinoid system and provide novel and synthetically accessible structural scaffolds for the design of cannabinoid receptor ligands sharing pharmacological properties with kavapyrones, a preliminary SAR analysis was performed on five commercially available natural kavalactones and nine kavalactone-analogues properly synthesized. These compounds were investigated for assessing their cannabinoid receptor binding affinity and capability of inhibiting the activity of the two major metabolic enzymes of the endocannabinoid system, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL). Among the molecules tested, only yangonin exhibited affinity for the human recombinant CB₁ receptor with a K(i)=0.72 μM and selectivity vs. the CB₂ receptor (K(i)>10 μM). None of the compounds exhibited strong inhibitory effects on the two enzymes analyzed. The CB₁ receptor affinity of yangonin suggests that the endocannabinoid system might contribute to the complex human psychopharmacology of the traditional kava drink and the anxiolytic preparations obtained from the kava plant.

N-acyl-vanillamide (NAVAM) analogues of the natural pungent principle of capsicum, capsaicin, were developed several years ago as potential non-pungent analgesic compounds. N-oleoyl-vanillamide (olvanil) and N-arachidonoy-vanillamide (arvanil), in particular, were described in several publications and patents to behave as potent anti-hyperalgesic compounds in experimental models of chronic and inflammatory pain, and to activate both "capsaicin receptors", i.e. the transient receptor potential of vanilloid type-1 (TRPV1) channel, and, either directly or indirectly, cannabinoid receptors of type-1. Here we report the biochemical and pharmacological characterization of a so far neglected NAVAM, N-palmitoyl-vanillamide (palvanil), and propose its possible use instead of capsaicin, as a possible topical analgesic. Palvanil exhibited a kinetics of activation of human recombinant TRPV1-mediated intracellular calcium elevation significantly slower than that of capsaicin (t(1/2) = 21 s and 8 s, respectively at 1 mu M). Slow kinetics of TRPV1 agonists were previously found to be associated with stronger potencies as TRPV1 desensitizing agents, which in turn are usually associated with lower pungency and stronger anti-hyperalgesic activity. Accordingly, palvanil desensitized the human recombinant TRPV1 to the effect of capsaicin (10 nM) with significantly higher potency than capsaicin (IC(50) = 0.8 nM and 3.8 nM, respectively), this effect reaching its maximum more rapidly (50 and 250 min, respectively). Palvanil was also more potent than capsaicin at desensitizing the stimulatory effect of TRPV1 by low pH together with anandamide, which mimics conditions occurring during inflammation. In the eye-wiping assay carried out in mice, palvanil was not pungent and instead caused a strong and long-lasting inhibition of capsaicin-induced eye-wiping. Finally, intraplantar palvanil inhibited the second phase of the nociceptive response to formalin in mice. In conclusion, palvanil appears to be a non-pungent analogue of capsaicin with stronger desensitizing effects on TRPV1 and hence potentially higher anti-hyperalgesic activity.

Four positively charged compounds, previously shown to produce analgesic activity by interacting with prokineticin receptor or T-type calcium channels, were tested for their ability to inhibit capsaicin-induced elevation of intracellular Ca2+ in HEK-293 cells stably transfected with the human recombinant TRPV1, with the goal of identifying novel TRPV1 open-pore inhibitors. KYS-05090 showed the highest potency as a TRPV1 antagonist, even higher than that of the open-pore triazine inhibitor 8aA. The latter showed quite remarkable agonist/desensitizer activity at the rat recombinant TRPM8 channel. The activity of KYS-05090 and the other compounds was selective because none of these compounds was able to modulate the rat TRPA1 channel. Open-pore inhibitors of TRPV1 may be a new class of multi-target analgesics with lesser side effects, such as loss of acute pain sensitivity and hyperthermia, than most TRPV1 antagonists developed so far. (C) 2015 Elsevier Ltd. All rights reserved.

We have previously developed quinolone-3-carboxamides with the aim of obtaining new ligands for both cannabinoid receptors, CB1 and CB2. Our preliminary screening led to the identification of cannabinoid receptor ligands characterized by high affinity and, in some cases, also selectivity for CB(2) receptors. Specifically, three compounds, 1, 2 and 3 showed high affinity for CB2 as well as high selectivity over CB1 receptors. In addition, the activity shown by 1 against the formalin-induced nocifensive response in mice, reported in our previous paper, suggests that quinolone-3-carboxamides possess anti-nociceptive properties. In the present work, we have performed functional in vitro bioassays with the aim of investigating the functional activity in the [35S]GTPgammaS binding assay of the other two compounds that, like 1, behave as CB2 selective ligands, and their potential analgesic actions in vivo. We found that both 2 and 3 behave in vitro as CB2 inverse agonists and are able to decrease nociceptive behaviour in the late phase of the formalin test only at the highest dose tested, although, at lower doses, they prevent the anti-nociceptive effects of a selective CB2 partial agonist in the formalin test. These results identify in 2 and 3 two novel, potent and selective CB2 antagonists/inverse agonists and confirm previous reports in the literature that, in addition to agonists at cannabinoid CB2 receptors, also inverse agonists/antagonists at these receptors show promise as anti-inflammatory agents.

The activity of the endocannabinoid system, in terms of the levels of the endocannabinoids and of cannabinoid receptors, or of the functional coupling of the latter to a biological response, undergoes to remodelling during pathological conditions. In the CNS, these changes, depending also on the nature of the disorder, can be transient or long-lasting, occur only in those tissues involved in the pathological condition and usually aim at restoring the physiological homeostasis by reducing excitotoxicity, inflammation and neuronal death. However, during chronic disorders, prolonged activation of the endocannabinoid system might also contribute to the symptoms of the pathology. Whilst acute changes of the tissue levels of the endocannabinoids reflect the "on demand" nature of their biosynthesis and release, and hence are effected mostly through regulation of the biosynthetic enzymes, chronic changes seem to be mostly due to longer-lasting alterations in the expression of anabolic and catabolic enzymes. The possibility of obtaining therapeutic advantage from endocannabinoid plasticity in neuropsychiatric and neurological disorders is discussed in this review article.