A cannabigerol-rich Cannabis sativa extract, devoid of [INCREMENT]9-tetrahydrocannabinol, elicits hyperphagia in rats.

Nonpsychoactive phytocannabinoids (pCBs) from Cannabis sativa may represent novel therapeutic options for cachexia because of their pleiotropic pharmacological activities, including appetite stimulation. We have recently shown that purified cannabigerol (CBG) is a novel appetite stimulant in rats. As standardized extracts from Cannabis chemotypes dominant in one pCB [botanical drug substances (BDSs)] often show greater efficacy and/or potency than purified pCBs, we investigated the effects of a CBG-rich BDS, devoid of psychoactive [INCREMENT]-tetrahydrocannabinol, on feeding behaviour. Following a 2 h prefeed satiation procedure, 16 male Lister-hooded rats were administered CBG-BDS (at 30-240 mg/kg) or vehicle. Food intake, meal pattern microstructure and locomotor activity were recorded over 2 h. The total food intake was increased by 120 and 240 mg/kg CBG-BDS (1.53 and 1.36 g, respectively, vs. 0.56 g in vehicle-treated animals). Latency to feeding onset was dose dependently decreased at all doses, and 120 and 240 mg/kg doses increased both the number of meals consumed and the cumulative size of the first two meals. No significant effect was observed on ambulatory activity or rearing behaviour. CBG-BDS is a novel appetite stimulant, which may have greater potency than purified CBG, despite the absence of [INCREMENT]-tetrahydrocannabinol in the extract.

Cannabigerol is a novel, well-tolerated appetite stimulant in pre-satiated rats.

RATIONALE: The appetite-stimulating properties of cannabis are well documented and have been predominantly attributed to the hyperphagic activity of the psychoactive phytocannabinoid, ∆(9)-tetrahydrocannabinol (∆(9)-THC). However, we have previously shown that a cannabis extract devoid of ∆(9)-THC still stimulates appetite, indicating that other phytocannabinoids also elicit hyperphagia. One possible candidate is the non-psychoactive phytocannabinoid cannabigerol (CBG), which has affinity for several molecular targets with known involvement in the regulation of feeding behaviour. OBJECTIVES: The objective of the study was to assess the effects of CBG on food intake and feeding pattern microstructure. METHODS: Male Lister hooded rats were administered CBG (30-120 mg/kg, per ora (p.o.)) or placebo and assessed in open field, static beam and grip strength tests to determine a neuromotor tolerability profile for this cannabinoid. Subsequently, CBG (at 30-240 mg/kg, p.o.) or placebo was administered to a further group of pre-satiated rats, and hourly intake and meal pattern data were recorded over 2 h. RESULTS: CBG produced no adverse effects on any parameter in the neuromotor tolerability test battery. In the feeding assay, 120-240 mg/kg CBG more than doubled total food intake and increased the number of meals consumed, and at 240 mg/kg reduced latency to feed. However, the sizes or durations of individual meals were not significantly increased. CONCLUSIONS: Here, we demonstrate for the first time that CBG elicits hyperphagia, by reducing latency to feed and increasing meal frequency, without producing negative neuromotor side effects. Investigation of the therapeutic potential of CBG for conditions such as cachexia and other disorders of eating and body weight regulation is thus warranted.

Neuromotor tolerability and behavioural characterisation of cannabidiolic acid, a phytocannabinoid with therapeutic potential for anticipatory nausea.

RATIONALE: Anticipatory nausea (AN) is a poorly controlled side effect experienced by chemotherapy patients. Currently, pharmacotherapy is restricted to benzodiazepine anxiolytics, which have limited efficacy, have significant sedative effects and induce dependency. The non-psychoactive phytocannabinoid, cannabidiolic acid (CBDA), has shown considerable efficacy in pre-clinical AN models, however determination of its neuromotor tolerability profile is crucial to justify clinical investigation. Provisional evidence for appetite-stimulating properties also requires detailed investigation. OBJECTIVES: This study aims to assess the tolerability of CBDA in locomotor activity, motor coordination and muscular strength tests, and additionally for ability to modulate feeding behaviours. METHODS: Male Lister Hooded rats administered CBDA (0.05-5 mg/kg; p.o.) were assessed in habituated open field (for locomotor activity), static beam and grip strength tests. A further study investigated whether these CBDA doses modulated normal feeding behaviour. Finally, evidence of anxiolytic-like effects in the habituated open field prompted testing of 5 mg/kg CBDA for anxiolytic-like activity in unhabituated open field, light/dark box and novelty-suppressed feeding (NSF) tests. RESULTS: CBDA had no adverse effects upon performance in any neuromotor tolerability test, however anxiolytic-like behaviour was observed in the habituated open field. Normal feeding behaviours were unaffected by any dose. CBDA (5 mg/kg) abolished the increased feeding latency in the NSF test induced by the 5-HT1AR antagonist, WAY-100,635, indicative of anxiolytic-like effects, but had no effect on anxiety-like behaviour in the novel open field or light/dark box. CONCLUSIONS: CBDA is very well tolerated and devoid of the sedative side effect profile of benzodiazepines, justifying its clinical investigation as a novel AN treatment.

Are cannabidiol and Δ(9) -tetrahydrocannabivarin negative modulators of the endocannabinoid system? A systematic review.

Based upon evidence that the therapeutic properties of Cannabis preparations are not solely dependent upon the presence of Δ(9) -tetrahydrocannabinol (THC), pharmacological studies have been recently carried out with other plant cannabinoids (phytocannabinoids), particularly cannabidiol (CBD) and Δ(9) -tetrahydrocannabivarin (THCV). Results from some of these studies have fostered the view that CBD and THCV modulate the effects of THC via direct blockade of cannabinoid CB1 receptors, thus behaving like first-generation CB1 receptor inverse agonists, such as rimonabant. Here, we review in vitro and ex vivo mechanistic studies of CBD and THCV, and synthesize data from these studies in a meta-analysis. Synthesized data regarding mechanisms are then used to interpret results from recent pre-clinical animal studies and clinical trials. The evidence indicates that CBD and THCV are not rimonabant-like in their action and thus appear very unlikely to produce unwanted CNS effects. They exhibit markedly disparate pharmacological profiles particularly at CB1 receptors: CBD is a very low-affinity CB1 ligand that can nevertheless affect CB1 receptor activity in vivo in an indirect manner, while THCV is a high-affinity CB1 receptor ligand and potent antagonist in vitro and yet only occasionally produces effects in vivo resulting from CB1 receptor antagonism. THCV has also high affinity for CB2 receptors and signals as a partial agonist, differing from both CBD and rimonabant. These cannabinoids illustrate how in vitro mechanistic studies do not always predict in vivo pharmacology and underlie the necessity of testing compounds in vivo before drawing any conclusion on their functional activity at a given target.

Evaluation of the potential of the phytocannabinoids, cannabidivarin (CBDV) and Δ(9) -tetrahydrocannabivarin (THCV), to produce CB1 receptor inverse agonism symptoms of nausea in rats.

BACKGROUND AND PURPOSE: The cannabinoid 1 (CB1 ) receptor inverse agonists/antagonists, rimonabant (SR141716, SR) and AM251, produce nausea and potentiate toxin-induced nausea by inverse agonism (rather than antagonism) of the CB1 receptor. Here, we evaluated two phytocannabinoids, cannabidivarin (CBDV) and Δ(9) -tetrahydrocannabivarin (THCV), for their ability to produce these behavioural effect characteristics of CB1 receptor inverse agonism in rats. EXPERIMENTAL APPROACH: In experiment 1, we investigated the potential of THCV and CBDV to produce conditioned gaping (measure of nausea-induced behaviour) in the same manner as SR and AM251. In experiment 2, we investigated the potential of THCV and CBDV to enhance conditioned gaping produced by a toxin in the same manner as CB1 receptor inverse agonists. KEY RESULTS: SR (10 and 20 mg·kg(-1) ) and AM251 (10 mg·kg(-1) ) produced conditioned gaping; however, THCV (10 or 20 mg·kg(-1) ) and CBDV (10 or 200 mg·kg(-1) ) did not. At a subthreshold dose for producing nausea, SR (2.5 mg·kg(-1) ) enhanced lithium chloride (LiCl)-induced conditioned gaping, whereas Δ(9) -tetrahydrocannabinol (THC, 2.5 and 10 mg·kg(-1) ), THCV (2.5 or 10 mg·kg(-1) ) and CBDV (2.5 or 200 mg·kg(-1) ) did not; in fact, THC (2.5 and 10 mg·kg(-1) ), THCV (10 mg·kg(-1) ) and CBDV (200 mg·kg(-1) ) suppressed LiCl-induced conditioned gaping, suggesting anti-nausea potential. CONCLUSIONS AND IMPLICATIONS: The pattern of findings indicates that neither THCV nor CBDV produced a behavioural profile characteristic of CB1 receptor inverse agonists. As well, these compounds may have therapeutic potential in reducing nausea.

Cannabinoid 1 receptors are critical for the innate immune response to TLR4 stimulation.

Sickness behaviors are host defense adaptations that arise from integrated autonomic outputs in response to activation of the innate immune system. These behaviors include fever, anorexia, and hyperalgesia intended to promote survival of the host when encountering pathogens. Cannabinoid (CB) receptor activation can induce hypothermia and attenuate LPS-evoked fever. The aim of the present study was to examine the role of CB1 receptors in the LPS-evoked febrile response. CB1 receptor-deficient (CB1(-/-)) mice did not display LPS-evoked fever; likewise, pharmacological blockade of CB1 receptors in wild-type mice blocked LPS-evoked fever. This unresponsiveness is not limited to thermogenesis, as the animals were not hyperalgesic after LPS administration. A Toll-like receptor (TLR)3 agonist and viral mimetic polyinosinic:polycytidylic acid evoked a robust fever in CB1(-/-) mice, suggesting TLR3-mediated responses are functional. LPS-evoked c-Fos activation in areas of the brain associated with the febrile response was evident in wild-type mice but not in CB1(-/-) mice. Liver and spleen TLR4 mRNA were significantly lower in CB1(-/-) mice compared with wild-type mice, and peritoneal macrophages from CB1(-/-) mice did not release proinflammatory cytokines in response to LPS. These data indicate that CB1 receptors play a critical role in LPS-induced febrile responses through inhibiting TLR4-mediated cytokine production.

Naphthalen-1-yl-(4-pentyloxynaphthalen-1-yl)methanone (SAB378), a peripherally restricted cannabinoid CB1/CB2 receptor agonist, inhibits gastrointestinal motility but has no effect on experimental colitis in mice.

The endocannabinoid system is involved in the regulation of gastrointestinal (GI) motility and inflammation. Using the peripherally restricted cannabinoid (CB)(1)/CB(2) receptor agonist naphthalen-1-yl-(4-pentyloxynaphthalen-1-yl)methanone (SAB378), we investigated the role of peripheral cannabinoid receptors in the regulation of GI motility and the development of colitis in mice. The actions of SAB378 on whole gut transit, upper GI transit, colonic propulsion, and locomotor activity were investigated in C57BL/6N, CB(1) receptor knockout, and CB(2) receptor knockout mice. The potential for SAB378 to modify inflammation was studied by using dextran sulfate sodium (DSS) and 2,4,6-trinitrobenzene sulfonic acid (TNBS) models of experimental colitis. SAB378 did not modify locomotor activity. SAB378 slowed all parameters of GI motility, and these effects were significantly reduced by the CB(1) receptor antagonist N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3 carboxamide (AM251), but not by the CB(2) receptor antagonist 6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H- indol-3-yl](4-methoxyphenyl)methanone (AM630). SAB378 did not inhibit GI transit or colonic propulsion in CB(1) receptor knockout mice, whereas its effects were observed in CB(2) receptor knockout mice. SAB378 did not reduce the degree of colitis induced by DSS or TNBS. The actions of SAB378 on GI motility are mediated by peripherally located CB(1) receptors. SAB378 was not effective against two models of experimental colitis, which may indicate that peripheral cannabinoid receptor stimulation alone may not be sufficient to mediate the anti-inflammatory effects of cannabinoids.

Distribution and function of monoacylglycerol lipase in the gastrointestinal tract.

The endogenous cannabinoid system plays an important role in the regulation of gastrointestinal function in health and disease. Endocannabinoid levels are regulated by catabolic enzymes. Here, we describe the presence and localization of monoacylglycerol lipase (MGL), the major enzyme responsible for the degradation of 2-arachidonoylglycerol. We used molecular, biochemical, immunohistochemical, and functional assays to characterize the distribution and activity of MGL. MGL mRNA was present in rat ileum throughout the wall of the gut. MGL protein was distributed in the muscle and mucosal layers of the ileum and in the duodenum, proximal colon, and distal colon. We observed MGL expression in nerve cell bodies and nerve fibers of the enteric nervous system. There was extensive colocalization of MGL with PGP 9.5 and calretinin-immunoreactive neurons, but not with nitric oxide synthase. MGL was also present in the epithelium and was highly expressed in the small intestine. Enzyme activity levels were highest in the duodenum and decreased along the gut with lowest levels in the distal colon. We observed both soluble and membrane-associated enzyme activities. The MGL inhibitor URB602 significantly inhibited whole gut transit in mice, an action that was abolished in cannabinoid 1 receptor-deficient mice. In conclusion, MGL is localized in the enteric nervous system where endocannabinoids regulate intestinal motility. MGL is highly expressed in the epithelium, where this enzyme may have digestive or other functions yet to be determined.

Cannabinoid CB2 receptors in the enteric nervous system modulate gastrointestinal contractility in lipopolysaccharide-treated rats.

Enhanced intestinal transit due to lipopolysaccharide (LPS) is reversed by cannabinoid (CB)2 receptor agonists in vivo, but the site and mechanism of action are unknown. We have tested the hypothesis that CB2 receptors are expressed in the enteric nervous system and are activated in pathophysiological conditions. Tissues from either saline- or LPS-treated (2 h; 65 microg/kg ip) rats were processed for RT-PCR, Western blotting, and immunohistochemistry or were mounted in organ baths where electrical field stimulation was applied in the presence or absence of CB receptor agonists. Whereas the CB2 receptor agonist JWH133 did not affect the electrically evoked twitch response of the ileum under basal conditions, in the LPS-treated tissues JWH133 was able to reduce the enhanced contractile response in a concentration-dependent manner. Rat ileum expressed CB2 receptor mRNA and protein under physiological conditions, and this expression was not affected by LPS treatment. In the myenteric plexus, CB2 receptors were expressed on the majority of neurons, although not on those expressing nitric oxide synthase. LPS did not alter the distribution of CB2 receptor expression in the myenteric plexus. In vivo LPS treatment significantly increased Fos expression in both enteric glia and neurons. This enhanced expression was significantly attenuated by JWH133, whose action was reversed by the CB2 receptor antagonist AM630. Taking these facts together, we conclude that activation of CB2 receptors in the enteric nervous system of the gastrointestinal tract dampens endotoxin-induced enhanced intestinal contractility.

Area postrema neurons are modulated by the adipocyte hormone adiponectin.

Adiponectin is an adipocyte-derived peptide hormone involved in energy homeostasis and the pathogenesis of obesity, including hypertension. Area postrema (AP) lacks a blood-brain barrier and is a critical homeostatic integration center for humoral and neural signals. Here we investigate the role of AP in adiponectin signaling. We show that rat AP expresses AdipoR1 and AdipoR2 adiponectin receptor mRNA. We used current-clamp electrophysiology to investigate whether adiponectin influenced membrane properties of AP neurons and found that approximately 60% of rat AP neurons tested were sensitive to adiponectin. Additional electrophysiology experiments coupled with single-cell reverse transcription-PCR indicated that all neurons that expressed both subtypes of receptor were sensitive to adiponectin, whereas neurons expressing only one subtype were predominantly insensitive. Last, microinjection of adiponectin into AP caused significant increases in arterial blood pressure, with no change in heart rate, suggesting that adiponectin acts at AP to provide a possible link between control of energy homeostasis and cardiovascular function.

Identification and functional characterization of brainstem cannabinoid CB2 receptors.

The presence and function of CB2 receptors in central nervous system (CNS) neurons are controversial. We report the expression of CB2 receptor messenger RNA and protein localization on brainstem neurons. These functional CB2 receptors in the brainstem were activated by a CB2 receptor agonist, 2-arachidonoylglycerol, and by elevated endogenous levels of endocannabinoids, which also act at CB1 receptors. CB2 receptors represent an alternative site of action of endocannabinoids that opens the possibility of nonpsychotropic therapeutic interventions using enhanced endocannabinoid levels in localized brain areas.

Characterization of cannabinoid modulation of sensory neurotransmission in the rat isolated mesenteric arterial bed.

The present study investigated the effects of different classes of cannabinoid (CB) receptor ligands on sensory neurotransmission in the rat isolated mesenteric arterial bed. Electrical field stimulation of the mesenteric bed evoked frequency-dependent vasorelaxation due to the activation of capsaicin-sensitive sensory nerves and release of calcitonin gene-related peptide (CGRP). The CB(1)/CB(2) cannabinoid agonists WIN55,212 [(R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone] and CP55,940 [(-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl) cyclohexanol] (0.01-1 microM) attenuated sensory neurogenic relaxation in a concentration-dependent manner. At 0.1 microM, WIN55,212 and CP55,940 were largely ineffective in the presence of the CB(1) antagonists SR141716A [N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichloro phenyl)-4-methyl-3-pyrazole-carboxamide] and LY320135 [[6-methoxy-2-(4-methoxyphenyl)benzo[b]-thien-3-yl][4-cyanophenyl] methanone] (1 microM), but their inhibitory actions remained in the presence of the CB(2)-selective antagonist SR144528 [N-[1S)-endo-1,3,3,-trimetyl bicyclo [2.2.1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide] (1 microM). The CB(1)/CB(2) agonist Delta(9)-tetrahydrocannabinol (THC) (1 microM) attenuated sensory neurogenic relaxations, as did the CB(2) agonist JWH-015 [(2-methyl-1-propyl-1H-indol-3-yl)-1-naphthalenylmethanone]. The inhibitory actions of both THC and JWH-015 were still evident in the presence of SR141716A (1 microM) and SR144528 (1 microM). None of the cannabinoid agonists investigated had an effect on vasorelaxation elicited by exogenous CGRP, indicating a prejunctional mechanism. These data demonstrate that different classes of cannabinoid agonists attenuate sensory neurotransmission via a prejunctional site and provide evidence for mediation by a CB(1) and/or a non-CB(1)/CB(2) receptor.

Noladin ether, a putative endocannabinoid, attenuates sensory neurotransmission in the rat isolated mesenteric arterial bed via a non-CB1/CB2 G(i/o) linked receptor.

1 Noladin ether has recently been reported to be an endocannabinoid, with selectivity for the cannabinoid (CB) CB1 receptor. In the present study, we investigated the effects of noladin ether in the rat isolated mesenteric arterial bed, cultured dorsal root ganglia (DRG) cells and human vanilloid (TRPV1)-receptor-expressing HEK293 cells (TRPV1-HEK293 cells). 2 Electrical field stimulation of the mesenteric bed evoked frequency-dependent vasorelaxation due to the action of calcitonin gene-related peptide (CGRP) released from sensory nerves. Noladin ether (0.1-3 microm) attenuated sensory neurogenic relaxation in a concentration-dependent manner. Noladin ether (1 microm) reduced vasorelaxation at a submaximal frequency (8 Hz), from 57.3+/-6.8 to 23.3+/-3.8% (P<0.05, n=4). 3 The inhibitory effects of noladin ether were unaffected by the CB1 antagonists SR141716A and LY320135, and the CB2 antagonist SR144528 (1 microm). 4 Noladin ether had no effect on vasorelaxation elicited by exogenous CGRP or capsaicin. These data suggest that noladin ether is acting at a prejunctional site and no interaction with TRPV1 is involved. 5 In mesenteric beds from pertussis toxin (PTX)-pretreated rats, the inhibitory actions of noladin ether on sensory neurotransmission were abolished, indicating the involvement of G(i/o) protein-coupled receptors. 6 Noladin ether evoked a concentration-dependent increase in intracellular Ca2+ concentration in TRPV1-HEK293 cells at 10 microm (36.5+/-3.2% of maximal capsaicin-induced response), but it was a less potent agonist than both capsaicin and anandamide and at 1 microm it was essentially inactive. Noladin ether (1 microm) had no effect on capsaicin-evoked Ca2+ responses in DRG cells, and produced no response alone, indicating it neither modulates nor acts directly on TRPV1 receptors. 7 These data demonstrate that noladin ether attenuates sensory neurotransmission in rat mesenteric arteries via a non-CB1 non-CB2 PTX-sensitive prejunctional site, independently of TRPV1 receptors.