Discriminative stimulus effects of the cannabinoid CB1 antagonist SR 141716A in rhesus monkeys pretreated with Δ9-tetrahydrocannabinol

Rationale Drug discrimination can be used to examine tolerance and dependence in agonist-treated animals by establishing an appropriate antagonist as a discriminative stimulus.Objective Establish intravenous SR 141716A as a discriminative stimulus in four rhesus monkeys pretreated with a relatively small dose of Δ⁹-tetrahydrocannabinol (Δ⁹-THC).Methods Rhesus monkeys received i.v. Δ⁹-THC (0.32 mg/kg) and discriminated i.v. SR 141716A (1 mg/kg) from vehicle while responding under a fixed ratio (FR) 5 schedule of stimulus-shock termination.Results The discriminative stimulus effects of SR 141716A were dose-dependent (ED₅₀=0.33 mg/kg) and were mimicked by the CB₁ antagonist AM 251 (ED₅₀=0.98 mg/kg), but not by a benzodiazepine (midazolam) or an N-methyl-D-aspartate antagonist (ketamine). An additional dose (0.32 mg/kg in addition to 0.32 mg/kg administered before the session) of Δ⁹-THC shifted the SR 141716A dose–effect curve 3-fold rightward. Omitting Δ⁹-THC before test sessions resulted in responding on the SR 141716A lever that was attenuated by subsequent administration of Δ⁹-THC (ED₅₀=0.13 mg/kg), CP 55940 (ED₅₀=0.013 mg/kg), and WIN 55212-2 (ED₅₀=0.35 mg/kg); midazolam and ketamine did not attenuate responding on the SR 141716A lever. SR 141716A (1 mg/kg) shifted the Δ⁹-THC and CP 55940 dose–effect curves 3.4-fold rightward; the WIN 55212-2 dose–effect curve was not significantly modified by a dose of 1 mg/kg of SR 141716A.Conclusions SR 141716A can be established as a discriminative stimulus in animals pretreated with Δ⁹-THC, and this assay is selective for cannabinoid activity. Differential antagonism of cannabinoids by SR 141716A might indicate that the mechanism of action of WIN 55212-2 is not identical to other cannabinoids. This study demonstrates that, under the appropriate conditions, drug discrimination has utility for examining cannabinoid dependence and withdrawal.     

Apparent affinity estimates of rimonabant in combination with anandamide and chemical analogs of anandamide in rhesus monkeys discriminating Δ9-tetrahydrocannabinol

Rationale  Anandamide and Δ⁹-tetrahydrocannabinol (Δ⁹-THC) sometimes produce different discriminative stimulus effects and, therefore, appear to differ in their mechanism of action. In order to understand the widespread use of cannabis and the therapeutic potential of cannabinoids, mechanisms responsible for behavioral effects need to be identified. Objective  Drug discrimination was used to compare the mechanism of action of Δ⁹-THC, anandamide, and two structural analogs of anandamide in rhesus monkeys. Materials and methods  Monkeys discriminated Δ⁹-THC (0.1 mg/kg i.v.) from vehicle. Δ⁹-THC, anandamide, methanandamide, and arachidonylcyclopropylamide (ACPA) were administered i.v. alone and in combination with at least one dose of rimonabant. Schild analysis and single-dose apparent affinity estimates were used to estimate the potency of rimonabant as an antagonist of each cannabinoid; these values were compared to examine whether the same receptors mediated discriminative stimulus effects. Results  Δ⁹-THC, ACPA, methanandamide, and anandamide produced greater than 96% of responses on the Δ⁹-THC lever. The ED₅₀ values were 0.024 mg/kg for Δ⁹-THC, 0.14 mg/kg for ACPA, 0.28 mg/kg for methanandamide, and 1.7 mg/kg for anandamide. The duration of action of Δ⁹-THC was 4–6 h and longer than the duration of action ACPA, methanandamide, and anandamide (i.e., each less than 50 min). Rimonabant surmountably antagonized the discriminative stimulus effects of each agonist, and the apparent affinity estimates (pA ₂ and pK _B values) were 6.24–6.83. Conclusions  Rimonabant can produce surmountable antagonism of the behavioral effects of not only Δ⁹-THC but also anandamide, methanandamide, and ACPA, and the interactions appear simple, competitive, and reversible. These cannabinoid agonists act at the same receptors to produce discriminative stimulus effects.     

Systemic or intrahippocampal cannabinoid administration impairs spatial memory in rats

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 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). ⁹-THC and WIN-55,212-2 disrupted mazechoice 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 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 µg/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.     

Cannabinoids of diverse structure inhibit two DOI-induced 5-HT(2A) receptor-mediated behaviors in mice

We have recently shown that the selective cannabinoid CB(1) receptor antagonist SR 141716A produces robust frequencies of head-twitch response (HTR) and ear-scratch response (ESR) in drug-naive mice. Both behaviors were potently blocked by the selective 5-HT(2A/C) receptor antagonist SR 46349B. Selective 5-HT(2A/C) agonists such as DOI also produce these behaviors in mice. The purpose of the present study was to: (1) investigate whether Delta(9)-tetrahydrocannabinol (Delta(9)-THC) and its analogs [Delta(8)-tetrahydrocannabinol (Delta(8)-THC), HU-210, CP 55,940, and WIN 55,212-2] can prevent the DOI-induced behaviors and (2) to see whether any correlation exists in the ID(50) potency order of these cannabinoids in inhibiting the DOI-induced HTR and ESR relative to their published ED(50) potency profiles in producing the tetrad of behaviors in mice. Thus, at 0 min, different groups of mice were injected intraperitoneally with either vehicle or varying doses of the following cannabinoids: Delta(9)-THC (0.25-20 mg/kg), Delta(8)-THC (2.5-20 mg/kg), HU-210 (0.02-0.5 mg/kg), CP 55,940 (0.004-0.5 mg/kg), and WIN 55,212-2 (0.5-10 mg/kg). Twenty minutes later, each mouse received an intraperitoneal injection of DOI (1 mg/kg) and the frequencies of DOI-induced behaviors (mean +/- S.E.M.) were recorded for the next 20 min. The tested cannabinoids reduced the frequencies of both DOI-induced HTR and ESR in a dose-dependent fashion. HU-210 was the most potent inhibitor of HTR, whereas CP 55,940 was most effective against ESR. The ID(50) potency order of cannabinoids in blocking the HTR is: HU-210 > CP 55,940 > WIN 55,212-2 > Delta(9)-THC > Delta(8)-THC, which is identical to their published order of potency in producing the tetrad of behaviors in mice. On the other hand, they had the following ID(50) potency order against the ESR: CP 55,940 > HU-210 > WIN 55,212-2 > Delta(9)-THC > Delta(8)-THC. The tested cannabinoids were 3-30 times more potent in preventing the ESR than the HTR. The data show that cannabinoids inhibit 5-HT(2A) receptor-mediated functions in a potent but differential manner.     

Cannabinoid CB1 receptor-mediated inhibition of the neurogenic vasopressor response in the pithed rat

The effects of two cannabinoid receptor agonists, R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]-pyrrolo[1, 2, 3-de]-1,4-benzoxazin-yl]-(1-naphthalenyl)methanone (WIN 55,212-2) and (-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol (CP-55,940), were studied on (i) the vasopressor response elicited in pithed rats by electrical stimulation of the sympathetic outflow and (ii) the release of ³H-noradrenaline and the vasoconstriction elicited in isolated rat tail arteries by transmural electrical stimulation. In pithed rats, the electrical (1Hz for 10s) stimulation of the preganglionic sympathetic nerve fibres increased diastolic blood pressure by about 30mmHg. This neurogenic vasopressor response (which under the conditions of our study was almost exclusively due to the release of catecholamines) was decreased by WIN 55-212,2 and CP-55,940 in a dose-dependent manner (inhibition by WIN 55,212-2 and CP-55,940, 0.1μmol/kg each, about 25–30%). The inhibition was identical in adrenalectomized rats and in animals with intact adrenals. The inhibitory action of WIN 55,212-2 and CP-55,940 was abolished by a dose of 0.03μmol/kg of the CB₁ receptor antagonist N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlo- rophenyl)-4-methyl-3-pyrazole-carboxamide (SR 141716), which, by itself, had no effect. WIN 55,212-2, CP-55,940 and SR 141716 failed to affect the vasopressor response to exogenous noradrenaline (1nmol/kg), which also increased diastolic blood pressure by about 30mmHg. In isolated rat tail arteries, the electrically (0.4Hz) evoked tritium overflow and vasoconstriction were not modified by WIN 55,212-2 and CP-55,940 (1μmol/l each). In conclusion, the neurogenic vasopressor response in the pithed rat can be modulated via cannabinoid CB₁ receptors probably located presynaptically on the postganglionic sympathetic nerve fibres innervating resistance vessels. Received: 4 April 1997 / Accepted: 10 May 1997     

Role of the cannabinoid system in the effects induced by nicotine on anxiety-like behaviour in mice

Rationale Acute behavioural effects and motivational responses induced by nicotine can be modulated by the endocannabinoid system supporting the existence of a physiological interaction between these two systems. Objectives The present study was designed to examine the possible involvement of the cannabinoid system in the anxiolytic- and anxiogenic-like responses induced by nicotine in mice. Methods Animals were only exposed once to nicotine. The acute administration of low (0.05) or high (0.8 mg/kg, s.c.) doses of nicotine produced opposite effects in the elevated plus-maze, i.e. anxiolytic- and anxiogenic-like responses, respectively. The effects of the pretreatment with the CB1 cannabinoid receptor antagonist, rimonabant (0.25, 0.5 and 1 mg/kg, i.p.), and the cannabinoid agonist, Δ9-tetrahydrocannabinol (Δ9-THC, 0.1 mg/kg, ip), were evaluated on the anxiolytic- and anxiogenic-like responses induced by nicotine. Results Rimonabant completely abolished nicotine-induced anxiolytic-like effects and increased the anxiogenic-like responses of nicotine, suggesting an involvement of CB1 receptors in these behavioural responses. On the other hand, Δ9-THC failed to modify nicotine anxiolytic-like responses but attenuated its anxiogenic-like effects. In addition, the association of non-effective doses of Δ9-THC and nicotine produced clear anxiolytic-like responses. Conclusions These results demonstrate that the endogenous cannabinoid system is involved in the regulation of nicotine anxiety-like behaviour in mice and provide new findings to support the use of cannabinoid antagonists in the treatment of tobacco addiction.     

Central mediation of the cannabinoid cue: activity of a selective CB1 antagonist, SR 141716A

Active cannabimimetic drugs are known to bind to two receptor subtypes: one, called CB1, is mainly localised in the central nervous system while the other (CB2) is expressed preferentially in the immune system. SR 141716A has been demonstrated to have a nanomolar affinity for CB1 receptor subtypes and a micromolar affinity for CB2 receptors. Moreover, it is an effective antagonist at these receptors both in vitro (antagonism of cannabinoid activity in vas deferens) and in vivo (suppression of the hypothermia elicited by WIN 55,212-2). The present experiments were thus undertaken to investigate the role of CB1 receptors in cannabinoid discrimination. Rats were trained to discriminate WIN 55,212-2 (0.3mg/kg s.c.) from saline in a standard operant (FR10) food rewarded discrimination procedure. Acquisition of the discrimination required 16 days on average and the ED(50) of WIN 55,212-2 was 0.032mg/kg s.c. CP55,940 and delta-9-tetrahydrocannabinol (Delta(9)-THC) generalised to the WIN 55,212-2 stimulus with the respective ED(50)s of 0.007mg/kg (s.c.) and 0.64mg/kg (p.o.). Pretreatment with SR 141716A antagonised the cue elicited by WIN 55,212-2 (ED(50) = 1.6mg/kg) as well as the generalisation to CP 55,940 (ED(50) = 0.08mg/kg) and to Delta(9)-THC (ED(50) = 0.15mg/kg). SR 140098 is a CB1 antagonist as potent as SR 141716A in vitro. This compound is unlikely to pass into the brain since it failed to displace [(3)H]-CP55, 940 from rat brain membranes ex vivo, and to reverse WIN 55,212-2-induced hypothermia. SR 140098, in contrast to SR 141716A, did not antagonise the WIN 55,212-2 stimulus. Taken together, the present results demonstrate that the brain CB1 receptor subtype mediates the cannabinoid cue.     

Effects of Δ<Superscript>9</Superscript>-THC and WIN-55,212-2 on place preference in the water maze in rats

Abstract Rationale. Cannabinoids such as Δ⁹-tetrahydrocannabinol (Δ⁹-THC) or WIN-55,212–2 (WIN-2) have psychoactive effects on cognition. As a result, the reinforcing properties of Δ⁹-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 Δ⁹-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 Δ⁹-THC induced place preference in the water maze. When trained in the swim procedure, however, WIN-2 was neutral, but Δ⁹-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 Δ⁹-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 Δ⁹-THC may be aversively related to a particular context has implications for previous work reporting deficits in spatial learning. Electronic Publication     

Receptor mechanism and antiemetic activity of structurally-diverse cannabinoids against radiation-induced emesis in the least shrew

Xenobiotic cannabinoid CB1/CB2-receptor agonists appear to possess broad-spectrum antiemetic activity since they prevent vomiting produced by a variety of emetic stimuli including the chemotherapeutic agent cisplatin, serotonin 5-HT3-receptor agonists, dopamine D2/D3-receptor agonists and morphine, via the stimulation of CB1-receptors. The purpose of this study was to evaluate whether structurally-diverse cannabinoids [Delta9-THC, (delta-9-tetrahydrocannabinol); (Delta8-THC, delta-8-tetrahydrocannabinol); WIN55,212-2, (R (+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)), methyl] pyrolol [1,2,3-de]-1,4 benzoxazinyl]-(1-naphthalenyl) methenone mesylate); and CP55,940, ((-)-3-[2-hydroxy-4-(1,1-dimethylheptyl]-4-[3-hydroxypropyl] cyclohexane-1-ol)), can prevent radiation-induced emesis. Exposure to total body radiation (0, 5, 7.5 and 10 Gy) caused robust emesis in the least shrew (Cryptotis parva) in a dose-dependent manner (ED50=5.99 (5.77-6.23) Gy) and all animals vomited at the highest tested dose of radiation. In addition, the radiation exposure reduced locomotor behaviors to a significant but mild degree in a non-dose-dependent fashion up to one hour post-treatment. Radiation-induced emesis (10 Gy) was blocked in a dose-dependent manner by the CB1/CB2-receptor agonists with the following ID50 potency order: CP55,940 (0.11 (0.09-0.12) mg/kg)>WIN55,212,2 (3.65 (3.15-4.23) mg/kg)=Delta8-THC (4.36 (3.05-6.22) mg/kg)>Delta9-THC (6.76 (5.22-8.75) mg/kg). Although the greater antiemetic potency and efficacy of Delta8-THC relative to its isomer Delta9-THC is unusual as the latter cannabinoid possesses higher affinity and potency for cannabinoid receptors in functional assays, the current data support the results of a clinical study in children suggestive of complete protection from emesis by Delta8-THC. This effect has not been clinically observed for Delta9-THC in cancer patients receiving chemo- or radiation-therapy. Cannabinoids prevented the induced emesis via the stimulation of cannabinoid CB1-receptors because the CB1 (SR141716A)--and not the CB2 (SR144528)--receptor antagonist reversed both the observed reduction in emesis frequency and shrew emesis protection afforded by either Delta9-THC or CP55,940 against radiation-induced emesis. These findings further suggest that the least shrew can be utilized as a versatile and inexpensive small animal model to rapidly screen the efficacy of investigational antiemetics for the prevention of radiation-induced emesis.     

Contrasting effects of different cannabinoid receptor ligands on mouse ingestive behaviour

This study characterized the effects of seven diverse cannabinoid receptor agonists (and one antagonist) on ingestive behaviour in nondeprived adult, male CD1 mice. Microstructural analysis of licking for a range of concentrations of condensed milk (10, 15 and 20%) was carried out following administration of vehicle or: Δ-tetrahydrocannabinol (Δ-THC) at 1, 3 or 6 mg/kg; CP55,940 at 10, 30 or 50 μg/kg; Win 55,212-2 at 0.5, 1 or 3 mg/kg; HU-210 at 0.01, 0.03 or 0.1 mg/kg; methanandamide at 1, 3 or 6 mg/kg; arachidonyl-2'-chloroethylamide at 1, 3 or 6 mg/kg and JWH133 at 1, 3 or 6 mg/kg. The cannabinoid receptor antagonist/inverse agonist rimonabant was also tested at 0.3, 1 or 3 mg/kg. Test sessions comprised three 30 s presentations of the milk concentrations separated by 10 s interpresentation intervals. The nonselective CB1 receptor agonists Δ-THC, CP55,940 and Win 55,212-2 increased the number of licks for condensed milk, primarily by a significant increase in bout number. The potent and nonselective CB1 receptor agonist HU-210 and the selective CB1 receptor agonists methanandamide and arachidonyl-2'-chloroethylamide did not significantly affect licking behaviour but did significantly increase the latency to start licking. The CB1 receptor antagonist rimonabant produced effects that were opposite in direction to those produced by Δ-THC, CP55,940 and Win 55,212-2. Finally, the selective CB2 receptor agonist JWH133 had no significant effects on behaviour. These data add to reports that cannabinoid agonists can enhance the appetitive aspects of feeding, but they also demonstrate that not all CB1 receptor agonists do this, and therefore the relationship between action at CB1 receptors and appetitive feeding effects is not straightforward.     

Δ9-THC training dose as a determinant for (R)-methanandamide generalization in rats

 The objective of this study was to examine if (R)-methanandamide, a metabolically stable chiral analog of the endogenous ligand anandamide, is a cannabimimetic with a lower efficacy than Δ⁹-THC. Employing a two-lever choice drug discrimination procedure, rats were trained to discriminate between 1.8, 3.0, or 5.6 mg/kg Δ⁹-tetrahydrocannabinol (Δ⁹-THC) and vehicle. Different training doses were used in order to create assays with different efficacy demands. Generalization tests with 18 mg/kg (R)-methanandamide yielded around 90% Δ⁹-THC responses in the two lower Δ⁹-THC training dose conditions. However, only around 60% Δ⁹-THC responses occurred in the 5.6 mg/kg Δ⁹-THC training dose condition in tests with 18 mg/kg (R)-methanandamide; a higher dose (30 mg/kg) produced even fewer Δ⁹-THC-appropriate responses in this group. Morphine did not substitute for Δ⁹-THC. In conclusion, the data with Δ⁹-THC and (R)-methanandamide indicate that cannabinoid agonists can have varying degrees of intrinsic activity at a receptor site, or may produce their behavioral actions through multiple mechanisms, or both. Received: 9 May 1998 / Final version: 28 May 1998     

An endocannabinoid signaling system modulates anxiety-like behavior in male Syrian hamsters

Rationale  An endocannabinoid signaling system has not been identified in hamsters. Objective  We examined the existence of an endocannabinoid signaling system in Syrian hamsters using neuroanatomical, biochemical, and behavioral pharmacological approaches. Materials and methods  The distribution of cannabinoid receptors was mapped, and membrane fatty-acid amide hydrolase (FAAH) activity and levels of fatty-acid amides were measured in hamster brain. The impact of cannabinoid CB₁ receptor blockade and inhibition of FAAH was evaluated in the elevated plus maze, rota-rod test, and models of unconditioned and conditioned social defeat. Results  A characteristic heterogeneous distribution of cannabinoid receptors was detected in hamster brain using [³H]CP55,940 binding and autoradiography. The FAAH inhibitor URB597 inhibited FAAH activity (IC₅₀ = 12.8 nM) and elevated levels of fatty-acid amides (N-palmitoyl ethanolamine and N-oleoyl ethanolamine) in hamster brain. Anandamide levels were not reliably altered. The cannabinoid agonist WIN55,212-2 (1– 10 mg/kg i.p.) induced CB₁-mediated motor ataxia. Blockade of CB₁ with rimonabant (5 mg/kg i.p.) induced anxiogenic-like behavior in the elevated plus maze. URB597 (0.1–0.3 mg/kg i.p.) induced CB₁-mediated anxiolytic-like effects in the elevated plus maze, similar to the benzodiazepine diazepam (2 mg/kg i.p.). Diazepam (2–6 mg/kg i.p.) suppressed the expression, but not the acquisition, of conditioned defeat. By contrast, neither URB597 (0.3–3.0 mg/kg i.p.) nor rimonabant (5 mg/kg i.p.) altered unconditioned or conditioned social defeat or rota-rod performance. Conclusions  Endocannabinoids engage functional CB₁ receptors in hamster brain to suppress anxiety-like behavior and undergo enzymatic hydrolysis catalyzed by FAAH. Our results further suggest that neither unconditioned nor conditioned social defeat in the Syrian hamster is dependent upon cannabinoid CB₁ receptor activation.     

Comparison of cannabinoid ligands affinities and efficacies in murine tissues and in transfected cells expressing human recombinant cannabinoid receptors.

Affinities and efficacies of several reference cannabinoid ligands were investigated at central and peripheral cannabinoid receptors in three different species (rat, mouse, and human). The tested compounds belong to different chemical classes such as classical and non-classical terpene derivatives (Delta(8)-THC, Delta(9)-THC, HU 210, CP 55,940, CP 55,244, CP 55,243 and CP 47,947), aminoalkylindole (WIN 55,212-2, WIN 55,212-3) and diarylpyrazole cannabinoids (SR 141716A, SR 144528). As cannabinoid receptors have been shown to be mainly coupled to Gi/o type G- proteins, and by using the [(35)S]-GTPgammaS nucleotide binding modulation, we characterized the functional activity of these ligands which can act as agonists (positive intrinsic activity), partial agonists (partial positive intrinsic activity), antagonists (no intrinsic activity), or inverse agonists (negative intrinsic activity). To our knowledge, some derivatives (Delta(8)-THC, WIN 55,212-3, CP 55,243 and CP 47,947) have never been characterized in [(35)S]-GTPgammaS binding assays and up to now, this study represents the largest survey of reference cannabinoids performed in unique experimental conditions and in the same laboratory.     

Agmatine enhances cannabinoid action in the hot-plate assay of thermal nociception

Agmatine–cannabinoid interactions are supported by the close association between cannabinoid CB₁ receptors and agmatine immunoreactive neurons and evidence that shared brain mechanisms underlie the pharmacological effects of agmatine and cannabinoid agonists. In the present study, we used the hot-plate assay of thermal nociception to determine if agmatine alters cannabinoid action through activation of imidazoline sites and/or alpha₂-adrenoceptors. WIN 55212-2 (1, 2 or 3 mg/kg, i.p.) or CP55,940 (1, 2 or 3 mg/kg, i.p.) administration increased hot-plate response latency. Agmatine (50 or 100 mg/kg, i.p.) was ineffective. Administration of agmatine (50 mg/kg, i.p.) with WIN 55212-2 (1, 2 or 3 mg/kg, i.p.) or CP55,940 (1, 2 or 3 mg/kg, i.p.) produced response-latency enhancement. Regression analysis indicated that agmatine increased the potency of WIN 55212-2 and CP55,940 by 3- and 4.4-fold, respectively, indicating synergy for both drug interactions. Idazoxan, a mixed imidazoline site/alpha₂-adrenoceptor antagonist, but not yohimbine (5 mg/kg, i.p.), a selective alphia₂-adrenoceptor antagonist, blocked response-latency enhancement produced by a combination of WIN 55212-2 (2 mg/kg) and agmatine. Response-latency enhancement produced by WIN 55212-2 (2 mg/kg) was blocked by SR 141716A (5 mg/kg, i.p.), a cannabinoid CB₁ receptor antagonist; attenuated by idazoxan (2 and 5 mg/kg); and not affected by yohimbine (5 mg/kg). These results demonstrate a synergistic interaction between agmatine and cannabinoid agonists and suggest that agmatine administration enhances cannabinoid action in vivo.     

Stimulation of voluntary ethanol intake by cannabinoid receptor agonists in ethanol-preferring sP rats

RATIONALE: Recent studies have shown that the cannabinoid CB1 receptor antagonist, SR 141716, is capable of reducing voluntary ethanol intake in rodents, suggesting the involvement of the CB1 receptor in the neural circuitry mediating the positive reinforcing properties of ethanol. OBJECTIVES: The present study extended to the agonists the investigation on the pharmacological manipulation of ethanol intake by cannabinoid agents. METHODS: Selectively bred, Sardinian alcohol-preferring (sP) rats were offered ethanol and water under the two-bottle free choice procedure with unlimited access for 24 h/day. RESULTS: The acute administration of WIN 55,212-2 (0.5-2 mg/kg; IP) and CP 55,940 (3-30 microg/kg; IP) induced a significant, dose-dependent increase in ethanol intake. Conversely, water consumption and intake of regular food and a highly palatable sucrose solution were not affected by treatment with WIN 55,212-2 and CP 55,940. The stimulatory effect of WIN 55,212-2 and CP 55,940 on ethanol intake was completely prevented by administration of SR 141716 (0.3 mg/kg; IP) and the opioid receptor antagonist, naloxone (0.1 mg/kg; IP). CONCLUSIONS: Administration of WIN 55,212-2 and CP 55,940 promoted voluntary ethanol intake in sP rats. This effect was mediated by stimulation of the cannabinoid CB1 receptor and required the activation of the endogenous opioid system. The results of the present study add further support to the hypothesis that the cannabinoid CB1 receptor is part of the neural substrate regulating ethanol intake. These results are also discussed in terms of WIN 55,212-2 and CP 55,940 administration possibly fixing to a higher level the hedonic set-point mechanism regulating ethanol drinking behavior in sP rats.     

Discriminative stimulus functions in rats of AM1346, a high-affinity CB1R selective anandamide analog

Objective  To characterize in vivo the high-affinity CB₁ cannabinoid receptor (CB₁R) selective anandamide analog AM1346 [alkoxyacid amide of N-eicosa-tetraenylamine] using drug discrimination. Substitution tests involved Δ⁹-tetrahydrocannabinol (Δ⁹-THC) and R-(+)-methanandamide (mAEA), a metabolically stable analog of anandamide (AEA), as well as the CB₁R antagonist/inverse agonist rimonabant; d-amphetamine and morphine were also examined to assess pharmacological specificity. Materials and methods  Rats were initially trained to discriminate between i.p.-injected vehicle and 3 mg/kg AM1346 (group 3 mg/kg; t′ = 20 min); subsequently, the rats were retrained with 5.6 mg/kg AM1346 (group 5.6 mg/kg; t′ = 20 min). Results  Dose-generalization curves of AM1346, Δ⁹-THC, and mAEA suggested the following order of potency: Δ⁹-THC > AM1346 > mAEA both for rats discriminating between 3 and 5.6 mg/kg AM1346 from vehicle. In group 3 mg/kg, challenge by 1 mg/kg rimonabant resulted in parallel shifts to the right of the dose-generalization curves for Δ⁹-THC and AM1346, suggesting surmountable antagonism. Surmountable antagonism was not demonstrated with rimonabant–mAEA combinations. A long duration of effect was indicated when 3 mg/kg AM1346 was examined after different time intervals following i.p. administration (group 3 mg/kg). The in vivo half-life was close to 5 h. Neither d-amphetamine nor morphine generalized in either of groups 3 mg/kg and 5.6 mg/kg, suggesting pharmacological specificity. Conclusion  Unlike mAEA, the surmountable antagonism between rimonabant and AM1346 showed that the structural features of AEA can be modified to produce novel ligands that reduce the dissociation between the discriminative stimulus and rate decreasing effects of CB₁R agonists derived from an AEA template.     

The role of cannabinoid receptors in intestinal motility, defaecation and diarrhoea in rats

We have studied the effects of the cannabinoid receptor agonists (R)-(+)[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2, 3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone (WIN 55,212-2, 0. 3-5 mg/kg, i.p.) and (-)-cis-3-[2-hydroxy-4-(1, 1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol) (CP 55,940, 0.03-1 mg/kg, i.p.), the cannabinoid CB(1) receptor antagonist (N-piperidin-1-yl)-5-(4-chlorophenyl)-1-2, 4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141716A, 0. 3-5 mg/kg, i.p.) and the cannabinoid CB(2) receptor antagonist N-[-(1S)-endo-1,3,3-trimethyl bicyclo [2.2.1] heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazo le- 3-carboxamide (SR144528, 1 mg/kg, i.p.) on intestinal motility, defaecation and castor-oil (1 ml/100 g rat, orally)-induced diarrhoea in the rat. SR141716A, but not SR144528, increased defaecation and upper gastrointestinal transit, while WIN 55,212-2 and CP 55,940 decreased upper gastrointestinal transit but not defaecation. WIN 55,212-3 (5 mg/kg), the less active enantiomer of WIN 55,212-2, was without effect. A per se non-effective dose of SR141716A (0.3 mg/kg), but not of SR144528 (1 mg/kg) or the opioid receptor antagonist, naloxone (2 mg/kg i.p.), counteracted the inhibitory effect of both WIN 55,212-2 (1 mg/kg) and CP 55,940 (0.1 mg/kg) on gastrointestinal motility. WIN 55,212-2 did not modify castor-oil-induced diarrhoea, while CP 55,940 produced a transient delay in castor-oil-induced diarrhoea at the highest dose tested (1 mg/kg), an effect counteracted by SR141715A (5 mg/kg). These results suggest that (i) intestinal motility and defaecation could be tonically inhibited by the endogenous cannabinoid system, (ii) exogenous activation of cannabinoid CB(1) receptors produces a reduction in intestinal motility in the upper gastrointestinal tract but not in defaecation, (iii) endogenous or exogenous activation of cannabinoid CB(2) receptors does not affect defaecation or intestinal motility and (iv) the cannabinoid receptor agonist, CP 55, 940, possesses a weak and transient antidiarrhoeal effect while the cannabinoid receptor agonist, WIN 55,212-2, does not possess antidiarrhoeal activity.     

Cannabinoid CB1 receptor-mediated inhibition of noradrenaline release in the human and guinea-pig hippocampus

We examined the question of whether cannabinoid receptors modulating noradrenaline release are detectable in the brain of humans and experimental animals. For this purpose, hippocampal slices from humans, guinea-pigs, rats and mice and cerebellar, cerebrocortical and hypothalamic slices from guinea-pigs were incubated with [³H]noradrenaline and then superfused. Tritium overflow was evoked either electrically (0.3 or 1Hz) or by introduction of Ca²⁺ ions (1.3μM) into Ca²⁺-free, K⁺-rich medium (25μM) containing tetrodotoxin 1μM. Furthermore, the cAMP accumulation stimulated by forskolin 10μM was determined in guinea-pig hippocampal membranes. We used the following drugs: the cannabinoid receptor agonists (–)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol (CP-55,940) and R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazin-yl]-(1-naphthalenyl)methanone (WIN 55,212-2), the inactive S(–)-enantiomer of the latter (WIN 55,212-3) and the CB₁ receptor antagonist N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-carboxamide (SR 141716). The electrically evoked tritium overflow from guinea-pig hippocampal slices was reduced by WIN 55,212-2 (pIC_30% 6.5) but not affected by WIN 55,212-3 up to 10μM. The concentration-response curve of WIN 55,212-2 was shifted to the right by SR 141716 (0.032μM) (apparent pA₂ 8.2), which by itself did not affect the evoked overflow. WIN 55,212-2 1μM also inhibited the Ca²⁺-evoked tritium overflow in guinea-pig hippocampal slices and the electrically evoked overflow in guinea-pig cerebellar, cerebrocortical and hypothalamic slices as well as in human hippocampal slices but not in rat and mouse hippocampal slices. SR 141716 (0.32μM) markedly attenuated the WIN 55,212-2-induced inhibition in guinea-pig and human brain slices. SR 141716 0.32μM by itself increased the electrically evoked tritium overflow in guinea-pig hippocampal slices but failed to do so in slices from the other brain regions of the guinea-pig and in human hippocampal slices. The cAMP accumulation stimulated by forskolin was reduced by CP-55,940 and WIN 55,212-2. The concentration-response curve of CP 55,940 was shifted to the right by SR 141716 (0.1μM; apparent pA₂ 8.3), which by itself did not affect cAMP accumulation. In conclusion, cannabinoid receptors of the CB₁ subtype occur in the human hippocampus, where they may contribute to the psychotropic effects of cannabis, and in the guinea-pig hippocampus, cerebellum, cerebral cortex and hypothalamus. The CB₁ receptor in the guinea-pig hippocampus is located presynaptically, is activated by endogenous cannabinoids and may be negatively coupled to adenylyl cyclase. Received: 5 June 1997 / Accepted: 6 August 1997     

Antiemetic and motor-depressive actions of CP55,940: cannabinoid CB1 receptor characterization,distribution, and G-protein activation

Dibenzopyran (Delta(9)-tetrahydrocannabinol) and aminoalkylindole [R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrolol[1,2,3-de]-1,4-benzoxazin-yl]-(1-naphthalenyl) methanone mesylate; (WIN55,212-2)] cannabinoids suppress vomiting produced by cisplatin via cannabinoid CB(1) receptors. This study investigates the antiemetic potential of the "nonclassical" cannabinoid CP55,940 [1alpha,2beta-(R)-5alpha]-(-)-5-(1,1-dimethyl)-2-[5-hydroxy-2-(3-hydroxypropyl) cyclohexyl-phenol] against cisplatin-induced vomiting and assesses the presence and functionality of cannabinoid CB(1) receptors in the least shrew (Cryptotis parva) brain. CP55,940 (0.025-0.3 mg/kg) reduced both the frequency of cisplatin-induced emesis (ID(50)=0.025 mg/kg) and the percentage of shrews vomiting (ID(50)=0.09 mg/kg). CP55,940 also suppressed shrew motor behaviors (ID(50)=0.06- 0.21 mg/kg) at such doses. The antiemetic and motor-suppressant actions of CP55,940 were countered by SR141716A [N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methylpyrazole-3-carboxamide], indicating both effects are cannabinoid CB(1) receptor-mediated. Autoradiographic studies with [3H]-SR141716A and [35S]-GTPgammaS binding revealed that the distribution of the cannabinoid CB(1) receptor and its activation pattern are similar to rodent brain and significant levels are present in brain loci (e.g., nucleus tractus solitarius (NTS)) that control emesis. The affinity rank order of structurally diverse cannabinoid ligands for cannabinoid CB(1) receptor in shrew brain is similar to rodent brain: HU-210=CP55,940=SR141716A>/=WIN55,212-2>/=delta-9-tetrahydrocannabinol>methanandamide=HU-211=cannabidiol=2-arachidonoylglycerol. This affinity order is also similar and is highly correlated to the cannabinoid EC(50) potency rank order for GTPgammaS stimulation except WIN55,212-2 and delta-9-tetrahydrocannabinol potency order were reversed. The affinity and the potency rank order of tested cannabinoids were significantly correlated with their antiemetic ID(50) potency order against cisplatin-induced vomiting (CP55,940>WIN55,212-2=delta-9-tetrahydrocannabinol) as well as emesis produced by 2-arachidonoylglycerol or SR141716A (CP55,940>WIN55,212-2>delta-9-tetrahydrocannabinol).     

Cannabinoid CB(1) antagonist SR 141716A attenuates reinstatement of heroin self-administration in heroin-abstinent rats

Rats with a previous history of heroin self-administration were studied to assess interactions occurring between cannabinoids and opioids in an animal model of reinstatement of heroin-seeking behaviour. Rats were trained to self-administer heroin and after a long-term extinction were primed with one of the following non-contingent non-reinforced drug administrations: saline (or vehicle), heroin, synthetic cannabinoid CB₁ receptor agonists (WIN 55,212-2 or CP 55,940), opioid antagonist (naloxone) or CB₁ antagonist (SR 141716A), alone or in combination. After primings, lever-pressing activity was recorded and compared to those observed during previous phases of training and extinction. Results of this study showed that (i) priming injections of heroin (0.1 mg/kg) as well as CB₁ agonists WIN 55,212-2 (0.15 or 0.30 mg/kg) and CP 55,940 (0.05 or 0.1 mg/kg) completely restore heroin-seeking behaviour; (ii) primings of naloxone (1 mg/kg) and SR 141716A (0.3 mg/kg) had no effect when administered alone; (iii) heroin-induced reinstatement was fully prevented by pre-treatment with either naloxone or SR 141716A; (iv) pre-treatment with SR 141716A significantly reduced WIN 55,212-2 and CP 55,940 priming effects. These results suggest that cannabinoid CB₁ receptors play an important role in the mechanisms underlying relapse to heroin-seeking and depict CB₁ antagonists as possible therapeutic agents for use in the prevention of relapse to heroin abuse.