Agonist AM251 Research Articles

Detailed analysis of food-reinforced operant lever pressing distinguishes effects of a cannabinoid CB1 inverse agonist and dopamine D1 and D2 antagonists

Overt similarities exist between the effects of systemic cannabinoid CB1 inverse agonists and dopamine (DA) antagonists on appetitive behavior. The present set of studies was undertaken to apply a fine-grained analysis of food-reinforced operant lever pressing in rats in order to compare the pattern of effects produced by administration of the CB1 inverse agonist AM 251 and those induced by the DA D1 antagonist SKF 83566, and the D2 antagonist raclopride. Three groups of rats were trained on a fixed-ratio 5 (FR5) schedule and administered these compounds over a range of doses expected to suppress responding. All three drugs produced a dose-related suppression of total lever pressing. In addition to main effects of dose, regression analyses were performed to determine which of several response timing- and rate-related variables correlated most strongly with overall responding in each group. It was found that total session time spent pausing from responding was significantly better at predicting responding in the AM 251 group, while both DA antagonists produced significantly stronger regression coefficients (versus AM 251) from fast responding measures. These results suggest that, while several similarities exist, CB1, D1, and D2 antagonists are not identical in their pattern of suppression of food-maintained lever pressing.

Abstract 232: PAX3-FOXO1a target genes in alveolar rhabdomyosarcoma: Possible therapeutic targets

Abstract Rhabdomyosarcoma (RMS) is a pediatric soft tissue tumor that is derived from skeletal muscle and expresses many muscle specific proteins. Most commonly RMS presents in one of two histological subtypes; Embryonal RMS (ERMS) or Alveolar RMS (ARMS). The ARMS subtype is more aggressive, more prone to metastasis and has the poorest prognosis. Over 75% of ARMS tumors possess a t(2;13)(q35;q14), or the variant t(1;13)(p36;q14) chromosomal translocation which generates a PAX3-FOXO1a or PAX7-FOXO1a fusion protein, respectively. Using murine primary myoblasts (a likely cell of origin for rhabdomyosarcoma), PAX3 and PAX3-FKHR were ectopically expressed and microarray analysis was performed to identify differentially regulated genes. Several genes were identified and two genes, Cannabinoid Receptor 1 (Cnr1/Cb1) and the pro-apoptotic BH3-only protein Noxa (Pmaip1), were chosen for further study. In primary myoblasts, Cnr1 is required but not sufficient to enhance the invasive capacity of PAX3-FOXO1a expressing primary myoblasts. This appears to be mediated through Cnr1 constitutive activity. In vivo the Cnr1 inverse agonist AM251 prevents lung metastasis formation in immunocompromized mice. This implicates Cnr1 as a potential therapeutic target to inhibit the metastatic capacity of ARMS tumors. Noxa expression is increased in PAX3-FOXO1a expressing primary myoblasts in a p53-independent manner. Moreover, PAX3-FOXO1a expression, through the up regulation of Noxa, increases the sensitivity of primary myoblasts to bortezomib and GSI-1, which also induce apoptosis in a p53-independent, Noxa-dependent manner. In addition, the up regulation of Noxa increases cell sensitivity to the anti-apoptotic Bcl-2 family inhibitor ABT-737. Bortezomib treatment in vivo significantly inhibits the growth of subcutaneous ARMS model tumors and prolongs the survival of treated animals. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 232.

Asynchronous release of GABA via tonic cannabinoid receptor activation at identified interneuron synapses in rat CA1

The influence of local circuit interneurons is thought to play an important role in adjusting synaptic strength via endogenous cannabinoid type 1 (CB1) receptors. Using paired whole-cell recordings, combined with double immunofluorescence and biocytin labelling in acute slices of rat CA1 at postnatal day 18-23, we investigated the properties of Cholecystokinin (CCK)-positive stratum radiatum local circuit interneuron connections that utilised CB1 receptors. Three types of synaptic connections were studied, lacunosum-moleculare-radiatum perforant path-associated (LM-R PPA) to Shaffer collateral-associated (SCA) interneurons, SCA-SCA interneurons and SCA-pyramidal cells. These three synapses were differentially under tonic reduction of inhibition that was blocked by the CB1 receptor inverse agonist AM-251 (10 microM), which enhanced IPSPs. The strength of tonic reduction of inhibition was correlated with asynchronous release which was apparent at connections among interneurons. AM-251 increased the ratio of synchronous to asynchronous release (synchronicity ratio), while the CB receptor agonist anandamide (14 microM) decreased the synchronicity ratio. Fast and slow calcium chelators (BAPTA-AM and EGTA-AM) also increased the synchronicity ratio, accelerated inhibitory time courses and reduced IPSP amplitudes. These data suggest that CB1 receptors at connections among interneuron synapses play a role in tonic suppression of inhibition and govern the asynchronous release of GABA, modulating the time windows of inhibition. Effects of calcium chelators suggest that asynchronous release is a result of a long-lasting presynaptic calcium transients and/or a large distance between calcium source and sensor of exocytosis. These properties of specialised inhibitory neurons may have important modulatory roles in controlling spike timing among local circuit interneurons.

The CB1 inverse agonist AM251, but not the CB1 antagonist AM4113, enhances retention of contextual fear conditioning in rats

The effects of CB1 antagonist/inverse agonists on the acquisition and consolidation of conditioned fear remain uncertain. Recent studies suggest that the CB1 antagonist/inverse agonist AM251 affects acquisition or consolidation of both contextual and discretely cued fear memories. AM251 is frequently referred to as a CB1 antagonist; however in vitro signal transduction assays indicate that this drug also elicits inverse agonist activity at CB1 receptors. The present studies were undertaken to compare the effects of AM251 on conditioned fear with those produced by AM4113, a novel CB1 antagonist with minimal inverse agonist activity. All drugs were administered prior to conditioning. In retention tests conducted two weeks after conditioning, both AM251 (4.0 mg/kg) and AM4113 (6.0 mg/kg)-treated animals exhibited reduced freezing during a conditioned tone cue played within a novel context. In contextual fear retention tests, animals previously treated with 4.0 or 8.0 mg/kg AM251 exhibited enhanced freezing. By contrast, no dose of AM4113 had any significant effect on contextual fear memory, which is consistent with the lower signal transduction activity of AM4113 at CB1 receptors compared to AM251. These results suggest that CB1 neutral antagonists may be less likely than CB1 inverse agonists to facilitate the acquisition or consolidation of contextual fear that may contribute to some clinical disorders.

Differential effects of CB1 neutral antagonists and inverse agonists on gastrointestinal motility in mice

Cannabinoid type 1 (CB(1)) receptors are involved in the regulation of gastrointestinal (GI) motility and secretion. Our aim was to characterize the roles of the CB(1) receptor on GI motility and secretion in vitro and in vivo by using different classes of CB(1) receptor antagonists. Immunohistochemistry was used to examine the localization of CB(1) receptor in the mouse ileum and colon. Organ bath experiments on mouse ileum and in vivo motility testing comprising upper GI transit, colonic expulsion, and whole gut transit were performed to characterize the effects of the inverse agonist/antagonist AM251 and the neutral antagonist AM4113. As a marker of secretory function we measured short circuit current in vitro using Ussing chambers and stool fluid content in vivo in mouse colon. We also assessed colonic epithelial permeability in vitro using FITC-labeled inulin. In vivo, the inverse agonist AM251 increased upper GI transit and whole gut transit, but it had no effect on colonic expulsion. By contrast, the neutral antagonist AM4113 increased upper GI transit, but unexpectedly reduced both colonic expulsion and whole gut transit at high, but not lower doses. Cannabinoid type 1 receptors regulate small intestinal and colonic motility, but not GI secretion under physiological conditions. Cannabinoid type 1 inverse agonists and CB(1) neutral antagonists have different effects on intestinal motility. The ability of the neutral antagonist not to affect whole gut transit may be important for the future development of CB(1) receptor antagonists as therapeutic agents.

Potential anxiogenic effects of cannabinoid CB1 receptor antagonists/inverse agonists in rats: Comparisons between AM4113, AM251, and the benzodiazepine inverse agonist FG-7142

Cannabinoid CB1 inverse agonists suppress food-motivated behaviors, but may also induce psychiatric effects such as depression and anxiety. To evaluate behaviors potentially related to anxiety, the present experiments assessed the CB1 inverse agonist AM251 (2.0-8.0mg/kg), the CB1 antagonist AM4113 (3.0-12.0mg/kg), and the benzodiazepine inverse agonist FG-7142 (10.0-20.0mg/kg), using the open field test and the elevated plus maze. Although all three drugs affected open field behavior, these effects were largely due to actions on locomotion. In the elevated plus maze, FG-7142 and AM251 both produced anxiogenic effects. FG-7142 and AM251 also significantly increased c-Fos activity in the amygdala and nucleus accumbens shell. In contrast, AM4113 failed to affect performance in the plus maze, and did not induce c-Fos immunoreactivity. The weak effects of AM4113 are consistent with biochemical data showing that AM4113 induces little or no intrinsic cellular activity. This research may lead to the development of novel appetite suppressants with reduced anxiogenic effects.

L-type channel inhibition by CB1 cannabinoid receptors is mediated by PTX-sensitive G proteins and cAMP/PKA in GT1-7 hypothalamic neurons

Using immortalized hypothalamic GT1-7 neurons, which express the CB1 cannabinoid receptor (CB1R) and three Ca 2+ channel types (T, R and L), we found that the CB1R agonist WIN 55,212-2 inhibited the voltage-gated Ca 2+ currents by about 35%. The inhibition by WIN 55,212-2 (10 μM) was reversible and prevented by nifedipine (3 μM), suggesting a selective action on L-type Ca 2+ channels (LTCCs). WIN 55,212-2 action exhibited all the features of voltage-independent Ca 2+ channel modulation: (1) no changes of the activation kinetics, (2) equal depressive action at all potentials and (3) no facilitation following strong prepulses. At variance with WIN 55,212-2, the CB1R inverse agonist AM-251 (10 μM) caused 20% increase of Ca 2+ currents. The inhibition of LTCCs by WIN 55,212-2 was prevented by overnight PTX-incubation and by intracellular perfusion with GDP-β-S. The latter caused also a 20% Ca 2+ current up-regulation. WIN 55,212-2 action was also prevented by application of the PKA-blocker H89 or by loading the neurons with 8-CPT-cAMP. Our results suggest that LTCCs in GT1-7 neurons are partially inhibited at rest due to a constitutive CB1R activity removed by AM-251 and GDP-β-S. Activation of CB1R via PTX-sensitive G proteins and cAMP/PKA pathway selectively depresses LTCCs that critically control the synchronized spontaneous firing and pulsatile release of gonadotropin-releasing hormone in GT1-7 neurons.

Functional CB2 type cannabinoid receptors at CNS synapses

To date, it has been thought that cannabinoid receptors in CNS are primarily of the CB1R subtype, with CB2R expressed only in glia and peripheral tissues. However, evidence for the expression of CB2 type cannabinoid receptors at neuronal sites in the CNS is building through anatomical localization of receptors and mRNA in neurons and behavioural studies of central effects of CB2R agonists. In the medial entorhinal area of the rat, we found that blockade of CB1R did not occlude suppression of GABAergic inhibition by the non-specific endogenous cannabinoid 2-AG, suggesting that CB1R could not account fully for the effects of 2-AG. Suppression could be mimicked using the CB2R agonist JWH-133 and reversed by the CB2R inverse agonist AM-630, indicating the presence of functional CB2R. When we reversed the order of drug application AM-630 blocked the effects of the CB2R agonist JWH-133, but not the CB1R inverse agonist LY320135. JTE-907, a CB2R inverse agonist structurally unrelated to AM-630 elicited increased GABAergic neurotransmission at picomolar concentrations. Analysis of mIPSCs revealed that CB2R effects were restricted to action potential dependent, but not action potential independent GABA release. These data provide pharmacological evidence for functional CB2R at CNS synapses.

Effects of Chronic Exposure to the CB1 Agonist AM 411 in Squirrel Monkeys

Cannabinoid CB1 agonists produce tolerance that may be associated with dependence and increased sensitivity to the behavioral effects of the CB1 antagonist SR141716A, perhaps indicative of antagonist‐precipitated withdrawal. In the present studies, the effects of the long‐acting CB1 agonist AM 411 and the CB1 antagonist SR141716A on operant responding were examined in squirrel monkeys before and during chronic AM 411 treatment (1.0 mg/kg/day, im). Prechronically, AM 411 (0.01‐1 mg/kg) produced dose‐related decreases in responding; the highest dose decreased behavior to approximately 20% of control values. In contrast, SR 141716A (1‐10 mg/kg) had little effect on behavior; the highest dose decreased responding only to >80% of control values. Re‐determination of drug effects during the chronic regimen revealed a >10‐fold rightward shift in the dose‐response function for AM 411, indicative of tolerance. On the other hand, SR 141716A (0.32‐3.2 mg/kg) produced dose‐related decreases in behavior; the highest dose decreased responding to <50% of control values. These data are consistent with the view that chronic CB1 receptor activation produces tolerance to the effects of CB1 agonists and enhances sensitivity to the behaviorally disruptive effects of CB1 antagonists. The increased sensitivity to the effects of SR 141716A may be indicative of antagonist‐precipitated withdrawal (supported by DA19205).

Influence of intracerebroventricular or intraperitoneal administration of cannabinoid receptor agonist (WIN 55,212-2) and inverse agonist (AM 251) on the regulation of food intake and hypothalamic serotonin levels

The effect of intracerebroventricular or intraperitoneal administration of cannabinoid receptor agonist WIN 55,212-2 or inverse agonist AM 251 on food intake and extracellular levels of serotonin and acetic acid 5-hydroxy-indol from presatiated rats was studied. Compared to the vehicle-injected control, the intracerebroventricular administration of WIN 55,212-2 was associated with a significant increase in food intake, whereas the administration of AM 251 caused a significant reduction in this respect. These results were accompanied by considerable reductions or increases in serotonin and acetic acid 5-hydroxy-indol levels compared to the vehicle-injected control and the baseline values for the different experimental groups studied. Intraperitoneal administration of WIN 55,212-2 at doses of 1 and 2 mg/kg promoted hyperphagia up to 6 h after injection, whereas administration of a higher dose (5 mg/kg) significantly inhibited food intake and motor behaviour in partially satiated rats. Administration of any of the AM 251 doses studied (0.5, 1, 2, 5 mg/kg) led to a significant decrease in the amount of food ingested from 2 h after the injection, compared to the vehicle-injected control group, with the most striking effect being observed when the 5 mg/kg dose was injected.

Regulation of Bone Mass, Osteoclast Function, and Ovariectomy-Induced Bone Loss by the Type 2 Cannabinoid Receptor

The endocannabinoid system has recently been shown to play a role in the regulation of bone metabolism. The type 2 cannabinoid receptor (CB2) has been reported to regulate bone mass, but conflicting results have been reported with regard to its effects on bone resorption and osteoclast function. Here we investigated the role that CB2 plays in regulating bone mass and osteoclast function using a combination of pharmacological and genetic approaches. The CB2-selective antagonist/inverse agonist AM630 inhibited osteoclast formation and activity in vitro, whereas the CB2-selective agonists JWH133 and HU308 stimulated osteoclast formation. Osteoclasts generated from CB2 knockout mice (CB2-/-) were resistant to the inhibitory effects of AM630 in vitro, consistent with a CB2-mediated effect. There was no significant difference in peak bone mass between CB2-/- mice and wild-type littermates, but after ovariectomy, bone was lost to a greater extent in wild-type compared with CB2-/- mice. Furthermore, AM630 protected against bone loss in wild-type mice, but the effect was blunted in CB2-/- mice. We conclude that CB2 regulates osteoclast formation and bone resorption in vitro and that under conditions of increased bone turnover, such as after ovariectomy, CB2 regulates bone loss. These observations indicate that CB2 regulates osteoclast formation and contributes to ovariectomy-induced bone loss and demonstrate that cannabinoid receptor antagonists/inverse agonists may be of value in the treatment of bone diseases characterized by increased osteoclast activity.

The cannabinoid CB1 receptor inverse agonist AM 251 and antagonist AM 4113 produce similar effects on the behavioral satiety sequence in rats

Cannabinoid CB1 inverse agonists such as rimonabant and AM 251 hold therapeutic promise as appetite suppressants, but the extent to which non-motivational factors contribute to their anorectic effects is not fully known. Examination of the behavioral satiety sequence (BSS) in rats, the orderly progression from eating to post-prandial grooming and then resting, has revealed that these compounds preserve the order of events but differ markedly from natural satiation. The most notable difference is that grooming (particularly scratching) is profoundly enhanced at anorectic doses, while eating and resting are diminished, raising the possibility that the anorectic effect is simply secondary to the grooming effect. In the current design, the neutral CB1 antagonist AM 4113, which has been found to lack some of the undesirable effects of AM 251, produced nearly identical effects on the BSS as AM 251. The possibility that competition from enhanced grooming could account for the anorectic effect of AM 4113 was examined by yoking the pattern of disruptions caused by grooming in the AM 4113-treated group to forced locomotion in a different group fed in a modified running wheel. This response competition did not significantly reduce food intake. It was concluded that AM 4113, a CB1 neutral antagonist, produces the same effects on the BSS as AM 251, but that response competition from enhanced grooming may not be a sufficient explanation for the anorectic effects of CB1 antagonists/inverse agonists.

Discriminative‐stimulus effects of the novel CB1 agonist AM 4054 in squirel monkeys

Discriminative‐stimulus (Sd) properties of cannabinergic (CB) ligands other than the CB‐1 partial agonist Δ9–THC have not been studied in nonhuman primates. The present studies were conducted to systematically examine the Sd effects of the novel CB‐1 full agonist AM 4054 in squirrel monkeys (n=4) trained under a two‐lever procedure to discriminate i.m. injections of saline from 0.01 mg/kg AM 4054. Results show that the AM 4054 Sd was acquired with varying speed among monkeys. Following acquisition, ≤ 5% and ≥ 97% drug‐lever responding occurred following injection with, respectively, saline and AM 4054. Under test conditions, AM 4054 produced dose‐related (0.001–0.01 mg/kg) increases in drug‐lever responding, with little or no effect on response rate and >90% substitution following 0.0056 mg/kg AM 4054. The Sd effects of 0.01 mg/kg AM 4054 were fully evident 1 hr after injection and dissipated gradually over 12 hr. Dose‐related substitution for AM 4054 also occurred with the CB‐1 agonists Δ9–THC (0.03–3 mg/kg) and methanandamide (0.3–10 mg/kg). In antagonism experiments, SR 141716A dose‐dependently reduced the Sd effects of 0.01 mg/kg AM 4054, with full attenuation after 1.0 mg/kg of the CB‐1 antagonist. Together, these data demonstrate that the discriminative stimulus effects of AM 4054 are CB‐1‐mediated and overlap those of the CB1 agonists Δ9–THC and AM 356 (supported by NIH/NIDA DA19205 and DA7252).

Cannabinoid CB1 antagonists and dopamine antagonists produce different effects on a task involving response allocation and effort-related choice in food-seeking behavior

Cannabinoid CB1 antagonists/inverse agonists suppress food-motivated behaviors and are being evaluated as potential appetite suppressants. It has been suggested that the effects of CB1 antagonism on food motivation could be related to actions on mesolimbic dopamine (DA). If this were true, then the effects of interference with cannabinoid CB1 transmission should closely resemble the effects of interference with DA transmission. To directly compare the effects of DA antagonists with those of CB1 antagonists/inverse agonists, the present studies employed a concurrent lever-pressing/chow-intake procedure. With this task, interference with DA transmission shifts choice behavior such that lever pressing for a preferred food is decreased but chow intake is increased. Rats treated with IP injections of the DA D1 antagonist SCH39166 (ecopipam; 0.05-0.2 mg/kg) or the D2 antagonist eticlopride (0.025-0.1 mg/kg) showed substantial decreases in lever pressing and concomitant increases in chow consumption. In contrast, IP administration of the CB1 neutral antagonist AM4113 (4.0-16.0 mg/kg) or the CB1 antagonist/inverse agonist AM251 (2.0-8.0 mg/kg) decreased operant responding for pellets, but there was no corresponding increase in chow intake. These effects of CB1 antagonists/inverse agonists were similar to those produced by the appetite suppressant fenfluramine and by prefeeding. In contrast, low doses of DA antagonists leave primary food motivation intact, but shift behaviors toward food reinforcers that can be obtained with lower response costs. These results suggest that the effects of interference with CB1 transmission are readily distinguishable from those of reduced DA transmission.

Acute anorectic response to cannabinoid CB1 receptor antagonist/inverse agonist AM 251 in rats: indirect behavioural mediation

Despite a large and consistent literature on the suppressant effects of cannabinoid CB1 receptor antagonists/inverse agonists (e.g. rimonabant, AM 251) on food intake and weight gain in rodents, surprisingly little is known about the behavioural selectivity of such effects. In this study, ethological scoring was used to characterize the acute behavioural effects of the rimonabant analogue AM 251 (1.5 and 3.0 mg/kg, intraperitoneally) in nondeprived male rats during a 1-h test with palatable mash. Data were also collected on daily weight gain and on retest food intake 7 days after dosing. Results showed that the higher dose of AM 251 significantly inhibited mash consumption (32% decrease relative to vehicle control), reduced time spent feeding during the test and suppressed body weight gain over the 48-h period that followed acute dosing. No effects on mash consumption were observed when the animals were retested drug-free 1 week after drug treatment. Detailed video analysis of the test sessions showed that, over the dose range tested, AM 251 did not significantly interfere with the vast majority of noningestive behaviours. Both doses of the compound, however, significantly increased the incidence of and the time spent on scratching, whereas the higher dose additionally increased both the number and duration of grooming episodes. The latter effect in particular disrupted the normal structure of behaviour (behavioural satiety sequence) with atypically high levels of grooming displacing feeding during the middle part of the test session. Overall, the behavioural profile of AM 251 in a free-feeding context is very similar to (but approximately two-fold less potent than) that recently reported for the parent molecule, rimonabant. Together, these data strongly suggest that the acute anorectic response to CB1 receptor antagonists/inverse agonists is indirectly mediated via major alterations to other components of the behavioural repertoire.

AM 251 and β-Funaltrexamine reduce fat intake in a fat-preferring strain of mouse

This experiment aimed to examine the role of the CB1 and Opioid mu receptors on fat preference by administering the CB1 inverse agonist AM 251 (5 mg/kg i.p.) and the opioid mu antagonist β-Funaltrexamine (15 mg/kg s.c.) for 4 days to fat-preferring C57BL/6 mice fed a two-choice high-fat/low-fat diet. Both drugs were found to significantly reduce total energy intake, high-fat diet intake and fat preference during treatment.

Zileuton added to low-dose inhaled beclomethasone for the treatment of moderate to severe persistent asthma

To assess the therapeutic effects of oral zileuton tablets combined with low-dose beclomethasone compared to doubling the dose of beclomethasone, in improving lung function and reducing asthma symptoms. Randomized, active-control, double-blind, parallel, multi-center study of zileuton (400 or 600 mg QID)+200 microg beclomethasone dipropionate (BDP) BID versus placebo+BDP 400 microg BID in asthmatics with baseline FEV(1) percent predicted values between 40% and 80% following a single-blind ICS (BDP 200 microg BID) 2-week run-in. During the 3-month double-blind treatment period, assessments included safety, daytime and nighttime symptoms, acute asthma exacerbations, beta(2)-agonist use, AM and PM peak expiratory flow (PEF) and FEV(1). The addition of a 5-lipoxygenase (5-LO) inhibitor added to a low-dose of BDP showed no significant difference in FEV(1) compared to doubling the dose of BDP. FEV(1) improved in all 3 treatment groups, with mean increases of 10% with zileuton 600 mg QID+BDP 200 microg BID, 12% with zileuton 400mg QID+BDP 200 microg BID, and 11% with BDP 400 microg BID by study end. Within each treatment group, there were significant improvements in asthma symptoms and AM and PM PEF compared to baseline. No significant differences were observed between groups with regards to salbutamol use, acute asthma exacerbations, the requirement for oral/parenteral corticosteroids and adverse clinical events. The addition of a 5-LO inhibitor added to low-dose beclomethasone may be an alternative to higher-doses of ICS in patients unable to achieve sufficient asthma control on low-dose ICS therapy.

The cannabinoid agonist WIN 55,212-2 inhibits TNF-α-induced neutrophil transmigration across ECV304 cells

Cannabinoids are known to possess both anti-inflammatory and neuroprotective effects. In the present study, we have investigated the ability of cannabinoids to inhibit the transmigration of neutrophils in response to chemotaxic stimuli. The cannabinoid receptor agonist WIN 55,212-2 (( R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)-pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate) significantly decreased the number of migrating neutrophils across a monolayer of tumour necrosis factor alpha (TNF-α) activated ECV304 cells at concentrations ≥ 1 μM. In contrast, the agonists HU210 and CP 55,940 (0.01–1 μM) and the endocannabinoid anandamide (0.1–10 μM) were without significant effect on the response to TNF-α. The ability of WIN 55,212-2 to reduce the neutrophil transmigration was still seen in the presence of the cannabinoid CB 1 receptor antagonist/inverse agonist AM251 (0.1–1 μM) and the cannabinoid CB 2 receptor antagonist/inverse agonist AM630 (0.1–1 μM). TNF-α treatment of ECV304 cells caused release of interleukin-8 (IL-8), but WIN 55,212-2 did not affect either the ability of neutrophils to migrate across chemotaxis plates in response to an IL-8 stimulus, or to change the percentage of CXC 1 and CXC 2 receptors expressed by the neutrophils. WIN 55,212-2 at a concentration of 1 μM, but not at lower concentrations, produced a significant inhibition of IL-8 release from ECV304 cells in response to TNF-α-stimulation. Thus WIN 55,212-2 reduces the transmigration of neutrophils across a monolayer of TNF-α-activated ECV304 cells by an indirect action upon the release of IL-8 and/or other chemokine release from the ECV304 cells, and that this effect is brought about mainly by a cannabinoid CB receptor-independent mechanism.

Effects of the CB1 cannabinoid receptor inverse agonist AM251 on food intake and body weight in mice lacking μ-opioid receptors

We used μ-opioid receptor-deficient (MOR−/−) mice to determine the effects of the CB1 cannabinoid receptor inverse agonist AM251 on feeding in the absence of MOR signaling. A single dose of AM251 at 0.6, 2 or 6 mg/kg caused similar dose-dependent suppression of food intake in wild-type and MOR−/− mice. Administration of AM251 at 3 mg/kg/day for 9 consecutive days also led to a similar reduction (∼8%) in body weight in wild-type and MOR−/− mice. Our results suggest that MOR signaling is not necessary for cannabinoid-mediated effects on feeding and that physiological antagonism of opioid receptor tone might be required for the observed synergistic effects of a CB1 inverse agonist and an opioid receptor antagonist on feeding.

Suppression of food intake and food-reinforced behavior produced by the novel CB1 receptor antagonist/inverse agonist AM 1387

Cannabinoid CB1 receptor antagonist/inverse agonists are becoming increasingly recognized for their potential therapeutic utility as appetite suppressants. In the current paper we characterize the biochemical and behavioral effects of AM 1387, which is a novel CB1 antagonist. AM 1387 exhibited binding affinity and selectivity for the CB1 over the CB2 receptor. Moreover, AM 1387 decreased GTPγS (EC 50: 22.82 nM) and increased forskolin-stimulated cAMP (EC 50: 274.6 nM), as did the CB1 inverse agonist AM 251 (GTPγS EC 50: 25.82 nM; cAMP EC 50: 363.8 nM), indicating that AM1387 also has inverse agonist properties in vitro. In the behavioral characterization in rats, AM 1387 suppressed lever pressing for food on two operant schedules (fixed-ratio 1 and 5). Timecourse of the effect on fixed-ratio 5 responding was then determined, and the half-life ( t 1/2 = 4.87 h) was found to be threefold shorter than what has been shown for SR 141716A, and fourfold shorter than AM 251. Finally, AM 1387 was found to suppress food intake using three diets of differing macronutrient composition and palatability. It was concluded that AM 1387 may be a useful tool for examining the effects of CB1 receptor antagonism or inverse agonism on food intake.