The influence of beta-arrestin2 on cannabinoid CB1 receptor coupling to G-proteins and subcellular localization and relative levels of beta-arrestin1 and 2 in mouse brain.

CONTEXT: Beta-arrestins are known to couple to some G-protein-coupled receptors (GPCRs) to regulate receptor internalization, G-protein coupling and signal transduction, but have not been investigated for most receptors, and for very few receptors in vivo. Previous studies have shown that beta-arrestin2 deletion enhances the efficacy of specific cannabinoid agonists. OBJECTIVE: The present study hypothesized that brain cannabinoid CB1 receptors are regulated by beta-arrestin2. METHODS: Beta-arrestin2+/+ and -/- mice were used. Western blotting was used to determine the relative levels of each beta-arrestin subtype in mouse brain. Receptor binding was measured to determine whether deletion of beta-arrestin2 influences agonist binding to brain CB1 receptors, or the subcellular localization of CB1 in brain membranes subjected to differential centrifugation. A variety of cannabinoid agonists from different chemical classes were investigated for their ability to activate G-proteins in the presence and absence of beta-arrestin2 in cerebellum, hippocampus and cortex. RESULTS: No differences were found in the density of beta-arrestin1 or cannabinoid CB1 receptors in several brains of beta-arrestin2+/+ versus -/- mice. Differences between genotypes were found in the proportion of high- and low-affinity agonist binding sites in brain areas that naturally express higher levels of beta-arrestin2. Cortex from beta-arrestin2-/- mice contained less CB1 in the P1 fraction and more CB1 in the P2 fraction compared to beta-arrestin2+/+. Of the agonists assayed for activity, only Δ(9)-tetrahydrocannabinol (THC) exhibited a difference between genotypes, in that it was less efficacious in beta-arrestin2-/- than +/+ mouse membranes. CONCLUSION: Beta-arrestin2 regulates cannabinoid CB1 receptors in brain.

The endogenous brain constituent N-arachidonoyl L-serine is an activator of large conductance Ca2+-activated K+ channels.

The novel endocannabinoid-like lipid N-arachidonoyl L-serine (ARA-S) causes vasodilation through both endothelium-dependent and -independent mechanisms. We have analyzed the vasorelaxant effect of ARA-S in isolated vascular preparations and its effects on Ca(2+)-activated K(+) currents in human embryonic kidney cells stably transfected with the alpha-subunit of the human, large conductance Ca(+)-activated K(+) (BK(Ca)) channel [human embryonic kidney (HEK) 293hSlo cells]. ARA-S caused relaxation of rat isolated, intact and denuded, small mesenteric arteries preconstricted with (R)-(-)-1-(3-hydroxyphenyl)-2-methylaminoethanol hydrochloride (pEC(50), 5.49 and 5.14, respectively), whereas it caused further contraction of vessels preconstricted with KCl (pEC(50), 5.48 and 4.82, respectively). Vasorelaxation by ARA-S was inhibited by 100 nM iberiotoxin. In human embryonic kidney cells stably transfected with the alpha-subunit of the human BK(Ca) channel cells, ARA-S and its enantiomer, N-arachidonoyl-D-serine, enhanced the whole-cell outward K(+) current with similar potency (pEC(50), 5.63 and 5.32, respectively). The potentiation was not altered by the beta(1) subunit or mediated by ARA-S metabolites, stimulation of known cannabinoid receptors, G proteins, protein kinases, or Ca(2+)-dependent processes; it was lost after patch excision or after membrane cholesterol depletion but was restored after cholesterol reconstitution. BK(Ca) currents were also enhanced by N-arachidonoyl ethanolamide (pEC(50), 5.27) but inhibited by another endocannabinoid, O-arachidonoyl ethanolamine (pIC(50), 6.35), or by the synthetic cannabinoid O-1918 [(-)-1,3-dimethoxy-2-(3-3,4-trans-p-menthadien-(1,8)-yl)-orcinol] (pIC(50), 6.59), which blocks ARA-S-induced vasodilation. We conclude the following. 1) ARA-S directly activates BK(Ca) channels. 2) This interaction does not involve cannabinoid receptors or cytosolic factors but is dependent on the presence of membrane cholesterol. 3) Direct BK(Ca) channel activation probably contributes to the endothelium-independent component of ARA-S-induced mesenteric vasorelaxation. 4) O-1918 is a BK(Ca) channel inhibitor.

N-arachidonyl maleimide potentiates the pharmacological and biochemical effects of the endocannabinoid 2-arachidonylglycerol through inhibition of monoacylglycerol lipase.

Inhibition of the metabolism of the endocannabinoids, anandamide (AEA) and 2-arachidonyl glycerol (2-AG), by their primary metabolic enzymes, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively, has the potential to increase understanding of the physiological functions of the endocannabinoid system. To date, selective inhibitors of FAAH, but not MAGL, have been developed. The purpose of this study was to determine the selectivity and efficacy of N-arachidonyl maleimide (NAM), a putative MAGL inhibitor, for modulation of the effects of 2-AG. Our results showed that NAM unmasked 2-AG activity in a tetrad of in vivo tests sensitive to the effects of cannabinoids in mice. The efficacy of 2-AG (and AEA) to produce hypothermia was reduced compared with Delta(9)-tetrahydrocannabinol; however, 2-AG differed from AEA by its lower efficacy for catalepsy. All tetrad effects were partially CB(1) receptor-mediated because they were attenuated (but not eliminated) by SR141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-H-pyrazole-3-carboxamide HCl] and in CB(1)(-/-) mice. In vitro, NAM increased endogenous levels of 2-AG in the brain. Furthermore, NAM raised the potency of 2-AG, but not AEA, in agonist-stimulated guanosine 5'-O-(3-[(35)S]thio)triphosphate binding assay, a measure of G-protein activation. These results suggest that NAM is an MAGL inhibitor with in vivo and in vitro efficacy. NAM and other MAGL inhibitors are valuable tools to elucidate the biological functions of 2-AG and to examine the consequences of dysregulation of this endocannabinoid. In addition, NAM's unmasking of 2-AG effects that are only partially reversed by SR141716A offers support for the existence of non-CB(1), non-CB(2) cannabinoid receptors.

Multiple pathways involved in the biosynthesis of anandamide.

Endocannabinoids, including anandamide (arachidonoyl ethanolamide) have been implicated in the regulation of a growing number of physiological and pathological processes. Anandamide can be generated from its membrane phospholipid precursor N-arachidonoyl phosphatidylethanolamine (NAPE) through hydrolysis by a phospholipase D (NAPE-PLD). Recent evidence indicates, however, the existence of two additional, parallel pathways. One involves the sequential deacylation of NAPE by alpha,beta-hydrolase 4 (Abhd4) and the subsequent cleavage of glycerophosphate to yield anandamide, and the other one proceeds through phospholipase C-mediated hydrolysis of NAPE to yield phosphoanandamide, which is then dephosphorylated by phosphatases, including the tyrosine phosphatase PTPN22 and the inositol 5' phosphatase SHIP1. Conversion of synthetic NAPE to AEA by brain homogenates from wild-type and NAPE-PLD(-/-) mice can proceed through both the PLC/phosphatase and Abdh4 pathways, with the former being dominant at shorter (<10 min) and the latter at longer (60 min) incubations. In macrophages, the endotoxin-induced synthesis of anandamide proceeds uniquely through the phospholipase C/phosphatase pathway.

Synthesis of 2-(5Z,8Z,11Z,14Z)-Icosa-5,8,11,14-tetraenamidoethyl-d(4) Dihydrogen Phosphate, Tetra-deuterated pAEA.

A labile intermediate phospho-anandamide (2-(5Z,8Z,11Z,14Z)-icosa-5,8,11,14-tetraenamidoethyl dihydrogen phosphate, pAEA) has been identified in mouse brain and macrophages, but its precise quantitation was difficult because of its low concentration and chemical instability. We report the synthesis of tetra-deuterated pAEA from 2-aminoethyl dihydrogen phosphate-1,1,2,2-d(4) and (5Z,8Z,11Z,14Z)-2,5-dioxopyrrolidin-1-yl icosa-5,8,11,14-tetraenoate. The compound will be used to quantitate the pAEA necessary for a novel biosynthetic pathway.

Cannabinoid CB(2) receptor activation decreases cerebral infarction in a mouse focal ischemia/reperfusion model.

Cannabinoid CB(2) Receptor (CB(2)) activation has been shown to have immunomodulatory properties without psychotropic effects. The hypothesis of this study is that selective CB(2) agonist treatment can attenuate cerebral ischemia/reperfusion injury. Selective CB(2) agonists (O-3853, O-1966) were administered intravenously 1 h before transient middle cerebral artery occlusion (MCAO) or 10 mins after reperfusion in male mice. Leukocyte/endothelial interactions were evaluated before MCAO, 1 h after MCAO, and 24 h after MCAO via a closed cranial window. Cerebral infarct volume and motor function were determined 24 h after MCAO. Administration of the selective CB(2) agonists significantly decreased cerebral infarction (30%) and improved motor function (P<0.05) after 1 h MCAO followed by 23 h reperfusion in mice. Transient ischemia in untreated animals was associated with a significant increase in leukocyte rolling and adhesion on both venules and arterioles (P<0.05), whereas the enhanced rolling and adhesion were attenuated by both selective CB(2) agonists administered either at 1 h before or after MCAO (P<0.05). CB(2) activation is associated with a reduction in white blood cell rolling and adhesion along cerebral vascular endothelial cells, a reduction in infarct size, and improved motor function after transient focal ischemia.

Significance of cannabinoid CB1 receptors in improgan antinociception.

UNLABELLED: Improgan is a congener of the H(2) antagonist cimetidine, which produces potent antinociception. Because a) the mechanism of action of improgan remains unknown and b) this drug may indirectly activate cannabinoid CB(1) receptors, the effects of the CB(1) antagonist/inverse agonist rimonabant (SR141716A) and 3 congeners with varying CB(1) potencies were studied on improgan antinociception after intracerebroventricular (icv) dosing in rats. Consistent with blockade of brain CB(1) receptors, rimonabant (K(d) = 0.23 nM), and O-1691 (K(d) = 0.22 nM) inhibited improgan antinociception by 48% and 70% after icv doses of 43 nmol and 25 nmol, respectively. However, 2 other derivatives with much lower CB(1) affinity (O-1876, K(d) = 139 nM and O-848, K(d) = 352 nM) unexpectedly blocked improgan antinociception by 65% and 50% after icv doses of 300 nmol and 30 nmol, respectively. These derivatives have 600-fold to 1500-fold lower CB(1) potencies than that of rimonabant, yet they retained improgan antagonist activity in vivo. In vitro dose-response curves with (35)S-GTPgammaS on CB(1) receptor-containing membranes confirmed the approximate relative potency of the derivatives at the CB(1) receptor. Although antagonism of improgan antinociception by rimonabant has previously implicated a mechanistic role for the CB(1) receptor, current findings with rimonabant congeners suggest that receptors other than, or in addition to CB(1) may participate in the pain-relieving mechanisms activated by this drug. The use of congeners such as O-848, which lack relevant CB(1)-blocking properties, will help to identify these cannabinoid-like, non-CB(1) mechanisms. PERSPECTIVE: This article describes new pharmacological characteristics of improgan, a pain-relieving drug that acts by an unknown mechanism. Improgan may use a marijuana-like (cannabinoid) pain-relieving mechanism, but it is shown presently that the principal cannabinoid receptor in the brain (CB(1)) is not solely responsible for improgan analgesia.

Novel, potent THC/anandamide (hybrid) analogs.

The structure-activity relationship (SAR) of the end pentyl chain in anandamide (AEA) has been established to be very similar to that of Delta(9)-tetrahydrocannabinol (Delta(9)-THC). In order to broaden our understanding of the structural similarities between AEA and THC, hybrid structures 1-3 were designed. In these hybrids the aromatic ring of THC-DMH was linked to the AEA moiety through an ether linkage with the oxygen of the phenol of THC. Hybrid 1 (O-2220) was found to have very high binding affinity to CB1 receptors (K(i)=8.5 nM), and it is interesting to note that the orientation of the side chain with respect to the oxygen in the phenol is the same as in THCs. To further explore the SAR in this series the terminal carbon of the side chain was modified by adding different substituents. Several such analogs were synthesized and tested for their CB1 and CB2 binding affinities and in vivo activity (tetrad tests). The details of the synthesis and the biological activity of these compounds are described.

New potent and selective inhibitors of anandamide reuptake with antispastic activity in a mouse model of multiple sclerosis.

We previously reported that the compound O-2093 is a selective inhibitor of the reuptake of the endocannabinoid anandamide (AEA). We have now re-examined the activity of O-2093 in vivo and synthesized four structural analogs (O-2247, O-2248, O-3246, and O-3262), whose activity was assessed in: (a) binding assays carried out with membranes from cells overexpressing the human CB(1) and CB(2) receptors; (b) assays of transient receptor potential of the vanilloid type-1 (TRPV1) channel functional activity (measurement of [Ca(2+)](i)); (c) [(14)C]AEA cellular uptake and hydrolysis assays in rat basophilic leukaemia (RBL-2H3) cells; (d) the mouse 'tetrad' tests (analgesia on a hot plate, immobility on a 'ring', rectal hypothermia and hypolocomotion in an open field); and (e) the limb spasticity test in chronic relapsing experimental allergic encephalomyelitis (CREAE) mice, a model of multiple sclerosis (MS). O-2093, either synthesized by us or commercially available, was inactive in the 'tetrad' up to a 20 mg kg(-1) dose (i.v.). Like O-2093, the other four compounds exhibited low affinity in CB(1) (K(i) from 1.3 to >10 microM) and CB(2) binding assays (1.310 microM), very low potency as fatty acid amide hydrolase (FAAH) inhibitors (IC(50)>25 microM) and were inactive in the 'tetrad' up to a 30 mg kg(-1) dose (i.v.). While O-2247 and O-2248 were poor inhibitors of [(14)C]AEA cellular uptake (IC(50)>40 microM), O-3246 and O-3262 were quite potent in this assay. O-3246, which exhibits only a very subtle structural difference with O-2093, is the most potent inhibitor of AEA uptake reported in vitro under our experimental conditions (IC(50)=1.4 microM) and is 12-fold more potent than O-2093. When injected intravenously O-3246 and O-3262, again like O-2093 and unlike O-2247 and O-2248, significantly inhibited limb spasticity in mice with CREAE. These data confirm the potential utility of selective AEA uptake inhibitors as anti-spasticity drugs in MS and, given the very subtle chemical differences between potent and weak inhibitors of uptake, support further the existence of a specific mechanism for this process.

Evaluation of the role of the arachidonic acid cascade in anandamide's in vivo effects in mice.

The pharmacological profiles of the endocannabinoid anandamide and exogenous cannabinoids (e.g., Delta9-tetrahydrocannabinol) are similar, but not exactly the same. One notable difference is that anandamide's in vivo effects in mice are not blocked by the brain cannabinoid (CB1) receptor antagonist SR141716A. The degree to which the rapid metabolism of anandamide to arachidonic acid might be involved in this unexpected lack of effect was the focus of this study. Mice were tested in a tetrad of tests sensitive to cannabinoids, consisting of spontaneous locomotion, ring immobility, rectal temperature and tail flick nociception. Anandamide and arachidonic acid produced a similar profile of effects, but neither drug was blocked by SR141716A. When hydrolysis of anandamide was inhibited by an amidase inhibitor (phenylmethyl sulfonyl fluoride; PMSF), however, SR141716A significantly attenuated anandamide's effects but did not completely block them. Similarly, the effects of the metabolically stable anandamide analog O-1812 were attenuated by SR141716A. The role of oxidative metabolism in anandamide's effects in the tetrad was also investigated through pharmacological modulation of cyclooxygenase and lipoxygenase, two major classes of enzymes that degrade arachidonic acid. Whereas the non-selective cyclooxygenase inhibitor ibuprofen blocked the in vivo effects of arachidonic acid, it did not alter anandamide's effects. Other modulators of the cyclooxygenase and lipoxygenase pathways also failed to block anandamide's effects. Together, these results offer partial support for a pharmacokinetic explanation of the failure of SR141716A to antagonize the effects of anandamide; however, they also suggest that non-CB1, non-CB2 receptors may be involved in mediation of anandamide's in vivo actions, particularly at higher doses.

CB1 cannabinoid receptor-mediated modulation of food intake in mice.

1 Marijuana's appetite-increasing effects have long been known. Recent research suggests that the CB(1) cannabinoid receptor antagonist SR141716A may suppress appetite. This study represents a further, systematic investigation of the role of CB(1) cannabinoid receptors in the pharmacological effects of cannabinoids on food intake. 2 Mice were food-restricted for 24 h and then allowed access to their regular rodent chow for 1 h. Whereas the CB(1) antagonist SR141716A dose-dependently decreased food consumption at doses that did not affect motor activity, Delta(9)-tetrahydrocannabinol (Delta(9)-THC) increased food consumption at doses that had no effect on motor activity. O-3259 and O-3257, structural analogs of SR141716A, produced effects similar to those of the parent compound. 3 Amphetamine (a known anorectic) and diazepam (a benzodiazepine and CNS depressant) decreased food consumption, but only at doses that also increased or decreased motor activity, respectively. The CB(2) cannabinoid receptor antagonist SR144528 and the nonpsychoactive cannabinoid cannabidiol did not affect food intake nor activity. 4 SR141716A decreased feeding in wild-type mice, but lacked pharmacological activity in CB(1) knockout mice; however, basal food intake was lower in CB(1) knockout mice. Amphetamine decreased feeding in both mouse genotypes. 5 These results suggest that SR141716A may affect the actions of endogenous cannabinoids in regulating appetite or that it may have effects of its own aside from antagonism of cannabinoid effects (e.g., decreased feeding behavior and locomotor stimulation). In either case, these results strongly suggest that CB(1) receptors may play a role in regulation of feeding behavior.

Task specificity of cross-tolerance between Delta9-tetrahydrocannabinol and anandamide analogs in mice.

Relatively few studies have compared the effects of tetrahydrocannabinols and anandamide-like cannabinoids following repeated dosing. Whereas pronounced tolerance develops to many of the in vivo pharmacological effects of Delta9-tetrahydrocannabinol with repeated dosing, tolerance to anandamide-induced effects is typically less noted. In the present study, we examined cross-tolerance between Delta9-tetrahydrocannabinol and anandamide-like compounds (anandamide, 2-methylanandamide, and O-1812) in a tetrad of in vivo tests sensitive to cannabinoid action, including spontaneous activity, tail flick, rectal temperature, and a ring immobility test of catalepsy. Six intraperitoneal injections of Delta9-tetrahydrocannabinol 10 mg/kg over a period of 4 days resulted in the development of pronounced tolerance to all of its in vivo effects. In contrast, task specificity was observed in cross-tolerance to anandamide and its analogs: antinociception (all three compounds), suppression of spontaneous activity (2-methylanandamide and O-1812), catalepsy (O-1812), and hypothermia (none of the compounds). Furthermore, when it occurred, the magnitude of cross-tolerance was notably smaller. These results suggest that anandamide-like cannabinoids may have a unique pharmacology that only partially overlaps with that of Delta9-tetrahydrocannabinol and other traditional cannabinoids. Although the basis for this unique pharmacology has not as yet been determined, it is possible that regional specificity of cannabinoid CB1 receptor downregulation and endocannabinoid release induced by repeated dosing with Delta9-tetrahydrocannabinol may play a role.

Inhibition of monoacylglycerol lipase and fatty acid amide hydrolase by analogues of 2-arachidonoylglycerol.

The pharmacology of monoacylglycerol lipase (MAGL) is not well understood. In consequence, the abilities of a series of analogues of 2-arachidonoylglycerol (2-AG) to inhibit cytosolic 2-oleoylglycerol and membrane-bound anandamide hydolysis by MAGL and fatty acid amide hydrolase (FAAH), respectively, have been investigated. 2-AG and its 1-regioisomer (1-AG) interacted with MAGL with similar affinities (IC(50) values 13 and 17 mum, respectively). Shorter homologues of 2-AG (2-linoleoylglycerol and 2-oleoylglycerol) had affinities for MAGL similar to 2-AG. This pattern was also seen when the arachidonoyl side chain of arachidonoyl trifluoromethylketone was replaced by an oleoyl side chain. Arachidonoyl serinol (IC(50) value 73 microM) was a weaker inhibitor of MAGL than 2-AG. The IC(50) values of noladin ether towards MAGL and FAAH were 36 and 3 microM, respectively. Arachidonoyl glycine interacted with FAAH (IC(50) value 4.9 microM) but only weakly interacted with MAGL (IC(50) value >100 microM). alpha-Methyl-1-AG had similar potencies towards MAGL and FAAH (IC(50) values of 11 and 33 microM, respectively). O-2203 (1-(20-cyano-16,16-dimethyl-eicosa-5,8,11,14-tetraenoyl) glycerol) and O-2204 (2-(20-hydroxy-16,16-dimethyl-eicosa-5,8,11,14-tetraenoyl) glycerol) were slightly less potent, but again affected both enzymes equally. alpha-Methyl-1-AG, O-2203 and O-2204 interacted only weakly with cannabinoid CB(1) receptors expressed in CHO cells (K(i) values 1.8, 3.7 and 3.2 microM, respectively, compared with 0.24 microM for 1-AG) and showed no evidence of central cannabinoid receptor activation in vivo at doses up to 30 mg kg(-1) i.v. It is concluded that compounds like alpha-Methyl-1-AG, O-2203 and O-2204 may be useful as leads for the discovery of selective MAGL inhibitors that lack direct effects upon cannabinoid receptors.

6"-Azidohex-2"-yne-cannabidiol: a potential neutral, competitive cannabinoid CB1 receptor antagonist.

Previous experiments with the mouse vas deferens have shown that cannabidiol produces surmountable antagonism of cannabinoid CB(1) receptor agonists at concentrations well below those at which it binds to cannabinoid CB(1) receptors and antagonizes alpha(1)-adrenoceptor agonists insurmountably. It also enhances electrically evoked contractions of this tissue. We have now found that subtle changes in the structure of cannabidiol markedly influence its ability to produce each of these effects, suggesting the presence of specific pharmacological targets for this non-psychoactive cannabinoid. Our experiments were performed with cannabidiol, 6"-azidohex-2"-yne-cannabidiol, abnormal-cannabidiol and 2'-monomethoxy- and 2',6'-dimethoxy-cannabidiol. Of these, 6"-azidohex-2"-yne-cannabidiol was as potent as cannabidiol in producing surmountable antagonism of (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo-[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone (R-(+)-WIN55212) in vasa deferentia. However, it produced this antagonism with a potency that matched its cannabinoid CB(1) receptor affinity, suggesting that, unlike cannabidiol, it is a competitive cannabinoid CB(1) receptor antagonist. Moreover, since it did not enhance the amplitude of electrically evoked contractions, it may be a neutral cannabinoid CB(1) receptor antagonist.

Assessment of structural commonality between tetrahydrocannabinol and anandamide.

In order to make further structural comparisons between tetrahydrocannabinol and anandamide, substituents at C1 and C3 of the phenolic ring of tetrahydrocannnabinol were altered. In order to examine the alignment of the phenolic hydroxyl of tetrahydrocannnabinol with the hydroxyl group of anandamide, 1-fluoro-1-deoxy-tetrahydrocannnabinol analogs were prepared. These analogs had low affinity for the CB(1) cannabinoid receptor and were considerably less potent than tetrahydrocannnabinol in producing pharmacological effects in mice. These results suggest that these two oxygen moieties do not overlap. Additionally, the fact that a fluorine group can only accept hydrogen bonds suggest that the phenolic oxygen at the C1 position of tetrahydrocannnabinol donates electrons for hydrogen bonding rather than the hydrogen of the hydroxyl group interacting with the receptor. Additionally, substitution of a fluorine for the hydroxyl group at C1 led to analogs with higher affinity for CB(2) than CB(1) cannabinoid receptors, thereby underscoring a fundamental difference in the binding properties of these two receptor subtypes.

SR-141716A-induced stimulation of locomotor activity. A structure-activity relationship study.

The central cannabinoid receptor (CB(1)) antagonist, SR-141716A, has been used extensively to ascertain that cannabinoids interact with the CB(1) receptor. SR-141716A has been shown to produce effects opposite of cannabinoids when administered alone. It has been theorized that SR-141716A may act as an inverse agonist at the CB(1) receptor or by disinhibiting an endogenous cannabinoid tone. In an effort to ascertain the exact interaction between SR-141716A and the CB(1) receptor, we have conducted a structure-activity relationship study to compare CB(1) receptor affinity of SR-141716A analogs with their ability to produce an increase in locomotor activity. SR-141716A produced a significant increase in locomotor activity in mice within the first hour of administration. Twenty SR-141716A analogs from five different chemical series were also tested. Our data implicate particular regions of the SR-141716A molecule that may be involved in stimulation and depression of locomotor activity. When the K(I) of the analogs was plotted against the percent stimulation that each analog produced, it is evident that there is no correlation between the ability of the analogs to stimulate locomotor activity and their affinity for the CB(1) receptor. [35S]GTPgammaS binding data indicate that SR-141716A and five of the analogs are inverse agonists. However, none of the analogs demonstrating inverse agonism produce stimulation of locomotor activity. It is therefore concluded that the SR-141716A-induced stimulation in locomotor activity is not the result of inverse agonist activity at the CB(1) receptor or by disinhibition of an endogenous tone.

A comparison of the discriminative stimulus effects of delta(9)-tetrahydrocannabinol and O-1812, a potent and metabolically stable anandamide analog, in rats.

Efforts to determine whether Delta(9)-tetrahydrocannabinol (Delta(9)-THC) and anandamide elicit similar discriminative stimulus effects have yielded conflicting results. The difficulty in establishing a discriminative cue to anandamide may be due to its metabolic instability. Rats were trained to discriminate either Delta(9)-THC or O-1812, a metabolically stable anandamide analog, from vehicle to avoid this issue. O-1812 and Delta(9)-THC substituted for each other; however, both drugs were more potent in the O-1812-trained rats. Further, O-1812 only substituted for Delta(9)-THC at response rate decreasing doses. The CB(1) antagonist, SR141716A, blocked the discriminative stimulus effects of both drugs but augmented their rate effects. O-1839, a VR(1) agonist, failed to substitute for either cannabinoid. These results suggest that the discriminative stimulus effects of Delta(9)-THC and O-1812 are similar, but subtle differences also exist.

The CB2 cannabinoid receptor-selective agonist O-3223 reduces pain and inflammation without apparent cannabinoid behavioral effects.

Although Δ(9)-tetrahydrocannabinol (THC) and other mixed CB(1)/CB(2) receptor agonists are well established to elicit antinociceptive effects, their psychomimetic actions and potential for abuse have dampened enthusiasm for their therapeutic development. Conversely, CB(2) receptor-selective agonists have been shown to reduce pain and inflammation, without eliciting apparent cannabinoid behavioral effects. In the present study, we developed a novel ethyl sulfonamide THC analog, O-3223, and compared its pharmacological effects to those of the potent, mixed CB(1)/CB(2) receptor agonist, CP55,940, in a battery of preclinical pain models. Competitive cannabinoid receptor binding experiments revealed that O-3223 was approximately 80-fold more selective for CB(2) than CB(1) receptors. Additionally, O-3223 behaved as a full CB(2) receptor agonist in [(35)S]GTPγS binding. O-3223 reduced nociceptive behavior in both phases of the formalin test, reduced thermal hyperalgesia in the chronic constriction injury of the sciatic nerve (CCI) model, and reduced edema and thermal hyperalgesia elicited by intraplantar injection of LPS. These effects were blocked by pretreatment with the CB(2) receptor-selective antagonist SR144528, but not by the CB(1) receptor antagonist, rimonabant. Unlike CP55,940, O-3223 did not elicit acute antinociceptive effects in the hot-plate test, hypothermia, or motor disturbances, as assessed in the rotarod test. These data indicate that the CB(2) receptor-selective agonist, O-3223, reduces inflammatory and neuropathic nociception, without affecting basal nociception or eliciting overt behavioral effects. Moreover, this compound can serve as a template to develop new CB(2) receptor agonists with increased receptor selectivity and increased potency in treating inflammatory and neuropathic pain.

Structural and pharmacological analysis of O-2050, a putative neutral cannabinoid CB(1) receptor antagonist.

Rimonabant, the prototypic antagonist of cannabinoid CB(1) receptors, has been reported to have inverse agonist properties at higher concentrations, which may complicate its use as a tool for mechanistic evaluation of cannabinoid pharmacology. Consequently, recent synthesis efforts have concentrated on discovery of a neutral antagonist using a variety of structural templates. The purpose of this study was to evaluate the pharmacological properties of the putative neutral cannabinoid CB(1) receptor antagonist O-2050, a sulfonamide side chain analog of Δ(8)-tetrahydrocannabinol. O-2050 and related sulfonamide cannabinoids exhibited good affinity for both cannabinoid CB(1) and CB(2) receptors. While the other sulfonamide analogs produced cannabinoid agonist effects in vivo (e.g., activity suppression, antinociception, and hypothermia), O-2050 stimulated activity and was inactive in the other two tests. O-2050 also decreased food intake in mice, an effect that was reminiscent of that produced by rimonabant. Unlike rimonabant, however, O-2050 did not block the effects of cannabinoid agonists in vivo, even when administered i.c.v. In contrast, O-2050 antagonized the in vitro effects of cannabinoid agonists in [(35)S]GTPγS and mouse vas deferens assays without having activity on its own in either assay. Further evaluation revealed that O-2050 fully and dose-dependently substituted for Δ(9)-tetrahydrocannabinol in a mouse drug discrimination procedure (a cannabinoid agonist effect) and that it inhibited forskolin-stimulated cyclic AMP signaling with a maximum efficacy of approximately half that of the full agonist CP55,940 [(-)-cis-3-[2-hydroxy-4(1,1-dimethyl-heptyl)phenyl]-trans-4-(3-hydroxy-propyl)cyclohexanol]. Together, these results suggest that O-2050 is not a viable candidate for classification as a neutral cannabinoid CB(1) receptor antagonist.

Evaluation of prevalent phytocannabinoids in the acetic acid model of visceral nociception.

Considerable preclinical research has demonstrated the efficacy of Delta(9)-tetrahydrocannabinol (Delta(9)-THC), the primary psychoactive constituent of Cannabis sativa, in a wide variety of animal models of pain, but few studies have examined other phytocannabinoids. Indeed, other plant-derived cannabinoids, including cannabidiol (CBD), cannabinol (CBN), and cannabichromene (CBC) elicit antinociceptive effects in some assays. In contrast, tetrahydrocannabivarin (THCV), another component of cannabis, antagonizes the pharmacological effects of Delta(9)-THC. These results suggest that various constituents of this plant may interact in a complex manner to modulate pain. The primary purpose of the present study was to assess the antinociceptive effects of these other prevalent phytocannabinoids in the acetic acid stretching test, a rodent visceral pain model. Of the cannabinoid compounds tested, Delta(9)-THC and CBN bound to the CB(1) receptor and produced antinociceptive effects. The CB(1) receptor antagonist, rimonabant, but not the CB(2) receptor antagonist, SR144528, blocked the antinociceptive effects of both compounds. Although THCV bound to the CB(1) receptor with similar affinity as Delta(9)-THC, it had no effects when administered alone, but antagonized the antinociceptive effects of Delta(9)-THC when both drugs were given in combination. Importantly, the antinociceptive effects of Delta(9)-THC and CBN occurred at lower doses than those necessary to produce locomotor suppression, suggesting motor dysfunction did not account for the decreases in acetic acid-induced abdominal stretching. These data raise the intriguing possibility that other constituents of cannabis can be used to modify the pharmacological effects of Delta(9)-THC by either eliciting antinociceptive effects (i.e., CBN) or antagonizing (i.e., THCV) the actions of Delta(9)-THC.