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Introduction: Highly purified cannabidiol (CBD) (approved as Epidiolex® in the United States) has demonstrated efficacy with an acceptable safety profile in patients with Lennox-Gastaut or Dravet syndrome in four randomized controlled trials. CBD possesses affinity for many target classes with functional effects relevant to the pathophysiology of many disease types, including epilepsy. Although the mechanism of action of CBD underlying the reduction of seizures in humans is unknown, transient receptor potential vanilloid 1 (TRPV1) represents a plausible target because (1) CBD activates and then desensitizes TRPV1, (2) TRPV1 is overexpressed in models of temporal lobe epilepsy and patients with epilepsy, (3) and TRPV1 modulates neuronal excitability. Methods: To investigate a potential role of TRPV1 in the anticonvulsive effects of CBD, the effect of CBD on seizure threshold was assessed using a mouse maximal electroshock threshold model of generalized seizure in TRPV1 knockout and wildtype mice. The dose dependence of the CBD effect was determined and compared with that of the positive comparator diazepam and vehicle. Results: At 50 and 100 mg/kg, CBD significantly (p

Acute or chronic alterations in energy status alter the balance between excitatory and inhibitory synaptic transmission and associated synaptic plasticity to allow for the adaptation of energy metabolism to new homeostatic requirements. The impact of such changes on endocannabinoid and cannabinoid receptor type 1 (CB1)-mediated modulation of synaptic transmission and strength is not known, despite the fact that this signaling system is an important target for the development of new drugs against obesity. We investigated whether CB1-expressing excitatory vs. inhibitory inputs to orexin-A–containing neurons in the lateral hypothalamus are altered in obesity and how this modifies endocannabinoid control of these neurons. In lean mice, these inputs are mostly excitatory. By confocal and ultrastructural microscopic analyses, we observed that in leptin-knockout (ob/ob) obese mice, and in mice with diet-induced obesity, orexinergic neurons receive predominantly inhibitory CB1-expressing inputs and overexpress the biosynthetic enzyme for the endocannabinoid 2-arachidonoylglycerol, which retrogradely inhibits synaptic transmission at CB1-expressing axon terminals. Patch-clamp recordings also showed increased CB1-sensitive inhibitory innervation of orexinergic neurons in ob/ob mice. These alterations are reversed by leptin administration, partly through activation of the mammalian target of rapamycin pathway in neuropeptide-Y-ergic neurons of the arcuate nucleus, and are accompanied by CB1-mediated enhancement of orexinergic innervation of target brain areas. We propose that enhanced inhibitory control of orexin-A neurons, and their CB1-mediated disinhibition, are a consequence of leptin signaling impairment in the arcuate nucleus. We also provide initial evidence of the participation of this phenomenon in hyperphagia and hormonal dysregulation in obesity.

The brain endocannabinoid system plays a crucial role in emotional processes. We have previously identified an important role for endocannabinoids in social play behavior, a highly rewarding form of social interaction in adolescent rats. Here, we tested the hypothesis that endocannabinoid modulation of social play behavior occurs in brain regions implicated in emotion and motivation. Social play increased levels of the endocannabinoid anandamide in the amygdala and nucleus accumbens (NAc), but not in prefrontal cortex or hippocampus of 4- to 5-week-old male Wistar rats. Furthermore, social play increased phosphorylation of CB1 cannabinoid receptors in the amygdala. Systemic administration of the anandamide hydrolysis inhibitor URB597 increased social play behavior, and augmented the associated elevation in anandamide levels in the amygdala, but not the NAc. Infusion of URB597 into the basolateral amygdala (BLA) increased social play behavior, and blockade of BLA CB1 cannabinoid receptors with the antagonist/inverse agonist SR141716A prevented the play-enhancing effects of systemic administration of URB597. Infusion of URB597 into the NAc also increased social play, but blockade of NAc CB1 cannabinoid receptors did not antagonize the play-enhancing effects of systemic URB597 treatment. Last, SR141716A did not affect social play after infusion into the core and shell subregions of the NAc, while it reduced social play when infused into the BLA. These data show that increased anandamide signaling in the amygdala and NAc augments social play, and identify the BLA as a prominent site of action for endocannabinoids to modulate the rewarding properties of social interactions in adolescent rats.

Chronic pain is associated with cognitive deficits. Palmitoylethanolamide (PEA) has been shown to ameliorate pain and pain-related cognitive impairments by restoring glutamatergic synapses functioning in the spared nerve injury (SNI) of the sciatic nerve in mice. SNI reduced mechanical and thermal threshold, spatial memory and LTP at the lateral entorhinal cortex (LEC)-dentate gyrus (DG) pathway. It decreased also postsynaptic density, volume and dendrite arborization of DG and increased the expression of metabotropic glutamate receptor 1 and 7 (mGluR1 and mGluR7), of the GluR1, GluR1s845 and GluR1s831 subunits of AMPA receptor and the levels of glutamate in the DG. The level of the endocannabinoid 2-arachidonoylglycerol (2-AG) was instead increased in the LEC. Chronic treatment with PEA, starting from when neuropathic pain was fully developed, was able to reverse mechanical allodynia and thermal hyperalgesia, memory deficit and LTP in SNI wild type, but not in PPAR alpha null, mice. PEA also restored the level of glutamate and the expression of phosphorylated GluR1 subunits, postsynaptic density and neurogenesis. Altogether, these results suggest that neuropathic pain negatively affects cognitive behavior and related LTP, glutamatergic synapse and synaptogenesis in the DG. In these conditions PEA treatment alleviates pain and cognitive impairment by restoring LIT and synaptic maladaptative changes in the LEC-DG pathway. These outcomes open new perspectives for the use of the N-acylethanolamines, such as PEA, for the treatment of neuropathic pain and its central behavioural sequelae.

Recent evidence points to the gut microbiota as a regulator of brain and behavior, although it remains to be determined if gut bacteria play a role in chronic pain. The endocannabinoid system is implicated in in- flammation and chronic pain processing at both the gut and central nervous system (CNS) levels. In the present study, we used low Vitamin D dietary intake in mice and evaluated possible changes in gut microbiota, pain processing and endocannabinoid system signaling. Vitamin D deficiency induced a lower microbial diversity characterized by an increase in Firmicutes and a decrease in Verrucomicrobia and Bacteroidetes. Concurrently, vitamin D deficient mice showed tactile allodynia associated with neuronal hyperexcitability and alterations of endocannabinoid system members (endogenous mediators and their receptors) at the spinal cord level. Changes in endocannabinoid (anandamide and 2-ara- chidonoylglycerol) levels were also observed in the duodenum and colon. Remarkably, the anti-inflammatory anandamide congener, palmitoylethanolamide, counteracted both the pain behaviour and spinal biochemical changes in vitamin D deficient mice, whilst increasing the levels of Akkermansia, Eubacterium and Enterobacteriaceae, as compared with vehicle-treated mice. Finally, induction of spared nerve injury in normal or vitamin D deficient mice was not accompanied by changes in gut microbiota composition. Our data suggest the existence of a link between Vitamin D deficiency – with related changes in gut bacterial composition – and altered nociception, possibly via molecular mechanisms involving the endocannabinoid and related mediator signaling systems.

The microbiota-gut-brain axis (MGBA) regulates the reciprocal interaction between chronic inflammatory bowel and psychiatric disorders. This interaction involves multiple pathways that are highly debated. We examined the behavioural, biochemical and electrophysiological alterations, as well as gut microbiota composition in a model of antibiotic-induced experimental dysbiosis. Inflammation of the small intestine was also assessed. Mice were exposed to a mixture of antimicrobials for 2 weeks. Afterwards, they received Lactobacillus casei DG (LCDG) or a vehicle for up to 7 days via oral gavage. Perturbation of microbiota was accompanied by a general inflammatory state and alteration of some endocannabinoidome members in the gut. Behavioural changes, including increased immobility in the tail suspension test and reduced social recognition were observed, and were associated with altered BDNF/TrkB signalling, TRPV1 phosphorylation and neuronal firing in the hippocampus. Moreover, morphological rearrangements of non-neuronal cells in brain areas controlling emotional behaviour were detected. Subsequent probiotic administration, compared with vehicle, counteracted most of these gut inflammatory, behavioural, biochemical and functional alterations. Interestingly, levels of Lachnospiraceae were found to significantly correlate with the behavioural changes observed in dysbiotic mice. Our findings clarify some of the biomolecular and functional modifications leading to the development of affective disorders associated with gut microbiota alterations.

After 4 millennia of more or less documented history of cannabis use, the identification of cannabinoids, and of Δ(9)-tetrahydrocannabinol in particular, occurred only during the early 1960s, and the cloning of cannabinoid CB1 and CB2 receptors, as well as the discovery of endocannabinoids and their metabolic enzymes, in the 1990s. Despite this initial relatively slow progress of cannabinoid research, the turn of the century marked an incredible acceleration in discoveries on the "endocannabinoid signaling system," its role in physiological and pathological conditions, and pain in particular, its pharmacological targeting with selective agonists, antagonists, and inhibitors of metabolism, and its previously unsuspected complexity. The way researchers look at this system has thus rapidly evolved towards the idea of the "endocannabinoidome," that is, a complex system including also several endocannabinoid-like mediators and their often redundant metabolic enzymes and "promiscuous" molecular targets. These peculiar complications of endocannabinoid signaling have not discouraged efforts aiming at its pharmacological manipulation, which, nevertheless, now seems to require the development of multitarget drugs, or the re-visitation of naturally occurring compounds with more than one mechanism of action. In fact, these molecules, as compared to "magic bullets," seem to offer the advantage of modulating the "endocannabinoidome" in a safer and more therapeutically efficacious way. This approach has provided so far promising preclinical results potentially useful for the future efficacious and safe treatment of chronic pain and inflammation.

Background Enhanced supraspinal glutamate levels following nerve injury are associated with pathophysiological mechanisms responsible for neuropathic pain. Chronic pain can interfere with specific brain areas involved in glutamate-dependent neuropsychological processes, such as cognition, memory, and decision-making. The medial prefrontal cortex (mPFC) is thought to play a critical role in pain-related depression and anxiety, which are frequent co-morbidities of chronic pain. Using an animal model of spared nerve injury (SNI) of the sciatic nerve, we assess bio-molecular modifications in glutamatergic synapses in the mPFC that underlie neuropathic pain-induced plastic changes at 30 days post-surgery. Moreover, we examine the effects of palmitoylethanolamide (PEA) administration on pain-related behaviours, as well as the cortical biochemical and morphological changes that occur in SNI animals. Results At 1 month, SNI was associated with mechanical and thermal hypersensitivity, as well as depression-like behaviour, cognitive impairments, and obsessive-compulsive activities. Moreover, we observed an overall glutamate synapse modification in the mPFC, characterized by changes in synaptic density proteins and amino acid levels. Finally, with regard to the resolution of pain and depressive-like syndrome in SNI mice, PEA restored the glutamatergic synapse proteins and changes in amino acid release. Conclusions Given the potential role of the mPFC in pain mechanisms, our findings may provide novel insights into neuropathic pain forebrain processes and indicate PEA as a new pharmacological tool to treat neuropathic pain and the related negative affective states. Graphical Abstract Palmitoylethanolamide: a new pharmacological tool to treat neuropathic pain and the related negative affective states. Electronic supplementary material The online version of this article (doi:10.1186/s13041-015-0139-5) contains supplementary material, which is available to authorized users.

Endocannabinoids (eCB) are key regulators of excitatory/inhibitory neurotransmission at cannabinoid-1-receptor (CB1 R)-expressing axon terminals. The most abundant eCB in the brain, that is 2-arachidonoylglycerol (2-AG), is hydrolyzed by the enzyme monoacylglycerol lipase (MAGL), whose chronic inhibition in the brain was reported to cause CB1 R desensitization. We employed the MAGL knock-out mouse (MAGL-/-), a genetic model of congenital and sustained elevation of 2-AG levels in the brain, to provide morphological and biochemical evidence for ?-arrestin2-mediated CB1 R desensitization in brain regions involved in the control of emotional states, that is, the prefrontal cortex (PFC), amygdala, hippocampus and cerebellar cortex. We found a widespread CB1 R/?-arrestin2 co-expression in the mPFC, amygdala and hippocampus accompanied by impairment of extracellular signal-regulated kinase signaling and elevation of vesicular glutamate transporter (VGluT1) at CB1 R-positive excitatory terminals in the mPFC, or vesicular GABA transporter (VGAT) at CB1 R-positive inhibitory terminals in the amygdala and hippocampus. The impairment of CB1 R signaling in MAGL-/- mice was also accompanied by enhanced excitatory drive in the basolateral amygdala (BLA)-mPFC circuit, with subsequent elevation of glutamate release to the mPFC and anxiety-like and obsessive-compulsive behaviors, as assessed by the light/dark box and marble burying tests, respectively. Collectively, these data provide evidence for a ?-arrestin2-mediated desensitization of CB1 R in MAGL-/- mice, with impact on the synaptic plasticity of brain circuits involved in emotional functions. In this study, the authors provide evidence that congenitally enhanced endocannabinoid levels in the neuronal circuits underlying anxiety-like behavioral states (mainly medial prefrontal cortex, amygdala and hippocampus) lead to CB1R desenistization and anxiety and depression. MAGL-/- mice, a model of congenital overactivity of the eCB system, exhibited a compensatory impairment of CB1R signaling in anxiety-associated brain areas and a subsequent change in excitatory/inhibitory tone associated with altered score in the marble burying and light/dark box test, in concomitance with anxiety and depression behavior states. These findings may have potential relevance to the understanding of the neurochemical effects of chronic CB1R overstimulation in cannabis abusers.

The molecular mechanism of action of dipyrone, a widely used antipyretic and non-opioid analgesic drug, is still not fully understood. Actions upon peripheral inflamed tissues as well as the central nervous system, especially upon the PAG-RVM axis, have been suggested. Dipyrone is a prodrug and its activity is due to its immediate conversion to its active metabolites. We tested the effect of two recently discovered metabolites of dipyrone, the arachidonoyl amides of 4-methylaminoantipyrine and 4-aminoantipyrine, on the neurons of the rostral ventromedial medulla (RVM), which are part of the descending pathway of antinociception. These compounds reduced the activity of ON-cells and increased the activity of OFF-cells. Both CB1 and TRPV1 blockade reversed these effects, suggesting that the endocannabinoid/endovanilloid system takes part in the analgesic effects of dipyrone.

Mesenchymal stromal cells are non-hematopoietic, multipotent progenitor cells producing cytokines, chemokines, and extracellular matrix proteins that support hematopoietic stem cell survival and engraftment, influence immune effector cell development, maturation, and function, and inhibit alloreactive T-cell responses. The immunosuppressive properties of human mesenchymal stromal cells have attracted much attention from immunologists, stem cell biologists and clinicians. Recently, the presence of the endocannabinoid system in hematopoietic and neural stem cells has been demonstrated. Endocannabinoids, mainly acting through the cannabinoid receptor subtype 2, are able to modulate cytokine release and to act as immunosuppressant when added to activated T lymphocytes. In the present study, we have investigated, through a multidisciplinary approach, the involvement of the endocannabinoids in migration, viability and cytokine release of human mesenchymal stromal cells. We show, for the first time, that cultures of human mesenchymal stromal cells express all of the components of the endocannabinoid system, suggesting a potential role for the cannabinoid CB2 receptor as a mediator of anti-inflammatory properties of human mesenchymal stromal cells, as well as of their survival pathways and their capability to home and migrate towards endocannabinoid sources. © 2013 Rossi et al.

We showed previously that inhibiting fatty acid amide hydrolase (FAAH), an endocannabinoid degrading enzyme, and transient receptor potential vanilloid type-1 (TRPV1) channels with the same molecule, the naturally occurring N -arachidonoyl-serotonin (AA-5-HT), produces more efficacious anti-nociceptive and anti-hyperalgesic actions than the targeting of FAAH or TRPV1 alone. We also reported the synthesis of some piperazinyl carbamates as “dual” FAAH inhibitors and either antagonists at TRPV1 or agonists/desensitizers of the transient receptor potential ankyrin type-1 (TRPA1) cannel, another target for analgesic drugs. We investigated here if two such compounds, the FAAH/TRPV1 blocker OMDM198 and the FAAH inhibitor/TRPA1 agonist, OMDM202, exert anti-nociceptive actions in the formalin test of pain in mice, and through what mechanism. Both compounds inhibited the second phase of the response to formalin, the effect being maximal at 3 mg/kg, i.p. Antagonism of CB1 or CB2 receptors with AM251 or AM630 (1 mg/kg, i.p.), respectively, reversed this effect. A TRPV1 agonist, palvanil (0.1 mg/kg, i.p.), also reversed the analgesic effect of OMDM198. OMDM202 action was also antagonized by a per se inactive dose of the selective TRPA1 blocker, AP-18 (0.05 mg/kg, i.p.), but not by a TRPV1 antagonist. AP-18 at higher doses (0.1–0.2 mg/kg) inhibited both the first and second phase of the formalin response. The effects of OMDM198 and OMDM202 were accompanied by elevation of anandamide levels in the spinal cord. OMDM198 (0.1–5.0 mg/kg, i.p.) also reversed carrageenan-induced oedema and thermal hyperalgesia in mice with efficacy similar to that of AA-5-HT. These data suggest that “dual” fatty acid amide hydrolase and transient receptor potential channel modulators should be clinically evaluated as novel analgesics.

N-Palmitoyl-vanillamide (palvanil) is a non-pungent capsaicinoid, found in low amounts in Capsicum and shown to rapidly desensitize transient receptor potential vanilloid type-1 (TRPV1) channels to the action of capsaicin and to exert analgesic effects after local administration. We have investigated here if systemic administration of palvanil to mice causes two typical adverse events of TRPV1 agonists, i.e. profound changes in body temperature and bronchoconstriction, and if it can still produce effective inhibition of inflammatory and chronic pain in different experimental models. Varying doses of palvanil were tested subcutaneously and acutely on body temperature in vivo or, or as a bolus, on bronchopulmunary function ex vivo, in comparison with capsaicin. Intraperitoneal palvanil was also tested against formalin-induced nocifensive behavior and carrageenan-induced oedema and thermal hyperalgesia, acutely, and against mechanical allodynia and thermal hyperalgesia in mice with spared nerve injury (SNI) of the sciatic nerve, after repeated administration over 7 days from SNI. Palvanil, at therapeutically relevant doses, produced significantly less hypothermia and bronchoconstriction than capsaicin. Palvanil (0.5-2.5 mg/kg) abolished formalin-induced nocifensive behavior and strongly attenuated SNI-induced mechanical allodynia and thermal hyperalgesia and carrageenan-induced oedema and thermal hyperalgesia. Systemic administration of the non-pungent capsaicinoid, palvanil, produces, at least in mice, much less of those side effects typical of TRPV1 agonists (hypothermia and bronchoconstriction), whilst being very effective at reducing pain and oedema. Thus, palvanil might be developed further as a novel pharmacological treatment for chronic abnormal pain.

Palmitoylethanolamide (PEA), a peroxisome proliferator-activated receptor-?? (PPAR-??) ligand, exerts antinociceptive and anti-inflammatory effects. PEA (3 and 6 nmol) was microinjected in the ventrolateral periaqueductal grey (VL PAG) of male rats and effects on nociceptive responses and ongoing and tail flick-related activities of rostral ventromedial medulla (RVM) ON and OFF cells were recorded. Intra-PAG microinjection of PEA reduced the ongoing activity of ON and OFF cells and produced an increase in the latency of the nociceptive reaction. These effects were prevented by a selective PPAR-?? antagonist, GW6471 and by a large-conductance Ca(2+)-activated K(+) channel inhibitor, charybdotoxin. Cannabinoid 1 (CB(1)) receptor blockade by AM251 increased the PEA-induced effect both on the ongoing activity of the ON cell and on the latency to tail flick without affecting the effect of PEA on the OFF cell. Conversely, a transient receptor potential vanilloid type 1 (TRPV(1)) blocker, I-RTX, had no effect on the ON cell activity and tail flick latency, whereas it blocked the PEA-induced decrease in ongoing activity of the OFF cell. PEA decreased the burst and increased the latency of tail flick-evoked onset of ON cell activity in a manner antagonised by GW6471 and charybdotoxin. AM251 and I-RTX, instead, enhanced these latter effects. In conclusion, intra-VL PAG PEA induces antinociceptive effects associated with a decrease in RVM ON and OFF cell activities. PPAR-?? receptors mediate, and CB(1) and TRPV(1) receptors antagonise, PEA-induced effects within the PAG-RVM circuitry.

Context: Cannabinoid CB1 receptor blockade decreases weight and hyperinsulinemia in obese animals and humans in a way greatly independent from food intake. Objective: The objective of this study was to investigate the regulation and function of the endocannabinoid system in adipocytes and pancreatic β-cells. Design, Setting, and Patients: Mouse 3T3-F442A adipocytes and rat insulinoma RIN-m5F β-cells, pancreas and fat from mice with diet-induced obesity, visceral and sc fat from patients with body mass index equal to or greater than 30 kg/m2, and serum from normoglycemic and type 2 diabetes patients were studied. Main Outcome Measure: Endocannabinoid enzyme and adipocyte protein expression, and endocannabinoid and insulin levels were measured. Results: Endocannabinoids are present in adipocytes with levels peaking before differentiation, and in RIN-m5F β-cells, where they are under the negative control of insulin. Chronic treatment of adipocytes with insulin is accompanied by permanently elevated endocannabinoid signaling, whereas culturing of RIN-m5F β-cells in high glucose transforms insulin down-regulation of endocannabinoid levels into up-regulation. Epididymal fat and pancreas from mice with diet-induced obesity contain higher endocannabinoid levels than lean mice. Patients with obesity or hyperglycemia caused by type 2 diabetes exhibit higher concentrations of endocannabinoids in visceral fat or serum, respectively, than the corresponding controls. CB1 receptor stimulation increases lipid droplets and decreases adiponectin expression in adipocytes, and it increases intracellular calcium and insulin release in RIN-m5F β-cells kept in high glucose. Conclusions: Peripheral endocannabinoid overactivity might explain why CB1 blockers cause weight-loss independent reduction of lipogenesis, of hypoadiponectinemia, and of hyperinsulinemia in obese animals and humans.

Fish oil (FO) and phytocannabinoids have received considerable attention for their intestinal anti-inflammatory effects. We investigated whether the combination of FO with cannabigerol (CBG) and cannabidiol (CBD) or a combination of all three treatments results in a more pronounced intestinal antiinflammatory action compared to the effects achieved separately. Colitis was induced in mice by 2,4-dinitrobenzenesulfonic acid (DNBS). CBD and CBG levels were detected and quantified by liquid chromatography coupled with time of flight mass spectrometry and ion trap mass spectrometry (LC-MS-IT-TOF). Endocannabinoids and related mediators were assessed by LC-MS. DNBS increased colon weight/colon length ratio, myeloperoxidase activity, interleukin-1β, and intestinal permeability. CBG, but not CBD, given by oral gavage, ameliorated DNBS-induced colonic inflammation. FO pretreatment (at the inactive dose) increased the antiinflammatory action of CBG and rendered oral CBD effective while reducing endocannabinoid levels. Furthermore, the combination of FO, CBD, and a per se inactive dose of CBG resulted in intestinal anti-inflammatory effects. Finally, FO did not alter phytocannabinoid levels in the serum and in the colon. By highlighting the apparent additivity between phytocannabinoids and FO, our preclinical data support a novel strategy of combining these substances for the potential development of a treatment of inflammatory bowel disease.

The endocannabinoid system (i.e., the cannabinoid receptors and their endogenous ligands) plays an important role in the physiological control of intestinal motility. However, its participation in intestinal pathological states is still poorly understood. In the present study, we investigated the possible role of the endocannabinoid system in the pathogenesis of paralytic ileus, a pathological state consisting of decreased intestinal motility following peritonitis, surgery, or other noxious situations. Ileus was induced by i.p. administration of acetic acid, and gastrointestinal propulsion was assessed by the charcoal method. Endocannabinoid levels were measured by isotope-dilution gas chromatography-mass spectrometry, whereas cannabinoid CB1 receptors were identified by immunohistochemistry. Acetic acid administration inhibited gastrointestinal transit (ileus), and this effect was accompanied by increased levels of the endocannabinoid anandamide compared with control mice and by overexpression of CB1 receptors in myenteric nerves. Furthermore, acetic acid-induced ileus was alleviated by the CB1 receptor antagonist SR141716A and worsened by VDM11, a selective inhibitor of anandamide cellular uptake (and hence inactivation). From these findings, it can be concluded that the intestinal hypomotility typical of paralytic ileus is due, at least in part, to the enhancement of anandamide levels and CB1 expression during this condition, and that selective, nonpsychotropic CB1 receptor antagonists could represent new drugs to treat this disorder.

Background and purpose N-acylethanolamine acid amidase (NAAA) is a lysosomal enzyme accountable for the breakdown of N-acylethanolamides (NAEs) and its pharmacological inhibition determines beneficial effects in inflammatory conditions. The knowledge of NAAA in cancer is fragmentary with an unclarified mechanism whereas its contribution to colorectal cancer (CRC) is unknown to date. Experimental approach CRC xenograft and azoxymethane models assessed the in vivo pharmacological effect of NAAA inhibition; tumor secretome was evaluated by an oncogenic array; CRC cell lines were used for in vitro studies; cell cycle was analyzed by cytofluorimetry; NAAA was knocked down with siRNA; human biopsies were obtained from surgically resected CRC patients; gene expression was revealed by RT-PCR; NAEs were measured by LC-MS. Key results The NAAA inhibitor AM9053 reduced CRC xenograft tumor growth and counteracted tumor development in the azoxymethane model. NAAA inhibition impacted the composition of the tumor secretome that negatively affected the expression of epidermal growth factor family members. In CRC cells, AM9053 reduced proliferation with a mechanism mediated by PPAR-α and TRPV1 and induced cell cycle arrest in the S phase with cyclin A2/CDK2 downregulation. NAAA knock-down mirrored the effects of NAAA pharmacological inhibition. NAAA expression was downregulated in human CRC tissues, with a consequential augmentation of NAEs levels and dysregulation of some of their targets. Conclusions and implications Our results provide unprecedented data on the functional importance of NAAA in CRC progression and its mechanism. We propose this enzyme as a valid drug target for the treatment of CRC growth and development.

To address the multifactorial nature of Alzheimer's Disease (AD), a multi-target-directed ligand approach was herein developed. As a follow-up of our previous studies, a small library of newly designed 2-arylbenzofuran derivatives was evaluated towards cholinesterases and cannabinoid receptors. The two most promising compounds, 8 and 10, were then assessed for their neuroprotective activity and for their ability to modulate the microglial phenotype. Compound 8 emerged as able to fight AD from several directions: it restored the cholinergic system by inhibiting butyrylcholinesterase, showed neuroprotective activity against A beta(1-42) oligomers, was a potent and selective CB2 ligand and had immunomodulatory effects, switching microglia from the pro-inflammatory M1 to the neuroprotective M2 phenotype. Derivative 10 was a potent CB2 inverse agonist with promising immunomodulatory properties and could be considered as a tool for investigating the role of CB2 receptors and for developing potential immunomodulating drugs addressing the endocannabinoid system. (C) 2019 Elsevier Masson SAS. All rights reserved.

In experimental colitis, administration of fish oil (FO) and the non-psychoactive cannabinoid, cannabidiol (CBD) reduces inflammation and ameliorates behavioural disturbances, two conditions associated with alterations in the gut microbiota (dysbiosis). We investigated the effect of combined FO/CBD administration on inflammation and dysbiosis in the dextran sulphate sodium (DSS) model of mouse colitis, which also causes behavioural disturbances. Colitis was induced in CD1 mice by 4% w/v DSS in drinking water for five consecutive days followed by normal drinking water. FO (20-75 mg/mouse) was administered once a day starting two days after DSS, whereas CBD (0.3-30 mg/kg), alone or after FO administration, was administered once a day starting three days after DSS, until day 8 (d8) or day 14 (d14). Inflammation was assessed at d8 and d14 (resolution phase) by measuring the Disease Activity Index (DAI) score, change in body weight, colon weight/length ratio, myeloperoxidase activity and colonic interleukin (IL)-1β (IL-1β), IL-10 and IL-6 concentrations. Intestinal permeability was measured with the fluorescein isothiocyanate-dextran. Behavioral tests (novel object recognition (NOR) and light/dark box test) were performed at d8. Fecal microbiota composition was determined by ribosomal 16S DNA sequencing of faecal pellets at d8 and d14. DSS-induced inflammation was stronger at d8 and accompanied by anxiety-like behaviour and impaired recognition memory at d8. FO (35, 50, 75 mg/mouse) alone reduced inflammation at d8, whereas CBD alone produced no effect at any of the doses tested; however, when CBD (3,10 mg/kg) was co-administered with FO (75 mg/mouse) inflammation was attenuated. FO (20 mg/mouse) and CBD (1 mg/kg) were ineffective when given alone, but when co-administered reduced all inflammatory markers and the impaired intestinal permeability at both d8 and d14 (but not the behavioural impairments). FO, CBD and their combination affected gut bacteria taxa that were not affected by DSS per se. Akkermansia muciniphila, a species suggested to afford anti-inflammatory action in colitis, was increased by DSS only at d14, but its levels were significantly elevated by all treatments at d8. FO and CBD co-administered at ineffective doses reduce colon inflammation, in a manner potentially strengthened by their independent elevation of Akkermansia muciniphila at d8.

Orexin-A (OX-A) protects the brain against oxidative stress-mediated ischemic injury. Since the endocannabinoid 2-arachidonoylglycerol (2-AG) and cannabinoid type-1 (CB1) receptors were previously shown to mediate some of the effects of OX-A exerted through the orexin-1 receptor (OX-1R), we investigated the involvement of 2-AG in OX-A-induced neuroprotection following oxygen and glucose deprivation (OGD) in mouse cortical neurons. OGD-induced reactive oxygen species (ROS) accumulation and neuronal death were prevented by both OX-A and arachidonyl-2&prime, chloroethylamide (ACEA), a synthetic CB1 receptor agonist, in a manner sensitive to OX-1R and CB1 receptor antagonists, SB334867 and AM251. OX-A stimulated 2-AG biosynthesis in cortical neurons. In neurons isolated from monoacylglycerol lipase (MAGL, a 2-AG hydrolyzing enzyme) null mice, 10-fold higher 2-AG concentrations were found and OGD failed to induce ROS production and cell death, whereas AM251 restored these noxious effects. OX-A-induced neuroprotection was mediated by the phosphoinositide-3-kinase/Akt (PI3K/Akt) survival pathway since both OX-A and ACEA induced phosphorylation of Akt and prevented OGD-induced cytochrome c release from the mitochondria, in a manner counteracted by SB334867 or AM251. Administration of OX-A reduced infarct volume and elevated brain 2-AG levels in a mouse model of transient ischemia. These results suggest that 2-AG and CB1 receptor mediate OX-A prevention of ischemia-induced neuronal apoptosis.

Traumatic brain injury (TBI) represents an important public health problem and is followed by neuroinflammation and neurological dysfunctions. It has been suggested that brain trauma is often associated to deep behavioral alterations and chronic pain-like syndrome. Despite inducing minimal brain damage, mild TBI (mTBI) leads to persistent behavioral changes, including anxiety, depression, social interaction impairment, and aggressiveness. The clinical management of these symptoms is still unsatisfactory and new pharmacological treatments are needed, especially for the aggressiveness and depression. In a mouse model of mTBI, we investigated the effect of 2-Pentadecyl-2-Oxazoline (PEA-OXA), a natural compound, that is a secondary metabolite, found in green and roasted coffee beans, on both the pain perception, and neuropsychiatric dysfunctions. We found that the compound acts as a α2 adrenergic antagonist and this mechanism is here described for the first time. Mild TBI mice, starting from 14-d post-trauma, developed anxious and aggressive behavior, whilst depressive-like behavior and impaired social interactions were observed from the 60th d onward. PEA-OXA normalized all the behavioral changes investigated. We also investigated the memory impairments through Morris Water Maze (MWM) test. Both sham and mTBI mice treated with PEA-OXA showed amelioration in the reversal task of the MWM. Nevertheless, the main symptom of the long-term mTBI is represented by the depressive-like behavior, which was completely reversed by PEA-OXA repeated administration. In humans, mTBI-induced depression precedes the appearance of dementias and is characterized by a massive deficit of GABAergic transmission in the cortices. We found that PEA-OXA normalized the GABA changes in the prefrontal cortex. In order to prove the α2-mediated effect of the PEA-OXA we have performed open field test in naïve animals by microinjecting into the medial prefrontal cortex the dexomedetomidine, a selective α2 agonist with or without PEA-OXA co-injection. We found that PEA-OXA antagonized the α2 agonist effect on the locomotor activity. Moreover, PEA-OXA microinjection into the medial prefrontal cortex induced an enhancement of dopamine release. Collectively, these data suggest that this natural compound, through its multi-target activity is able to: i) ameliorate behavioral alterations (i.e. depression), ii) selectively normalize cortical GABA levels, iii) rescue the impaired neuronal activity in the prefrontal cortex.

The discovery of extended catalytic versatilities is of great importance in both the chemistry and biotechnology fields. Fatty acid amide hydrolase (FAAH) belongs to the amidase signature superfamily and is a major endocannabinoid inactivating enzyme using an atypical catalytic mechanism involving hydrolysis of amide and occasionally ester bonds. FAAH inhibitors are efficacious in experimental models of neuropathic pain, inflammation, and anxiety, among others. We report a new multitarget drug, AGN220653, containing a carboxyamide-4-oxazole moiety and endowed with efficacious analgesic and anti-inflammatory activities, which are partly due to its capability of achieving inhibition of FAAH, and subsequently increasing the tissue concentrations of the endocannabinoid anandamide. This inhibitor behaves as a noncompetitive, slowly reversible inhibitor. Autoradiography of purified FAAH incubated with AGN220653, opportunely radiolabeled, indicated covalent binding followed by fragmentation of the molecule. Molecular docking suggested a possible nucleophilic attack by FAAH-Ser241 on the carbonyl group of the carboxyamide-4-oxazole moiety, resulting in the cleavage of the C-C bond between the oxazole and the carboxyamide moieties, instead of either of the two available amide bonds. MRM-MS analyses only detected the Ser241-assisted formation of the carbamate intermediate, thus confirming the cleavage of the aforementioned C-C bond. Quantum mechanics calculations were fully consistent with this mechanism. The study exemplifies how FAAH structural features and mechanism of action may override the binding and reactivity propensities of substrates. This unpredicted mechanism could pave the way to the future development of a completely new class of amidase inhibitors, of potential use against pain, inflammation, and mood disorders.

Onco-nephrology is an emerging field in medicine. Patients with cancer may suffer from kidney diseases because of the cancer itself and cancer-related therapy. It is critical for nephrologists to be knowledgeable of cancer biology and therapy in order to be fully integrated in the multidisciplinary team and optimally manage patients with cancer and kidney diseases. In a recent international meeting, the key issues in this challenging clinical interface were addressed, including many unresolved basic science questions, such as the high tumor incidence in kidney transplant recipients. To this end, 70 highly qualified faculty members were gathered from all over the world to discuss these issues in 8 plenary sessions, including 5 keynote lectures. In addition, 48 young nephrologists and oncologists were invited to present their original observations that were highlighted in 2 large poster sessions.

Patients with ulcerative colitis (UC) using marijuana have been reported to experience symptomatic benefit. Cannabidivarin (CBDV) is a safe non-psychoactive phytocannabinoid able to activate and desensitize TRPA1, a member of the TRP channels superfamily, which plays a pivotal role in intestinal inflammation. Here, we have investigated the potential intestinal anti-inflammatory effect of CBDV in mice and in biopsies from pediatric patients with active UC. Colonic inflammation was induced in mice by dinitrobenzenesulfonic acid (DNBS). The effect of orally administered CBDV on macroscopic and microscopic damage, inflammatory parameters (i.e. myeloperoxidase activity, intestinal permeability and cytokine production) and faecal microbiota composition, was evaluated 3 days after DNBS administration. TRPA1 expression was studied by RT-PCR in inflamed colons of mice as well as in mucosal colonic biopsies of children with active UC, whose response to incubation with CBDV was also investigated. CBDV attenuates, in a TRPA1-antagonist sensitive manner, DNBS-induced signs of inflammation including neutrophil infiltration, intestinal permeability, and cytokine (i.e. IL-1β, IL-6 and the chemokine MCP-1) production. CBDV also alters the dysregulation of gut microbiota associated to colitis. Finally, CBDV lessens cytokine expression in colonic biopsies from pediatric patients with ulcerative colitis, a condition in which TRPA1 was up-regulated. Our preclinical study shows that CBDV exerts intestinal anti-inflammatory effects in mice via TRPA1, and in children with active UC. Since CBDV has a favorable safety profile in humans, it may be considered for possible clinical trials in patients with UC.

Cigarette smokers with brain damage involving the insular cortex display cessation of tobacco smoking, suggesting that this region may contribute to nicotine addiction. In the present study, we speculated that molecules in the insular cortex that are sensitive to experimental traumatic brain injury (TBI) in mice might provide leads to ameliorate nicotine addiction. Using targeted lipidomics, we found that TBI elicited substantial increases of a largely uncharacterized lipid, N-acyl-glycine, N-oleoyl-glycine (OlGly), in the insular cortex of mice. We then evaluated whether intraperitoneal administration of OlGly would alter withdrawal responses in nicotine-dependent mice as well as the rewarding effects of nicotine, as assessed in the conditioned place preference paradigm (CPP). Systemic administration of OlGly reduced mecamylamine-precipitated withdrawal responses in nicotine-dependent mice and prevented nicotine CPP. However, OlGly did not affect morphine CPP, demonstrating a degree of selectivity. Our respective in vitro and in vivo observations that OlGly activated peroxisome proliferator-activated receptor alpha (PPAR-α) and the PPAR-α antagonist GW6471 prevented the OlGly-induced reduction of nicotine CPP in mice suggests that this lipid acts as a functional PPAR-α agonist to attenuate nicotine reward. These findings raise the possibility that the long chain fatty acid amide OlGly may possess efficacy in treating nicotine addiction.

Toll-like receptors (TLRs) are key receptors through which infectious and non-infectious challenges act with consequent activation of the inflammatory cascade that plays a critical function in various acute and chronic diseases, behaving as amplification and chronicization factors of the inflammatory response. Previous studies have shown that synthetic analogues of lipid A based on glucosamine with few chains of unsaturated and saturated fatty acids, bind MD-2 and inhibit TLR4 receptors. These synthetic compounds showed antagonistic activity against TLR4 activation in vitro by LPS, but little or no activity in vivo. This study aimed to show the potential use of N-palmitoyl-D-glucosamine (PGA), a bacterial molecule with structural similarity to the lipid A component of LPS, which could be useful for preventing LPS-induced tissue damage or even peripheral neuropathies. Molecular docking and molecular dynamics simulations showed that PGA stably binds MD-2 with a MD-2/(PGA)3 stoichiometry. Treatment with PGA resulted in the following effects: (i) it prevented the NF-kB activation in LPS stimulated RAW264.7 cells, (ii) it decreased LPS-induced keratitis and corneal pro-inflammatory cytokines, whilst increasing anti-inflammatory cytokines, (iii) it normalized LPS-induced miR-20a-5p and miR-106a-5p upregulation and increased miR-27a-3p levels in the inflamed corneas, (iv) it decreased allodynia in peripheral neuropathy induced by oxaliplatin or formalin, but not following spared nerve injury of the sciatic nerve (SNI), (v) it prevented the formalin- or oxaliplatin-induced myelino-axonal degeneration of sciatic nerve. SIGNIFICANCE STATEMENT We report that PGA acts as a TLR4 antagonist and this may be the basis of its potent anti-inflammatory activity. Being unique because of its potency and stability, as compared to other similar congeners, PGA can represent a tool for the optimization of new TLR4 modulating drugs directed against the cytokine storm and the chronization of inflammation.

Purpose PEA is an endogenous mediator released together with the endocannabinoid anandamide from membrane phospholipids. It is a plant derived compound with analgesic and anti-inflammatory properties. We verified whether the pathophysiology of experimental cystitis involves changes in the levels of PEA and of some of its targets, ie CB1 and CB2 receptors, and PPAR?. We also determined whether exogenously administered PEA could be proposed as a preventive measure for cystitis. Materials and Methods Cystitis was induced by cyclophosphamide in female rats. Nociceptive responses, voiding episodes, gross damage, myeloperoxidase activity, bladder weight, bladder PEA and endocannabinoid levels (measured by liquid chromatography-mass spectrometry) and the expression of PEA targets (measured by quantitative reverse transcriptase-polymerase chain reaction) were recorded. Results Cyclophosphamide induced pain behavior, bladder inflammation and voiding dysfunction associated with increased bladder levels of PEA, up-regulation of CB1 receptor mRNA expression, down-regulation of PPAR? mRNA and no change in CB2 receptor mRNA expression. Exogenously administered, ultramicronized PEA attenuated pain behavior, voids and bladder gross damage. The CB1 antagonist rimonabant and the PPAR? antagonist GW6471 counteracted the beneficial effect of PEA on gross damage. Also, GW6471 further decreased voiding episodes in rats treated with PEA. Conclusions The current study provides strong evidence for a protective role of PEA as well as an alteration in bladder levels of PEA and of some of its targets in cyclophosphamide induced cystitis.

The CB2 receptor is a therapeutic target of increasing importance for several diseases, including pain, inflammation, neurodegeneration, cancer and osteoporosis. While several compounds showing CB2-selective agonist or inverse agonist properties have been developed, only few CB2 receptor selective neutral antagonists are actually known. Such type of compounds could be useful to study more in depth the role of the CB2 receptor, because they lack the ability to counteract its “constitutive” activity. Here we describe the synthesis and biological activity of a series of biphenylic carboxamides as a new class of CB2 receptor selective ligands. In binding assays, one of these compounds showed good CB2 receptor affinity and selectivity (Ki = 11.48 nM; Selectivity Index = 130). Furthermore, in functional assays, the same compound showed a very interesting pharmacological profile as CB2 receptor selective neutral antagonist. These results pave the way to further developments, including structural optimization, with the aim to obtain more potent CB2 receptor ligands with this peculiar feature.

The endogenous fatty acid amide palmitoylethanolamide (PEA) has been shown to exert anti-inflammatory actions mainly through inhibition of the release of pro-inflammatory molecules from mast cells, monocytes and macrophages. Indirect activation of the endocannabinoid (eCB) system is among the several mechanisms of action that have been proposed to underlie the different effects of PEA in vivo. In this study, we used cultured rat microglia and human macrophages to evaluate whether PEA affects eCB signaling. PEA was found to increase CB2 mRNA and protein expression through peroxisome proliferator-activated receptor-α (PPAR-α) activation. This novel gene regulation mechanism was demonstrated through: (i) pharmacological PPAR-α manipulation, (ii) PPAR-α mRNA silencing, (iii) chromatin immunoprecipitation. Moreover, exposure to PEA induced morphological changes associated with a reactive microglial phenotype, including increased phagocytosis and migratory activity. Our findings suggest indirect regulation of microglial CB2R expression as a new possible mechanism underlying the effects of PEA. PEA can be explored as a useful tool for preventing/treating the symptoms associated with neuroinflammation in CNS disorders.

Background: Intestinal immune activation is involved in irritable bowel syndrome (IBS) pathophysiology. While most dietary approaches in IBS involve food avoidance, there are fewer indications on food supplementation. Palmithoylethanolamide, structurally related to the endocannabinoid anandamide, and polydatin are dietary compounds which act synergistically to reduce mast cell activation. Aim: To assess the effect on mast cell count and the efficacy of palmithoylethanolamide/polydatin in patients with IBS. Methods: We conducted a pilot, 12-week, randomised, double-blind, placebo-controlled, multicentre study assessing the effect of palmithoylethanolamide/polydatin 200mg/20mg or placebo b.d. on low-grade immune activation, endocannabinoid system and symptoms in IBS patients. Biopsy samples, obtained at screening visit and at the end of the study, were analysed by immunohistochemistry, enzyme-linked immunoassay, liquid chromatography and Western blot. Results: A total of 54 patients with IBS and 12 healthy controls were enrolled from five European centres. Compared with controls, IBS patients showed higher mucosal mast cell counts (3.2±1.3 vs. 5.3±2.7%, P=0.013), reduced fatty acid amide oleoylethanolamide (12.7±9.8 vs. 45.8±55.6 pmol/mg, P=0.002) and increased expression of cannabinoid receptor 2 (0.7±0.1 vs. 1.0±0.8, P=0.012). The treatment did not significantly modify IBS biological profile, including mast cell count. Compared with placebo, palmithoylethanolamide/polydatin markedly improved abdominal pain severity (P&nbsp

N-palmitoylethanolamine (PEA), an endogenous fatty acid ethanolamide, plays a key role in the regulation of the inflammatory response and pain through, among others, activation of nuclear peroxisome proliferator-activated receptors (PPAR-α). Endogenous cannabinoids play a protective role in several central nervous system (CNS) disorders, particularly those associated with neuronal hyperexcitability. We investigated the effects of PEA and the role of PPAR-α in absence epilepsy using the WAG/Rij rat model. PEA, anandamide (AEA), a PPAR-α antagonist (GW6471) and a synthetic CB1 receptor antagonist/inverse agonist (SR141716) were administered to WAG/Rij rats in order to evaluate the effects on epileptic spike-wave discharges (SWDs) on EEG recordings. We studied also the effects of PEA co-administration with SR141716 and GW6471 and compared these effects with those of AEA to evaluate PEA mechanism of action and focusing on CB1 receptors and PPAR-α. Both PEA and AEA administration significantly decreased SWDs parameters (absence seizures). In contrast, GW6471 was devoid of effects while SR141716 had pro-absence effects. The co-administration of SR141716 with PEA or AEA completely blocked the anti-absence effects of these compounds. GW6471 antagonized PEA's effects whereas it did not modify AEA's effects. Furthermore, we have also measured PEA, AEA and 2-AG (2-arachidonoylglycerol) brain levels identifying significant differences between epileptic and control rats such as decreased PEA levels in both thalamus and cortex that might contribute to absence epilepsy. Our data demonstrate that PEA has anti-absence properties in the WAG/Rij rat model and that such properties depend on PPAR-α and indirect activation of CB1 receptors. This article is part of the Special Issue entitled 'New Targets and Approaches to the Treatment of Epilepsy'.

Inflammatory bowel disease (IBD) is an incurable disease which affects millions of people in industrialised countries. Anecdotal and scientific evidence suggest that Cannabis use may have a positive impact in IBD patients. Here, we investigated the effect of cannabigerol (CBG), a non-psychotropic Cannabis-derived cannabinoid, in a murine model of colitis. Colitis was induced in mice by intracolonic administration of dinitrobenzene sulphonic acid (DNBS). Inflammation was assessed by evaluating inflammatory markers/parameters (colon weight/colon length ratio and myeloperoxidase activity), by histological analysis and immunohistochemistry; interleukin-1?, interleukin-10 and interferon-? levels by ELISA, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) by western blot and RT-PCR; CuZn-superoxide dismutase (SOD) activity by a colorimetric assay. Murine macrophages and intestinal epithelial cells were used to evaluate the effect of CBG on nitric oxide production and oxidative stress, respectively. CBG reduced colon weight/colon length ratio, myeloperoxidase activity, and iNOS expression, increased SOD activity and normalized interleukin-1?, interleukin-10 and interferon-? changes associated to DNBS administration. In macrophages, CBG reduced nitric oxide production and iNOS protein (but not mRNA) expression. Rimonabant (a CB1 receptor antagonist) did not change the effect of CBG on nitric oxide production, while SR144528 (a CB2 receptor antagonist) further increased the inhibitory effect of CBG on nitric oxide production. In conclusion, CBG attenuated murine colitis, reduced nitric oxide production in macrophages (effect being modulated by the CB2 receptor) and reduced ROS formation in intestinal epithelial cells. CBG could be considered for clinical experimentation in IBD patients. Graphical abstract

4-(Thiophen-2-yl)butanoic acid was identified as a cyclic substitute of the unsaturated alkyl chain of the natural ligand, capsaicin. Accordingly, a new class of amides was synthesized in good yield and high purity and their molecular recognition against the target was investigated by means of docking experiments followed by molecular dynamics simulations, in order to rationalize their geometrical and thermodynamic profiles. The pharmacological properties of these new compounds were expressed as activation (EC50) and desensitization (IC50) potencies. Several compounds were found to activate TRPV1 channels, and in particular, derivatives 1 and 10 behaved as TRPV1 agonists endowed with good efficacy as compared to capsaicin. The most promising compound 1 was also evaluated for its protective role against oxidative stress on keratinocytes and differentiated human neuroblastoma cell lines expressing the TRPV1 receptor as well as for its cytotoxicity and analgesic activity in vivo.

Pain perception can become altered in individuals with eating disorders and obesity for reasons that have not been fully elucidated. We show that leptin deficiency in ob/ob mice, or leptin insensitivity in the arcuate nucleus of the hypothalamus in mice with high-fat diet (HFD)-induced obesity, are accompanied by elevated orexin-A (OX-A) levels and orexin receptor-1 (OX1-R)-dependent elevation of the levels of the endocannabinoid, 2-arachidonoylglycerol (2-AG), in the ventrolateral periaqueductal gray (vlPAG). In ob/ob mice, these alterations result in the following: (i) increased excitability of OX1-R-expressing vlPAG output neurons and subsequent increased OFF and decreased ON cell activity in the rostral ventromedial medulla, as assessed by patch clamp and in vivo electrophysiology; and (ii) analgesia, in both healthy and neuropathic mice. In HFD mice, instead, analgesia is only unmasked following leptin receptor antagonism. We propose that OX-A/endocannabinoid cross talk in the descending antinociceptive pathway might partly underlie increased pain thresholds in conditions associated with impaired leptin signaling.Neuropsychopharmacology advance online publication, 15 July 2015; doi:10.1038/npp.2015.173.

Eleven compounds belonging to the chalcone family were tested for their ability to activate and subsequently desensitize the rat transient receptor potential ankyrin 1 cation channel, subfamily A, member 1 (TRPA1) in a heterologous expression system. Four of the tested compounds were more potent than the TRPA1 agonist mustard oil, and showed also a strong desensitizing effect. Some chalcone compounds were not pungent in the eye-wiping assay and quite remarkably inhibited in a long-lasting and dose-dependent manner the pain response in the formalin test. Chalcones can be considered as novel candidates for the development of antihyperalgesic preparations based on TRPA1 desensitization.

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Journal ofMedicinal Chemistry, 59, 2612–2632 (DOI: 10.1021/acs.jmedchem.5b01812), © 2016 American ChemicalSociety, after peer review and technical editing by the publisher. To access the final edited and published work,see http://pubsdc3.acs.org/articlesonrequest/AOR-teeM6WpzZVZ52Aqz9AMW This Manuscript version is made available under the CC-BY-NC-ND 4.0license.https://creativecommons.org/licenses/by-nc-nd/4.0/ ABSTRACT We report the discovery of compound4a, a potent β-lactam-based monoacylglycerol lipase (MGL) inhibitor characterized by an irreversible and stereoselective mechanism of action, high membrane permeability, high brain penetration evaluated using a human in vitro blood–brain barrier model, high selectivity in binding and affinity-based proteomic profiling assays, and low in vitro toxicity. Mode-of-action studies demonstrate that4a, by blocking MGL, increases 2-arachidonoylglycerol and behaves as a cannabinoid (CB1/CB2) receptor indirect agonist. Administration of4ain mice suffering from experimental autoimmune encephalitis ameliorates the severity of the clinical symptoms in a CB1/CB2-dependent manner. Moreover,4aproduced analgesic effects in a rodent model of acute inflammatory pain, which was antagonized by CB1 and CB2 receptor antagonists/inverse agonists.4aalso relieves the neuropathic hypersensitivity induced by oxaliplatin. Given these evidence,4a, as MGL selective inhibitor, could represent a valuable lead for the future development of therapeutic options for multiple sclerosis and chronic pain.

Purpose: Hedonic eating occurs independently from homeostatic needs prompting the ingestion of pleasurable foods that are typically rich in fat, sugar and/or salt content. In normal weight healthy subjects, we found that before hedonic eating, plasma levels of 2-arachidonoylglycerol (2-AG) were higher than before nonhedonic eating, and although they progressively decreased after food ingestion in both eating conditions, they were significantly higher in hedonic eating. Plasma levels of anandamide (AEA), oleoylethanolamide (OEA) and palmitoylethanolamide (PEA), instead, progressively decreased in both eating conditions without significant differences. In this study, we investigated the responses of AEA, 2-AG, OEA and PEA to hedonic eating in obese individuals. Methods: Peripheral levels of AEA, 2-AG, OEA and PEA were measured in 14 obese patients after eating favourite (hedonic eating) and non-favourite (nonhedonic eating) foods in conditions of no homeostatic needs. Results: Plasma levels of 2-AG increased after eating the favourite food, whereas they decreased after eating the non-favourite food, with the production of the endocannabinoid being significantly enhanced in hedonic eating. Plasma levels of AEA decreased progressively in nonhedonic eating, whereas they showed a decrease after the exposure to the favourite food followed by a return to baseline values after eating it. No significant differences emerged in plasma OEA and PEA responses to favourite and non-favourite food. Conclusion: Present findings compared with those obtained in our previously studied normal weight healthy subjects suggest deranged responses of endocannabinoids to food-related reward in obesity.

We synthesized new N-phenylethyl-1H-indole-2-carboxamides as the first SAR study of allosteric modulators of the CB 1 receptor. The presence of the carboxamide functionality was required in order to obtain a stimulatory effect. The maximum stimulatory activity on CB 1 was exerted by carboxamides 13 (EC 50 = 50 nM) and 21 (EC 50 = 90 nM) bearing a dimethylamino or piperidinyl group, respectively, at position 4 of the phenethyl moiety and a chlorine atom at position 5 of the indole.

New 1-aryl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamides were synthesized as cannabinoid (CB) receptor ligands. Compound 11 (CB(1)K(i) = 2.3 nM, CB(1) SI = 163.6) showed CB(1) receptor affinity and selectivity superior to Rimonabant and AM251. Acute administration of 2mg/kg 11 reduced sucrose, but not regular food, intake in rats. On the other hand, compound 23 (CB(2)K(i) = 0.51 nM, CB(2) SI = 30.0) showed significant affinity and selectivity for the CB(2) receptor. The results presented here show that the 1-aryl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamide may serve as an effective scaffold for the design of either CB(1) or CB(2) receptor ligands.

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.

Background & Aims: Cholera toxin (CT) is the most recognizable enterotoxin causing secretory diarrhea, a major cause of infant morbidity and mortality throughout the world. In this study, we investigated the role of the endogenous cannabinoid system (i.e., the cannabinoid receptors and their endogenous ligands) in CT-induced fluid accumulation in the mouse small intestine. Methods: Fluid accumulation was evaluated by enteropooling; endocannabinoid levels were measured by isotope-dilution gas chromatography mass spectrometry; CB 1 receptors were localized by immunohistochemistry and their messenger RNA (mRNA) levels were quantified by reverse-transcription polymerase chain reaction (PCR). Results: Oral administration of CT to mice resulted in an increase in fluid accumulation in the small intestine and in increased levels of the endogenous cannabinoid, anandamide, and increased expression of the cannabinoid CB 1 receptor mRNA. The cannabinoid receptor agonist CP55,940 and the selective cannabinoid CB 1 receptor agonist arachidonoyl-chloro-ethanolamide inhibited CT-induced fluid accumulation, and this effect was counteracted by the CB 1 receptor antagonist SR141716A, but not by the CB 2 receptor antagonist SR144528. SR141716A, per se, but not the vanilloid VR1 receptor antagonist capsazepine, enhanced fluid accumulation induced by CT, whereas the selective inhibitor of anandamide cellular uptake, VDM11, prevented CT-induced fluid accumulation. Conclusions: These results indicate that CT, along with enhanced intestinal secretion, causes overstimulation of endocannabinoid signaling with an antisecretory role in the small intestine.

BackgroundEndocannabinoid anandamide (AEA) inhibits intestinal motility and visceral pain, but it may also be proalgesic through transient receptor potential vanilloid-1 (TRPV1). AEA is degraded by fatty acid amide hydrolase (FAAH). This study explored whether dual inhibition of FAAH and TRPV1 reduces diarrhea and abdominal pain. MethodsImmunostaining was performed on myenteric plexus of the mouse colon. The effects of the dual FAAH/TRPV1 inhibitor AA-5-HT on electrically induced contractility, excitatory junction potential (EJP) and fast (f) and slow (s) inhibitory junction potentials (IJP) in the mouse colon, colonic propulsion and visceromotor response (VMR) to rectal distension were studied. The colonic levels of endocannabinoids and fatty acid amides were measured. Key ResultsCB1-positive neurons exhibited TRPV1; only some TRPV1 positive neurons did not express CB1. CB1 and FAAH did not colocalize. AA-5-HT (100nM-10M) decreased colonic contractility by similar to 60%; this effect was abolished by TRPV1 antagonist 5-IRTX, but not by CB1 antagonist, SR141716. AA-5-HT (1M-10M) inhibited EJP by similar to 30% and IJPs by similar to 50%. The effects of AA-5-HT on junction potentials were reversed by SR141716 and 5`-IRTX. AA-5-HT (20mg/kg; i.p.) inhibited colonic propulsion by similar to 30%; SR141716 but not 5`-IRTX reversed this effect. AA-5-HT decreased VMR by similar to 50%-60%; these effects were not blocked by SR141716 or 5`-IRTX. AA-5-HT increased AEA in the colon. Conclusions and InferencesThe effects of AA-5-HT on visceral sensation and colonic motility are differentially mediated by CB1, TRPV1 and non-CB1/TRPV1 mechanisms, possibly reflecting the distinct neuromodulatory roles of endocannabinoid and endovanilloid FAAH substrates in the mouse intestine.

Allergen exposure may induce changes in the brainstem secondary neurons, with neural sensitization of the nucleus solitary tract (NTS), which in turn can be considered one of the causes of the airway hyperresponsiveness, a characteristic feature of asthma. We evaluated neurofunctional, morphological, and biochemical changes in the NTS of naive or sensitized rats. To evaluate the cell firing activity of NTS, in vivo electrophysiological experiments were performed before and after capsaicin challenge in sensitized or naive rats. Immunohistochemical studies, endocannabinoid, and palmitoylethanolamide quantification in the NTS were also performed. This study provides evidence that allergen sensitization in the NTS induced: (1) increase in the neural firing response to intratracheal capsaicin application, (2) increase of endocannabinoid anandamide and palmitoylethanolamide, a reduction of 2-arachidonoylglycerol levels in the NTS, (3) glial cell activation, and (4) prevention by a Group III metabotropic glutamate receptor activation of neural firing response to intratracheal application of capsaicin in both naïve and sensitized rats. Therefore, normalization of ovalbumin-induced NTS neural sensitization could open up the prospect of new treatments based on the recovery of specific brain nuclei function and for extensive studies on acute or long-term efficacy of selective mGlu ligand, in models of bronchial hyperreactivity. © 2015 Giuseppe Spaziano et al.

In our ongoing program aimed at deeply investigating the endocannabinoid system (ES), a set of new alkyl-resorcinol derivatives was prepared focusing on the nature and the importance of the carboxamide functionality. Binding studies on CB 1 and CB 2 receptors, monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH) showed that some of the newly developed compounds behaved as very potent cannabinoid receptor ligands ( K i in the nanomolar range) while, however, none of them was able to inhibit MAGL and/or FAAH. Derivative 11 was a potent CB 1 and CB 2 ligand, with K i values similar to WIN 55,212, exhibiting a CB 1 and CB 2 agonist profile in vitro. In the formalin test of peripheral acute and inflammatory pain in mice, this compound showed a weak and delayed antinociceptive effect against the second phase of the nocifensive response, exhibiting, interestingly, a quite potent transient receptor potential ankyrin type-1 (TRPA1) channel agonist activity. Moreover, derivative 14 , characterized by lower affinity but higher CB 2 selectivity than 11 , proved to behave as a weak CB 2 competitive inverse agonist.

Little is known of the involvement of endocannabinoids and cannabinoid receptors in skeletal muscle cell differentiation. We report that, due to changes in the expression of genes involved in its metabolism, the levels of the endocannabinoid 2-arachidonoylglycerol (2-AG) are decreased both during myotube formation in vitro from murine C2C12 myoblasts and during mouse muscle growth in vivo. The endocannabinoid, as well as the CB1 agonist arachidonoyl-2-chloroethylamide, prevent myotube formation in a manner antagonized by CB1 knockdown and by CB1 antagonists, which, per se, instead stimulate differentiation. Importantly, 2-AG also inhibits differentiation of primary human satellite cells. Muscle fascicles from CB1 knockout embryos contain more muscle fibers, and postnatal mice show muscle fibers of an increased diameter relative to wild-type littermates. Inhibition of Kv7.4 channel activity, which plays a permissive role in myogenesis and depends on phosphatidylinositol 4,5-bisphosphate (PIP2), underlies the effects of 2-AG. We find that CB1 stimulation reduces both total and Kv7.4-bound PIP2 levels in C2C12 cells and inhibits Kv7.4 currents in transfected CHO cells. We suggest that 2-AG is an endogenous repressor of myoblast differentiation via CB1-mediated inhibition of Kv7.4 channels.

Although originally described as a signalling system encompassing the cannabinoid CB1 and CB2 receptors, their endogenous agonists (the endocannabinoids), and metabolic enzymes regulating the levels of such agonists, the endocannabinoid system is now viewed as being more complex, and including metabolically related endocannabinoid-like mediators and their molecular targets as well. The function and dysfunction of this complex signalling system in the molecular and cellular mechanisms of pain transduction and control has been widely studied over the last two decades. In this review article, we describe some of the latest advances in our knowledge on the role of the endocannabinoid system, in its most recent and wider conception, in pain pathways, by focusing on: (1) neuron-glia interactions; and (2) emerging data on endocannabinoid cross-talk with neurotrophins, such as nerve growth factor and brain-derived neurotrophic factor.

The relevance of CB2R-mediated therapeutic effects is well-known for the treatment of inflammatory and neuropathic pain and neurodegenerative disorders. In our search for new cannabinoid receptor modulators, we report the optimization of a series of 1,2-dihydro-2-oxopyridine-3-carboxamide derivatives as CB2R ligands. In particular, N-cycloheptyl-5-(4-methoxyphenyl)-1-(4- fluorobenzyl)-pyridin-2(1H)-on-3-carboxamide (17) showed high CB2R affinity (Ki = 1.0 nM), accompanied by interesting Ki(CB1R)/K i(CB2R) selectivity ratio (SI = 43.4). Compound 17 was also identified as a potent CB2R neutral antagonist/weak partial inverse agonist. Finally we found that the functionality activity of the series of 1,2-dihydro-2-oxopyridine is controlled by the presence of a substituent in position 5 of the heterocyclic nucleus. In fact when the hydrogen atom in position 5 of the unsubstituted compound 1 was replaced with a phenyl group (compound 18) the CB2R activity was shifted from agonism to inverse agonism whereas the introduction in the same position of p-methoxyphenyl group lead to compound 17 which showed a behavior as CB2R neutral antagonist/weak partial inverse agonist. © 2013 Elsevier Masson SAS. All rights reserved.

Lipid mediators variedly affect adipocyte differentiation. Anandamide stimulates adipogenesis via CB1 receptors and peroxisome proliferator-activated receptor γ. Anandamide may be converted by PTGS2 (COX2) and prostaglandin F synthases, such as prostamide/prostaglandin F synthase, to prostaglandin F2α ethanolamide (PGF2αEA), of which bimatoprost is a potent synthetic analog. PGF2αEA/bimatoprost act via prostaglandin F2αFP receptor/FP alt4 splicing variant heterodimers. We investigated whether prostamide signaling occurs in preadipocytes and controls adipogenesis. Exposure of mouse 3T3-L1 or human preadipocytes to PGF2αEA/bimatoprost during early differentiation inhibits adipogenesis. PGF2αEA is produced from anandamide in preadipocytes and much less so in differentiating adipocytes, which express much less PTGS2, FP, and its alt4 splicing variant. Selective antagonism of PGF2αEA receptors counteracts prostamide effects on adipogenesis, as does inhibition of ERK1/2 phosphorylation. Selective inhibition of PGF2αEA versus prostaglandin F2α biosynthesis accelerates adipogenesis. PGF2αEA levels are reduced in the white adipose tissue of high fat diet-fed mice where there is a high requirement for new adipocytes. Prostamides also inhibit zebrafish larval adipogenesis in vivo. We propose that prostamide signaling in preadipocytes is a novel anandamide-derived antiadipogenic mechanism.