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Dronabinol, a synthetic agonist at cannabinoid receptors, was reported to decrease the pruritus of cholestasis, in an uncontrolled observation. We hypothesized that the reported antipruritic effect of dronabinol might have resulted from an increased threshold to experience nociception (i.e. pruritus) by the drug. To test this hypothesis, we studied the effect of WIN 55, 212-2, a cannabinoid agonist, on the threshold to experience nociception, using a tail-flick assay in rats with cholestasis secondary to bile duct resection and in sham-resected controls. The administration of WIN 55, 212-2 was associated with a significant increase in the mean tail-flick latency in both groups as compared to baseline. Pruritus is a nociceptive stimulus; accordingly, drugs that increase the threshold to nociception in human beings may be a novel approach to the treatment of this symptom in patients with liver disease.

Synthetic cannabinoids have a promising future as treatments for nausea, appetite modulation, pain, and many neurological disorders. Transdermal delivery is a convenient and desirable dosage form for these drugs and health conditions. The aim of the present study was to investigate the in vitro transdermal permeation of two synthetic cannabinoids, WIN 55,212-2 and CP 55,940. Transdermal flux, drug content in the skin, and lag times were measured in split-thickness human abdominal skin in flow-through diffusion cells with receiver solutions of 4% bovine serum albumin (BSA) or 0.5% Brij 98. Differential thermal analysis (DSC) was performed in order to determine heats of fusion, melting points, and relative thermodynamic activities. The in vitro diffusion studies in 0.5% Brij 98 indicated that WIN 55,212-2 diffuses across human skin faster than CP 55,940. The WIN 55,212-2 skin disposition concentration levels were also significantly higher than that of CP 55,940. Correspondingly, CP 55,940 was significantly metabolized in the skin. WIN 55,212-2 flux and skin disposition were significantly lower into 4% BSA than into 0.5% Brij 98 receiver solutions. There was no significant difference in the flux, lag time, and drug content in the skin of CP 55,940 in 4% BSA versus 0.5% Brij 98 receiver solutions. The DSC studies showed that CP 55,940 had a significantly lower melting point, smaller heat of fusion, and corresponding higher calculated thermodynamic activity than the more crystalline WIN 55,212-2 mesylate salt. The permeation results indicated that WIN 55,212-2 mesylate, CP 55,940, and other potent synthetic cannabinoids with these physicochemical properties could be ideal candidates for the development of a transdermal therapeutic system.

Pain associated with cancer and chronic musculoskeletal disorders can be difficult to control. We used murine models of cancer and inflammatory muscle pain to examine whether the cannabinoid receptor agonist WIN55,212-2 reduces hyperalgesia originating in deep tissues. C3H/He mice were anesthetized and implanted with osteolytic NCTC clone 2472 cells into the humeri or injected with 4% carrageenan into the triceps muscles of both forelimbs. At the time of peak hyperalgesia, WIN55,212-2 (1-30mg/kg) or vehicle was administered intraperitoneally and forelimb grip force was measured 0.5-24h later. WIN55,212-2 produced time- and dose-related antihyperalgesia in both models. A 10mg/kg dose of WIN55,212-2 fully reversed carrageenan-evoked muscle hyperalgesia. However, 30mg/kg of WIN55,212-2 attenuated tumor-evoked hyperalgesia only approximately 50%. After controlling for the difference in magnitude of hyperalgesia between the two models, WIN55,212-2 was still more potent at reducing hyperalgesia in the inflammatory model. In the cancer pain model, the antihyperalgesic effect of WIN55,212-2 was partially blocked by pretreatment with the selective CB1 (SR141716A) but not the CB2 (SR144528) receptor antagonist. In contrast, both antagonists blocked antihyperalgesic effects of WIN55,212-2 on carrageenan-evoked muscle hyperalgesia. Catalepsy and loss of motor coordination, known side effects of cannabinoids, did not account for the antihyperalgesia produced by WIN55,212-2. These data show that cannabinoids attenuate deep tissue hyperalgesia produced by both cancer and inflammatory conditions. Interestingly, cannabinoids differentially modulated carrageenan- and tumor-evoked hyperalgesia in terms of potency and receptor subtypes involved suggesting that differences in underlying mechanisms may exist between these two models of deep tissue pain.

CB1 cannabinoid receptor agonists show a different profile compared to other drugs of abuse on the basis of experimental data that reveal their reinforcing properties. Thus, there are controversial data in the literature concerning the ability of CB 1 receptor agonists to reinforce behavioral responses in experimental animals, i.e. to lower self-stimulation thresholds, and to support self-administration or conditioned place preference. The aim of the present study was to examine the effects of WIN 55,212-2, a potent CB1 receptor agonist (graded doses 0.1, 0.3, 1 mg/kg, i.p.), on the rewarding efficacy of lateral hypothalamic self-stimulation and on the systemic cocaine-induced potentiation of brain-stimulation reward. WIN 55,212-2 did not affect lateral hypothalamic self-stimulation thresholds both in drug na ¨ ove rats and in rats pretreated with the drug, whereas it produced a significant, dose-dependent decrease in the maximal rate of responding, i.e. in the performance of the animals. Cocaine (5.0 mg/kg, i.p.) produced a significant reduction in self-stimulation threshold, without altering maximal rates of responding. Importantly, WIN 55,212-2 attenuated the effect of cocaine at the two higher doses tested. The effects of the CB1 receptor agonist were reversed by pretreatment with the selective CB1 receptor antagonist SR 141716A (0.02 mg/kg, i.p.) that did not by itself affect cocaine’s action. These results indicate that acute stimulation of CB 1 receptors per se does not affect baseline self-stimulation, but reduces the reinforcing effects induced by cocaine. Taken together these findings suggest that cannabinoids may interfere with brain-reward systems responsible for the expression of acute reinforcing properties of drugs of abuse, such as cocaine, and provide evidence that the cannabinoid system could be an interesting drug discovery and development target for the treatment of drug addiction. © 2002 Elsevier Science B.V. All rights reserved.

Drugs acting on brain cannabinoid CB(1) receptors exert complex actions on modulatory transmitters that are involved in attention and cognition; however, little is known about the precise pharmacological and anatomical mechanisms that govern these effects. Previously demonstrated effects of cannabinoids on acetylcholine (ACh) in the hippocampus prompted us to evaluate changes in the prefrontal cortex, a site associated with mnemonic and attentional functions. We utilized in vivo microdialysis, coupled with direct reverse perfusion of agents, to study the actions on cannabinoidergic drugs on ACh release within the rat frontal cortex. Systemic administration of the CB(1) receptor agonists Delta(9)-tetrahydrocannabinol (THC) or WIN 55,212-2 (WIN) dose- and time-dependently increased ACh release; these effects were blocked by pretreatment with the selective CB(1) receptor antagonist / partial inverse agonist SR141716A (SR). THC applied by reverse dialysis in the frontal cortex caused no change in ACh release, although intrastriatal infusions of THC decreased ACh efflux. These data indicate that cannabinoid agonists potentiate ACh release in the frontal cortex by activating cannabinoid receptors in brain regions other than the frontal cortex.

The expression of genes encoding the cannabinoid CB(1) and CB(2) receptors and fatty acid amide hydrolase (FAAH) and the lipolytic activity of cannabinoid agonists were investigated in rat adipose tissue.RT-PCR studies indicated that the genes encoding CB(1) and CB(2) receptors and FAAH are not expressed in epididymal adipocytes. In functional studies, the non-selective cannabinoid receptor agonist WIN 55,212-2 concentration-dependently (0.01-30 micro M) induced glycerol release above baseline ( E(max) 96.1+/-6.2% of isoprenaline-induced lipolytic response). The selective CB(2) agonist JWH-015 (0.01-30 micro M) had no lipolytic activity while the endocannabinoid 2-arachidonoylglycerol and the stable anandamide derivative, R(+)-methanandamide had, only a weak lipolytic effect at the highest concentrations employed (10 and 30 micro M). The concentration/response relationship for WIN 55,212-2-mediated lipolytic activity, mimicked by the S(-)-enantiomer WIN 55,212-3, was shifted significantly to the right by the CB(1) antagonist AM 251 only at 10 micro M, but was not modified by the beta-adrenoceptor antagonist propranolol (1 micro M). The protein kinase inhibitor H-89, but not the two adenylyl cyclase inhibitors (+/-) N(6)- R-phenylisopropyladenosine (R-PIA, 1 micro M, a selective A(1) adenosine receptor agonist) or SQ 22,536 (50 micro M) significantly reduced the glycerol efflux induced by WIN 55,212-2. Our data suggest that the cannabinoid drug WIN 55,212-2 may exert lipolytic activity in male rat adipocytes via an intracellular mechanism, not activated by CB(1) or CB(2) receptor stimulation, significantly reversed by H-89 but not clearly linked to stimulation of adenylyl cyclase.

Cannabinoids, the active components of marijuana, are presumed to affect memory by an action on long-term potentiation. However, these molecules also reduce hippocampal glutamatergic neurotransmission. To distinguish the two activities, we studied the effects of the synthetic cannabinoid (R)-(+)-[2,3-Dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo-[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphtalenylmethanone (Win 55,212-2, 10 microM) on CA1 hippocampal responses generated by the activation of two independent pathways, one of which was potentiated with a tetanic stimulation. The results of the study show the following: (a) the drug does not affect long-term potentiation provided that the responses are compared to those obtained in the unpotentiated control pathway; (b) Win 55,212-2 increases post-tetanic potentiation.

Cannabinoid receptors are found in moderate density throughout the cerebral cortex. The anterior cingulate cortex (ACC) is of particular interest due its high level of cannabinoid receptors and role in behaviors known to be modulated by cannabinoids. These studies were conducted to determine the cellular localization of cannabinoid receptors and to compare the level of cannabinoid receptor binding with receptor-mediated G-protein activity in the rat ACC. Either ibotenic acid or undercut lesions were made in ACC, and brains were processed for [ 3 H]WIN 55,212-2 and WIN 55,212-2-stimulated [ 35 S]GTPγS autoradiography. Both cannabinoid receptors and receptor-activated G-proteins were highest in laminae I and VI of ACC in control tissue. Although similar levels of receptor binding were found in these laminae, significantly higher levels of receptor-activated G-proteins were found in lamina VI. Ibotenic acid lesions that destroyed ACC neurons decreased [ 3 H]WIN 55,212-2 binding by 60–70% and eliminated WIN 55,212-2-stimulated [ 35 S]GTPγS binding. In contrast, deafferentation of the ACC with undercut lesions had no significant effect on cannabinoid receptor binding or G-protein activation. These results indicate that cannabinoid receptors in laminae I and VI of the ACC are located on somatodendritic elements or axons intrinsic to the ACC. In addition, differences in the relative levels of cannabinoid binding sites and activated G-proteins between cortical laminae indicate that the efficiency of cannabinoid receptors for G-protein activation may vary within a specific brain region.

The extent to which acute and repeated administration of the CB(1) agonist WIN 55,212-2 would affect the stimulatory properties of amphetamine was assessed in Lewis rats. In the first experiment, Lewis rats were treated with either 1 mg/kg of WIN 55,212-2 or vehicle and subsequently treated with 2 mg/kg amphetamine. Acute treatment with WIN 55,212-2 initially increased locomotor activity and then attenuated the stimulating effect of amphetamine on locomotion and exploration (as measured by rears). In a separate experiment, Lewis rats were given daily injections of either WIN 55,212-2 (1 mg/kg) or vehicle for 10 days and the effects of amphetamine were assessed at 1 and 3 days following the last chronic cannabinoid treatment. Those rats, which had been treated with WIN 55,212-2, had an enhanced response to amphetamine with rearing but not with ambulatory movements, suggesting the occurrence of behavioral cross-sensitization to the ability of amphetamine to increase rearing. These data add to the growing evidence that there is at least some overlap between those neural systems acted upon by cannabinoids and those that are believed to be involved in incentive properties associated with other drugs of abuse.

RATIONALE: Cannabinoids such as delta(9)-tetrahydrocannabinol (delta(9)-THC) or WIN-55,212-2 (WIN-2) have psychoactive effects on cognition. As a result, the reinforcing properties of delta(9)-THC or WIN-2 may confound learning and memory tests with false negative results. It therefore seems advisable to assess the reinforcing properties of the drugs in the same behavioural model used for learning experiments. OBJECTIVE: We therefore developed conditioned place preference protocols in the open-field water maze and tested both delta(9)-THC (2 mg/kg) and WIN-2 (1 mg/kg and 3 mg/kg). Given that previous reports on cannabinoids have revealed conflicting data and that this was a novel behavioural test, we also tested the benzodiazepine receptor agonist diazepam (2.5 mg/kg). Some methodical refinements were appropriate in order to determine the behavioural strategy implemented by the animals. METHODS: All animals were injected intraperitoneally 30 min prior to training/testing. In experiment 1, male hooded Lister rats injected with drug were repeatedly placed on the drug-related platform and subsequently tested for place preference. In experiment 2, rats were trained to swim to the drug platform on drug days and to the vehicle platform on vehicle days. A series of probe trials was introduced to delineate what had been learned. Experiment 3 studied the effect of WIN-2 on spatial learning in the water maze. RESULTS: Neither WIN-2 nor delta(9)-THC induced place preference in the water maze. When trained in the swim procedure, however, WIN-2 was neutral, but Delta(9)-THC resulted in place aversion. Conversely, diazepam consistently produced place preference in both procedures. WIN-2 (3 mg/kg), however, produced a small learning deficit in the spatial water maze task. CONCLUSION: It appears that the reinforcing properties of delta(9)-THC and WIN-2 in the doses used here are different, despite them both being agonists at cannabinoid receptors within the central nervous system. The fact that delta(9)-THC may be aversively related to a particular context has implications for previous work reporting deficits in spatial learning. Language: en

The effects of the cannabinoid receptor agonist WIN 55,212-2 on endogenous extracellular glutamate levels in the prefrontal cortex of the awake rat and in primary cultures of rat cerebral cortex neurons were investigated. In the prefrontal cortex WIN 55,212-2 (0.1 and 1 mg/kg i.p.) increased dialysate glutamate levels from of the awake rat, while the lower (0.01 mg/kg) and the higher (2 mg/kg) doses were ineffective. Furthermore, the WIN 55,212-2 (0.1 mg/kg)- induced increase of dialysate glutamate levels was counteracted by pretreatment with the selective CB(1) receptor antagonist SR141716A (0.1 mg/kg i.p.) and by the local perfusion with a low-calcium Ringer solution (Ca(2+) 0.2 mM). In primary cultures of rat cerebral cortex neurons, WIN 55,212-2 (0.01--100 nM) increased extracellular glutamate levels, displaying a bell-shaped concentration-response curve. The facilitatory effect of WIN 55,212-2 (1 nM) was fully counteracted by SR141716A (10 nM), by the replacement of the normal Krebs Ringer-bicarbonate buffer with a low Ca(2+) medium (0.2 mM) and by the IP(3) receptor antagonist xestospongin C (1 microM). These in vivo and in vitro findings suggest an increase in cortical glutamatergic transmission by CB(1) receptors, an effect that may underlie some of the psychoactive and behavioural actions of acute exposure to marijuana.

Cannabinoid use is known to disrupt learning and memory in a number of species. cholecystokinin (CCK) release and CCK receptors have been implicated in spatial memory processes in rodents. Rat hippocampal CCK interneurons express cannabinoid 1 receptors (CB1). The CB1 agonist R(+)WIN 55,212-2 (WIN+), at 1 and 10 micromol, strongly inhibited potassium-evoked CCK release from rat hippocampal slices, while the inactive isomer S(-)WIN,55,212-3 (WIN-) had no effect. CCK release from cerebral cortical slices was not altered by WIN+.

The effects of the cannabinoid receptor agonist WIN 55,212-2 (0.1-5 mg/kg i.p.) on endogenous extracellular gamma-aminobutyric acid (GABA) levels in the cerebral cortex of the awake rat was investigated by using microdialysis. WIN 55,212-2 (1 and 5 mg/kg i.p.) was associated with a concentration-dependent decrease in dialysate GABA levels (-16% +/- 4% and -26% +/- 4% of basal values, respectively). The WIN 55,212-2 (5 mg/kg i.p.) induced-inhibition was counteracted by a dose (0.1 mg/kg i.p.) of the CB(1) receptor antagonist SR141716A, which by itself was without effect on cortical GABA levels. These findings suggest that cannabinoids decrease cortical GABA levels in vivo, an action that might underlie some of the cognitive and behavioral effects of acute exposure to marijuana.

Previous reports have indicated that administration of the central cannabinoid receptor (CB 1 ) antagonist SR141716A decreases intake of highly palatable food and drink. Disruption of normal food intake has been reported only at high doses known to disrupt spontaneous behaviors. The present study was designed to determine if rates of responding for normal food were sensitive to the effects of cannabinoid receptor blockade. Adult, male Sprague–Dawley rats were trained to lever press for normal food pellets under a fixed-ratio 15 (FR 15) schedule of reinforcement. SR141716A (0.3–3.0 mg/kg) produced dose-dependent reductions in response rate. WIN 55,212-2 (0.3 mg/kg), a high efficacy cannabinoid agonist, given as a pre-treatment to SR141716A, significantly attenuated the rate-suppressing effects of SR141716A, suggesting a principal role of CB 1 receptors in mediating these behavioral effects. These data indicate that high palatability is not necessary to observe an anorectic effect of SR141716A.

Learning and memory are often correlated with cellular changes within the hippocampus, and drugs or environmental factors which affect learning and memory will thus often induce observable morphological changes in this structure. Like tetrahydrocannabinol (THC) itself, many synthetic cannabinoids such as the CB-1 receptor agonist WIN 55,212-2 will induce learning and memory changes. In the current study, we investigate whether or not these changes could be related to structural changes within the hippocampus. Adult male Sprague-Dawley rats were injected twice daily (12:00 and 0:00 h) subcutaneously with WIN 55,212-2 (2.0 mg/kg) in DMSO or DMSO for 21 days. On day 22, animals were perfused and stained immunochemically for the dendritic marker MAP-2, or with cresyl violet. Morphometric analysis showed dendritic rearrangement with increased staining of MAP-2 in CA3 and the lower blade of the dentate gyrus. However, a loss of staining was observed in CA1. Counting of cresyl violet stained sections showed an apparent increase in granule cell number in the lower blade of the dentate gyrus. This work shows the potential for cannabinoids to influence hippocampal morphology. The pattern of changes may be similar to that seen after ischemic or toxic damage, but may be opposite to changes seen in stress.

Rationale: Δ9-Tetrahydrocannabinol (Δ9-THC), the main psychoactive ingredient of marijuana, as well as synthetic cannabinoid (CB1) receptor agonists, has led to negative or equivocal results when tested with the intravenous self-administration procedure, the best validated behavioural model for evaluating abuse liability of drugs in experimental animals. We recently reported, however, that the synthetic CB1 receptor agonist WIN 55,212-2 is intravenously self-administered by drug-naive mice and that its self-administration is blocked by the cannabinoid CB1 receptor antagonist SR 141716A. Objective: To assess a reliable model of cannabinoid intravenous self-administration in rats. Long Evans male rats were allowed the opportunity to self-administer WIN 55,212-2 at doses ranging from 6.25 to 50 µg/kg per injection, under a fixed-ratio 1 (FR1) schedule of reinforcement and nose-pokes as the operant responses. The effect of either a change in the unit drug dose available or a pretreatment with the specific CB1 receptor antagonist SR 141716A were then investigated (maintenance phase). Finally, the extinction of the self-administration behaviour was evaluated. Results: Response rate depended on the drug dose available, with maximum rates occurring at 12.5 µg/kg per injection. Response rate increased following pretreatment with the specific CB1 receptor antagonist, SR 141716A. Moreover, operant behaviour rapidly extinguished following both the substitution of saline or vehicle for cannabinoid and the disconnection of the drug delivery pumps. Conclusion: Rats will intravenously self-administer the synthetic CB1 receptor agonist WIN 55,212-2 under specific experimental conditions, thus allowing further investigation of the neurobiological mechanisms underlying cannabinoid-taking behaviour.

Intravenous administration of the cannabinoid CB(1) receptor agonists (R-(+)-[2, 3-Dihydro-5-methyl-3[morpholinyl)methyl]-pyrrolo[1,2,3-de]-1, 4-benzoxazinyl]-(1-naphthalenyl)methanone mesylate), WIN 55,212-2 (10, 37.5, 75 and 150 microg/kg), and ((6aR)-trans-3-(1, 1-Dimethylheptyl)-6a,7,10,10a-tetrahydro-1-hydroxy-6, 6-dimethyl-6H-dibenzo[b,d]pyran-9-methanol), HU 210 (1 and 4 microg/kg) dose-dependently increased acetylcholine release in dialysates from the prefrontal cortex and the hippocampus of freely moving rats. Administration of the cannabinoid receptor antagonist [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2, 4-dichlorophenyl)-4-methyl-1H-pyrazole-3 carboxamide]HCl, SR 141716A, at a dose that per se did not affect basal acetylcholine release (2. 5 microg/kg), prevented the increase of acetylcholine release by WIN 55,212-2 (150 microg/kg i.v.) or by HU 210 (4 microg/kg i.v.) in both areas. These data demonstrate that, at low i.v. doses, the synthetic cannabinoid CB(1) receptor agonists, WIN 55,212-2 and HU 210 stimulate cortical and hippocampal acetylcholine release.

A decline in neurogenesis in the hippocampus may underlie age-related memory impairment in rats and humans. We now show that WIN 55,212-2 administration for 3 weeks can partially restore neurogenesis in the hippocampus of aged rats. Cannabinoid receptor stimulation therapy may thus provide clinical benefit for humans with age-associated memory impairment.

The influence of saturated and unsaturated fatty acid ethanolamides as well as delta9-tetrahydrocannabinol (delta9-THC), WIN 55,212-2 and cannabinoid CB1 receptor antagonist SR 141716 on sea urchin fertilization was studied. The ethanolamides of arachidonic, oleic and linoleic acids but not saturated fatty acid (C14-C20) derivatives inhibited fertilization when pre-incubated with sperm cells. Delta9-THC and WIN 55,212-2 also inhibited fertilization, delta9-THC being ten times as potent as WIN 55,212-2. Selective cannabinoid CB1 receptor antagonist SR 141716 also blocked fertilization and did not antagonize the action of delta9-THC. The obtained results indicate that different unsaturated fatty acid ethanolamides may control sea urchin fertilization, and that sea urchin sperm cell cannabinoid receptor may differ from the known cannabinoid receptor subtypes.

The cross-tolerance and convergent dependence between morphine and the cannabimimetic agent R(+)-[2,3-dihydro-5-methyl-3[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-+ ++benzoxazin-yl]-(1-naphthalenyl) methanone mesylate (WIN 55,212-2) were assessed in vitro on guinea-pig ileum. To induce tolerance and dependence the myenteric plexus-longitudinal muscle was incubated at 37 degrees C for 5 h with a fixed concentration representing the IC50 for each compound. Myenteric plexus-longitudinal muscle exposed to WIN 55,212-2 (5 x 10(-8) M) was less sensitive to its inhibitory effect on electrically evoked contractions than naive myenteric plexus-longitudinal muscle. The exposure to cannabinoid induced a parallel rightward shift in the lower part of the concentration-response curve of WIN 55,212-2 and a marked reduction in the maximal inhibitory effect of the drug. Myenteric plexus-longitudinal muscle tolerant to WIN 55,212-2 was subsensitive to the inhibitory effect of morphine on the twitch response. The cross-tolerance between WIN 55,212-2 and morphine was bidirectional. In fact, after 5 h the morphine (10(-7) M)-incubated myenteric plexus-longitudinal muscle was less sensitive to the inhibitory effect of WIN 55,212-2. The tissue tolerant to morphine or WIN 55,212-2 was tested for the presence of physical dependence. Naloxone (10(-5) M) produced a typical withdrawal contracture in morphine-tolerant myenteric plexus-longitudinal muscle which could be reduced by a 15-min pretreatment with WIN 55,212-2 (5 X 10(-8) M). In contrast, SR141716 (10(-6) M) [N-(piperidino)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyr azole-carboxamide], a concentration which fully antagonized the inhibitory effect of WIN 55,212-2 (10(-7) M) in control preparations, did not produce significant contracture in WIN 55,212-2-tolerant myenteric plexus-longitudinal muscle. The mechanisms underlying the cross-tolerance and convergent dependence remain to be ascertained.

The globus pallidus has been identified as a site of action for the motor effects of cannabinoids. A previous report from this laboratory demonstrated that systemic administration of the potent and selective cannabinoid receptor agonist (R)-(+)-[2,3-dihydro-5-methyl-3-[(4-morpholinyl) methyl]pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl] (1-napthalenyl) methanone (WIN 55,212-2) inhibits rat pallidal neurons and reverses the inhibition of pallidal activity produced by electrical stimulation of the striatum. The current study used in vivo single unit electrophysiology/micropressure ejection to investigate whether the effects of cannabinoids on spontaneous activity and evoked inhibition in the globus pallidus are locally mediated. Micropressure ejection of either WIN 55,212-2 or CP 55,940 into the globus pallidus inhibited spontaneous activity in the globus pallidus. Local administration of the cannabinoid receptor antagonist, N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-carboxamide (SR141716A), did not produce an effect on its own but blocked the effect of WIN 55,212-2 on spontaneous activity of pallidal neurons. The decrease in pallidal activity produced by WIN 55,212-2 was not blocked by coadministration of bicuculline, suggesting this effect is independent of GABAA receptors. Micropressure ejection of cannabinoids into the globus pallidus did not reverse the inhibitory effect of striatal stimulation in the globus pallidus. Taken together, these findings suggest that pallidal cannabinoid receptors mediate an inhibition of spontaneous activity in the globus pallidus.

The effect of cannabinoid receptor activation and blockade on the propulsive activity in the mouse small intestine was assessed in the present study by measuring the transit of an orally administered, non-absorbable marker. The cannabinoid receptor agonist WIN 55,212-2 ( R (+)-[2,3-dihydro-5-methyl-3[(morpholinyl)methyl]pyrrolo[1,2,3-de-1,4benzoxazin-yl]-(1-naphthalenyl)methanone mesylate) inhibited, while the selective cannabinoid CB 1 receptor antagonist SR 141716A ( N -piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-carboxamide) stimulated the marker transit. Furthermore, a per se non-effective dose of SR 141716A reversed WIN 55,212-2-induced reduction of the transit. The results of the present study suggest a role for cannabinoid CB 1 receptors in the control of propulsive activity in the mouse small intestine.

The present investigation examines WIN 55,212-2 and AM630 at the cloned human cannabinoid CB1 receptor stably expressed in Chinese hamster ovary (CHO) cells. The effect of various concentrations of WIN 55,212-2 and AM630 on basal [35S]GTPgammaS binding to cell membranes was determined. WIN 55,212-2 (100 microM) stimulated basal [35S]GTPgammaS binding 77.9% with an EC50 value of 0.36 microM. Conversely, AM630 (100 microM) inhibited basal [35S]GTPgammaS binding by 20.9% with an EC50 value of 0.90 microM. These results show that WIN 55,212-2 is an agonist and AM630 is an inverse agonist in this system.

The role of nitric oxide (NO) in the development of cannabinoid tolerance was examined by using N ω -nitro- l -arginine methyl ester ( l -NAME) as an inhibitor of NO synthase. R (+)-[2,3-Dihydro-5-methyl-3 [(morpholinyl)methyl]pyrrolo[1,2,3-de]- 1,4-benzoxazin-yl]-(1-naphthalenyl)methanone mesylate (WIN 55,212-2), a cannabinoid receptor agonist, or l -NAME plus WIN 55,212-2 was acutely or chronically injected i.p. to mice and analgesia, body temperature and immobility were measured. A single injection of WIN 55,212-2 induced time- and dose-dependent analgesia, hypothermia and catalepsy. l -NAME (50 mg/kg), which per se was ineffective, administered 20 min before WIN 55,212-2 did not modify the analgesic, hypothermic and cataleptic responses to the cannabinoid. When WIN 55,212-2 was administered once a day, the animals became completely tolerant to the analgesic, hypothermic and cataleptic effects within five, seven and nine days respectively. l -NAME injected once daily 20 min before WIN 55,212-2 inhibited the development of tolerance to the hypothermic and cataleptic actions but not to the analgesic action of WIN 55,212-2. Since l -NAME given chronically by itself did not modify the analgesia, hypothermia and catalepsy induced by acute administration of WIN 55,212-2, our findings suggest l -NAME acts with some selectivity on the mechanisms involved in cannabinoid tolerance.

The purpose of the present study was to investigate the disruptive effects of cannabinoids on working memory as assessed in the eight-arm radial-maze. Systemic administration of delta 9-THC, WIN-55,212-2, and CP-55,940 increased the number of errors committed in the radial-maze. CP-55,940 was the most potent cannabinoid in impairing memory (ED50 = 0.13 mg/kg). delta 9-THC and WIN-55,212-2 disrupted maze-choice accuracy at equipotent doses (ED50 values = 2.1 and 2.2 mg/kg, respectively). In addition, systemic administration of each of these agents retarded completion time. Whereas the doses of delta 9-THC and CP-55,940 required to retard maze performance were higher than those needed to increase error numbers, WIN-55,212-2 was equipotent in both of these measures. On the other hand, neither anandamide, the putative endogenous cannabinoid ligand, nor cannabidiol, an inactive naturally occurring cannabinoid, had any apparent effects on memory. A second aim of this study was to elucidate the neuroanatomical substrates mediating the disruptive effects of cannabinoids on memory. Intrahippocampal injections of CP-55,940 impaired maze performance in a dose-dependent manner (ED50 = 8 micrograms/rat), but did not retard the amount of time required to complete the maze. The effects of intrahippocampal CP-55,940 were apparently specific to cognition because no other cannabinoid pharmacological effects (e.g., antinociception, hypothermia, and catalepsy) were detected. This dissociation between choice accuracy in the radial-maze and other cannabinoid pharmacological effects suggests that the working memory deficits produced by cannabinoids may be mediated by cannabinoid receptors in the hippocampus.

The effects of a potent synthetic cannabinoid WIN 55,212-2 on nociceptive responses of wide dynamic range (WDR) neurons in the lumbar spinal cord were investigated in anesthetized rats. WDR neurons were identified by their responses to innocuous brushing and to a range of pressure stimuli from innocuous to noxious. Noxious pressure was applied to regions of the ipsilateral hind paw corresponding to the receptive field of the neuron. WIN 55,212-2 (125 micrograms/kg and 250 micrograms/kg, i.v.) produced a profound inhibition of firing evoked by the noxious pressure stimulus. By contrast, the cannabinoid did not alter the evoked activity of non-nociceptive neurons in response to non-noxious levels of stimulation. Treatment with either vehicle or the inactive enantiomer WIN 55,212-3 (250 micrograms/kg) failed to alter noxious stimulus-evoked activity of WDR neurons. These data provide direct evidence for cannabinoid-mediated inhibition of pain neurotransmission in the spinal dorsal horn. The site of action for these effects remains to be determined.

The effects of the novel high affinity cannabinoid receptor agonist (+)-WIN 55,212-2 ((R)-4,5-dihydro-2-methyl-4(4-morphoinylmethyl)-1-(1-naphthalen ylcarbonyl)-6H-pyrrolo[3,2,1-ij]quinolin-6-one) on severity of dystonia were investigated in mutant Syrian hamsters with primary generalized dystonia. Following injections of (+)-WIN 55,212-2 (1.0-5.0 mg/kg i.p.) a dose-dependent reduction of the severity of dystonia was observed. At antidystonic doses (2.5 and 5.0 mg/kg i.p.) (+)-WIN 55,212-2 caused a reduction of spontaneous motor activity and catalepsy. 1 mg/kg of (+)-WIN 55,212-2 exhibited neither antidystonic effects nor any side effects. However, the coadministration of 1.0 mg/kg (+)-WIN 55,212-2 with an ineffective dose of diazepam (0.1 mg/kg i.p.) exerted antidystonic effects in the absence of severe side effects. Although psychotropic effects of cannabinoids, such as (+)-WIN 55,212-2, limit the therapeutical utility of cannabinoids, the present data indicate that cannabinoids exert antidystonic effects and that low doses of cannabinoids may increase antidystonic efficacy of benzodiazepines.

1 Mice pretreated intraperitoneally for 2 days with delta-9-tetrahydrocannabinol (delta-9-THC) at a dose of 20 mg kg−1 day−1 and then challenged intravenously with this drug, 24 h after the second pretreatment, showed a 6 fold tolerance to the hypothermic effect of delta-9-THC. This pretreatment also induced tolerance to the hypothermic effects of the cannabimimetic agents, CP 55,940 (4.6 fold) and WIN 55,212-2 (4.9 fold), but not to the hypothermic effect of the putative endogenous cannabinoid, anandamide. 2 Vasa deferentia removed from mice pretreated intraperitoneally with delta-9-THC twice at a dose of 20 mg kg−1 day−1 were less sensitive to its inhibitory effect on electrically-evoked contractions than vasa deferentia obtained from control animals. The cannabinoid pretreatment induced a 30 fold parallel rightward shift in the lower part of the concentration-response curve of delta-9-THC and a marked reduction in the maximal inhibitory effect of the drug. It also induced tolerance to the inhibitory effects on the twitch response of CP 55,940 (8.7 fold), WIN 55,212-2 (9.6 fold) and anandamide (12.3 fold). 3 The results confirm that cannabinoid tolerance can be rapid in onset and support the hypothesis that it is mainly pharmacodynamic in nature. The finding that in vivo pretreatment with delta-9-THC can produce tolerance not only to its own inhibitory effect on the vas deferens but also to that of three other cannabimimetic agents, suggests that this tissue would be suitable as an experimental model for investigating the mechanisms responsible for cannabinoid tolerance. 4 Further experiments are required to establish why tolerance to anandamide-induced hypothermia was not produced by a pretreatment with delta-9-THC that did induce tolerance to the hypothermic effects of delta-9-THC, CP 55,940 and WIN 55,212-2 and to the inhibitory effects of delta-9-THC, CP 55,940, WIN 55,212-2 and anandamide on the twitch response of the vas deferens.

Background The gastric myoelectric activity (GMA) is the electrical pacesetter potential, which drives gastric motility. Cannabinoids have broad-spectrum antiemetic and antinauseant activity. Paradoxically, they inhibit intestinal peristalsis and reduce gastric motility but their effect on GMA remains unknown. Methods Ferrets were surgically implanted with radiotelemetry transmitters to record GMA, body temperature and heart rate. The effect of WIN 55,212–2 (1 mg kg−1, i.p.), an agonist at the cannabinoid type 1 and 2 receptors was examined in conscious, unrestrained ferrets. WIN 55,212–2 was also compared to the anandamide upregulator URB 597 (5 mg kg−1, i.p.) for a potential to modulate the emetic response and behavioral changes induced by apomorphine (0.25 mg kg−1, s.c.). Key Results WIN 55,212–2 decreased GMA frequency (8.1 ± 0.4 cpm, compared to 9.6 ± 0.1 cpm in vehicle-treated animals, n = 6, P

Background In the absence of pathology, cannabinoid-induced depression of gastrointestinal (GI) motility is thought to be mediated primarily by CB1 receptors, whereas the role of CB2 receptors is still unclear. The aim of this work was to radiographically analyze the acute effect of the mixed cannabinoid agonist WIN 55,212-2 (WIN) on GI motor function in the rat, focusing on the involvement of CB1 and CB2 receptors. Methods Male Wistar rats received different doses of WIN and both psychoactivity (cannabinoid tetrad) and GI motility (radiographic analysis) were tested. The duration of WIN effect on GI motility was also radiographically analyzed. Finally, the involvement of the different cannabinoid receptors on WIN-induced alterations of GI motility was analyzed by the previous administration of selective CB1 (AM251) and CB2 (SR144528 or AM630) antagonists. After administration of contrast medium, alterations in GI motility were quantitatively evaluated in serial radiographs by assigning a compounded value to each region of the GI tract. Key Results Low, analgesic doses of WIN delayed intestinal transit, but high, psychoactive doses were required to delay gastric emptying. Acute WIN effects on GI motility were confined to the first few hours after administration. AM251 partially counteracted the effect of WIN on GI motility. Surprisingly, SR144528 (but not AM630) enhanced WIN-induced delayed gastric emptying. Conclusions & Inferences X-ray analyses confirm that cannabinoids inhibit GI motility via CB1 receptors; in addition, cannabinoids could influence motility through interaction with a SR144528-sensitive site. Further studies are needed to verify if such site of action is the CB2 receptor.

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.

A significant complaint associated with spinal cord injury (SCI) is chronic pain, which includes symptoms such as cutaneous hypersensitivity and spontaneous unevoked pain and is difficult to treat with currently available drugs. One complication with current analgesics is tolerance, a decrease in efficacy with repeated treatment over time. One promising class of pharmacological treatment is cannabinoid (CB) receptor agonists. The current study assessed the efficacy of the CB receptor agonist WIN 55,212-2 (WIN) in a rat model of neuropathic SCI pain. Brief spinal compression leads to significant hindpaw hypersensitivity to tactile stimulation. WIN dose-dependently increased withdrawal thresholds and continued to demonstrate efficacy over a twice-daily 7-day treatment regimen. By contrast, the efficacy of morphine in SCI rats decreased over the same treatment period. Similarly, the antinociceptive efficacy of WIN to acute noxious heat in uninjured rats diminished over time. These data suggest that the sustained efficacy of a CB receptor agonist for pain could depend on the pain state. Such agonists may hold promise for long-term use in alleviating chronic SCI pain.

Marijuana and many of its constituent cannabinoids influence the central nervous system (CNS) in a complex and dose-dependent manner. Although CNS depression and analgesia are well documented effects of the cannabinoids, the mechanisms responsible for these and other cannabinoid-induced effects are not so far known. The hydrophobic nature of these substances has suggested that cannabinoids resemble anaesthetic agents in their action, that is, they nonspecifically disrupt cellular membranes. Recent evidence, however, has supported a mechanism involving a G protein-coupled receptor found in brain and neural cell lines, and which inhibits adenylate cyclase activity in a dose-dependent, stereoselective and pertussis toxin-sensitive manner. Also, the receptor is more responsive to psychoactive cannabinoids than to non-psychoactive cannabinoids. Here we report the cloning and expression of a complementary DNA that encodes a G protein-coupled receptor with all of these properties. Its messenger RNA is found in cell lines and regions of the brain that have cannabinoid receptors. These findings suggest that this protein is involved in cannabinoid-induced CNS effects (including alterations in mood and cognition) experienced by users of marijuana.

The effects of the synthetic cannabinoid WIN 55,212-2 on forskolin-stimulated and basal adenosine 3':5'-cyclic monophosphate (cyclic AMP) accumulation in globus pallidus slices were investigated. WIN 55,212-2 caused a concentration-dependent decrease in forskolin-stimulated cyclic AMP accumulation in globus pallidus slices (maximum inhibition 36% for 30 microM). This effect was blocked by the cannabinoid receptor antagonist SR 141716A (100 microM). WIN 55,212-2 alone caused a concentration-dependent increase in cyclic AMP levels in unstimulated slices (maximum increase 52.6% for 100 microM). This effect was also blocked by SR 141716A (100 microM). In either forskolin-stimulated or unstimulated conditions SR 1417161A (100 microM) did not affect cyclic AMP levels.