Brain Cannabinoid Receptors Research Articles

Age-related changes of anandamide metabolism in CB1 cannabinoid receptor knockout mice: correlation with behaviour.

Anandamide (N-arachidonoylethanolamine, AEA) and 2-arachidonoylglycerol (2-AG) are the most active endocannabinoids at brain (CB1) cannabinoid receptors. CD1 mice lacking the CB1 receptors ("knockout" [KO] mutants) were compared with wildtype (WT) littermates for their ability to degrade AEA through an AEA membrane transporter (AMT) and an AEA hydrolase (fatty acid amide hydrolase, FAAH). The age dependence of AMT and FAAH activity were investigated in 1- or 4-month-old WT and KO animals, and found to increase with age in KO, but not WT, mice and to be higher in the hippocampus than in the cortex of all animals. AEA and 2-AG were detected in nmol/mg protein (microm) concentrations in both regions, though the hippocampus showed approximately twice the amount found in the cortex. In the same regions, 2-AG failed to change across groups, while AEA was significantly decreased (approximately 30%) in hippocampus, but not in cortex, of old KO mice, when compared with young KO or age-matched WT animals. In the open-field test under bright light and in the lit-dark exploration model of anxiety, young KO mice, compared with old KO, exhibited a mild anxiety-related behaviour. In contrast, neither the increase in memory performance assessed by the object recognition test, nor the reduction of morphine withdrawal symptoms, showed age dependence in CB1 KO mice. These results suggest that invalidation of the CB1 receptor gene is associated with age-dependent adaptive changes of endocannabinoid metabolism which appear to correlate with the waning of the anxiety-like behaviour exhibited by young CB1 KO mice.

Cannabinoids on the brain.

Cannabis has a long history of consumption both for recreational and medicinal uses. Recently there have been significant advances in our understanding of how cannabis and related compounds (cannabinoids) affect the brain and this review addresses the current state of knowledge of these effects. Cannabinoids act primarily via two types of receptor, CB1 and CB2, with CB1 receptors mediating most of the central actions of cannabinoids. The presence of a new type of brain cannabinoid receptor is also indicated. Important advances have been made in our understanding of cannabinoid receptor signaling pathways, their modulation of synaptic transmission and plasticity, the cellular targets of cannabinoids in different central nervous system (CNS) regions and, in particular, the role of the endogenous brain cannabinoid (endocannabinoid) system. Cannabinoids have widespread actions in the brain: in the hippocampus they influence learning and memory; in the basal ganglia they modulate locomotor activity and reward pathways; in the hypothalamus they have a role in the control of appetite. Cannabinoids may also be protective against neurodegeneration and brain damage and exhibit anticonvulsant activity. Some of the analgesic effects of cannabinoids also appear to involve sites within the brain. These advances in our understanding of the actions of cannabinoids and the brain endocannabinoid system have led to important new insights into neuronal function which are likely to result in the development of new therapeutic strategies for the treatment of a number of key CNS disorders.

Evidence for a New G Protein-Coupled Cannabinoid Receptor in Mouse Brain

The purpose of these studies was to support the hypothesis that an undiscovered cannabinoid receptor exists in brain. [(35)S]GTP gamma S binding was stimulated by anandamide and WIN55212-2 in brain membranes from both CB(1)(+/+) and CB(1)(-/-) mice. In contrast, a wide variety of other compounds that are known to activate CB(1) receptors, including CP55940, HU-210, and Delta(9)-tetrahydrocannabinol, failed to stimulate [(35)S]GTP gamma S binding in CB(1)(-/-) membranes. In CB(1)(-/-) membranes, SR141716A affected both basal and anandamide- or WIN55212-2-induced stimulation of [(35)S]GTP gamma S binding only at concentrations greater than 1 microM. In CB(1)(+/+) membranes, SR141716A inhibited only 84% of anandamide and 67% of WIN55212-2 stimulated [(35)S]GTP gamma S binding with an affinity appropriate for mediation by CB(1) receptors (K(B) approximately 0.5 nM). The remaining stimulation seemed to be inhibited with lower potency (IC(50) approximately 5 microM) similar to that seen in CB(1)(-/-) membranes or in the absence of agonist. Further experiments determined that the effects of anandamide and WIN55212-2 were not additive, but that the effect of mu opioid, adenosine A1, and cannabinoid ligands were additive. Finally, assays of different central nervous system (CNS) regions demonstrated significant activity of cannabinoids in CB(1)(-/-) membranes from brain stem, cortex, hippocampus, diencephalon, midbrain, and spinal cord, but not basal ganglia or cerebellum. Moreover, some of these same CNS regions also showed significant binding of [(3)H]WIN55212-2, but not [(3)H]CP55940. Thus anandamide and WIN55212-2 seemed to be active in CB(1)(-/-) mouse brain membranes via a common G protein-coupled receptor with a distinct CNS distribution, implying the existence of an unknown cannabinoid receptor subtype in brain.

Conditioned place preference induced by the cannabinoid agonist CP 55,940: interaction with the opioid system.

Cannabinoids appear atypical as drugs of abuse since controversial data exist concerning the ability to lower the thresholds for electrical self-stimulation (Stark and Dews, 1980; Gardner et al., 1988; Gardner, 1992) and to support self-administration (Martellotta et al., 1998; Tanda et al., 2000) or conditioned place preference in animals (Lepore et al., 1995; Parker and Gillies, 1995; McGregor et al., 1996; Sañudo-Peña et al., 1997; Chaperon et al., 1998; Hutcheson et al., 1998; Mallet and Beninger, 1998; Cheer et al., 2000; Valjent and Maldonado, 2000). Opioids and cannabinoids share some pharmacological properties (Manzanares et al., 1999). The most interactions were found in antinociception (Welch and Stevens, 1992; Smith et al., 1994) and, to a lesser extent, in drug reinforcement (Chen et al., 1990; Vela et al., 1995; Tanda et al., 1997). In the present study we asked whether: (1) a potent synthetic cannabinoid receptor agonist, [(-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptil)-phenyl]-trans-4-(3-hydroxy propyl) cyclohexanol] (CP 55,940) (from 10 to 40 microg/kg), which binds to the brain cannabinoid receptors with high affinity (Herkenham et al., 1991), would induce conditioned place preference, in comparison with heroin (from 0.1 to 5 mg/kg); (2) what type of receptor was involved; (3) what kind of interaction there was between the two drugs, when given in combination, on reward. CP 55,940 elicited a conditioned place preference only at a dose of 20 microg/kg similar in intensity to that of heroin (2 mg/kg). The reinforcing properties of the cannabinoid agonist were fully antagonised by pretreatment with the brain cannabinoid receptor-1 (CB(1)) antagonist, [N-piperidino-5-(4-chlorophenyl) 1-(2,4-dichloro-phenyl)-4-methyl pyrazole-3-carboxamide hydrochloride] (SR 141716A) and naloxone. The combination of CP 55,940 and heroin, at the reinforcing doses, led to a reward which did not show any additive effect. Taken together these findings are important for understanding how the cannabinoids produce reward and the interconnection of the opioid and cannabinoid system in the motivation.

Pharmacological Properties of Cannabinoid Receptors in the Avian Brain: Similarity of Rat and Chicken Cannabinoid1 Receptor Recognition Sites and Expression of Cannabinoid2 Receptor‐Like Immunoreactivity in the Embryonic Chick Brain

Abstract: The pharmacological properties of brain cannabinoid receptors were investigated in brains of 35 day‐old chickens, since little is known about the avian cannabinoid system. The cannabinoid1 receptor‐selective antagonist ligand [3H]SR 141716A bound to chicken brain membranes with KD and Bmax values of 0.92±0.28 nM and 790±58 fmol/mg protein, respectively. The binding was inhibited by CP 55,940 with a pI50 value of 7.63±0.14 and by a series of compounds with the order of potency CP 55,940>R(+)WIN 55,212–2>R‐1 methanandamide≈DAK. S(−)WIN 55,212–3 and AM404 were without inhibitory effect at 1 μM. Similar results were found for rat brain membranes. For both rat and chicken brain membranes, addition of the non‐hydrolysable GTP analogues Gpp[NH]p and GTPγS shifted the CP 55,940 inhibition curve to the right, consistent with an intact coupling to G‐proteins in the preparations. Fatty acid amidohydrolase in chicken brain membranes was less sensitive to inhibition by phenylmethylsulphonyl fluoride and arachidonoyl serotonin than its rodent equivalent. However, when fatty acid amidohydrolase activity in the preparations was reduced by use of a lower assay membrane concentration, anandamide was found to inhibit the binding of [3H]SR 141716A to chicken membranes with a pI50 value of 6.39±0.16. Using a novel antibody raised to amino acids 346–359 from the C‐terminal tail of the human cannabinoid2 receptor, it was found that embryonic chick brain tissue (and embryonic chick neurones in primary culture) expressed a ∼53 kDa immunoreactive band. This immunoreactivity, which was prevented by preincubation of the antibody with the immunising peptide, was also seen in cells expressing the recombinant human cannabinoid2 receptor, but was not seen in adult chicken brain homogenates or in rat cerebellar homogenates. However, a “classical” cannabinoid2‐receptor component of [3H]WIN 55212‐2 binding (i.e. a fraction inhibited by low concentrations of the cannabinoid2‐receptor‐selective antagonist SR 144528) was not found.

Behavioral suppression induced by cannabinoids is due to activation of the arachidonic acid cascade in rats

Tetrahydrocannabinol (THC) is the principle psychoactive ingredient of marijuana and produces various psychoactive effects through the brain cannabinoid (CB1) receptor. The CB1 receptor belongs to the seven-transmembrane domain family of G-protein-coupled receptors and is involved in the arachidonic acid cascade in the brain. Few reports have attempted to clarify the functional role of endogenous cannabinoid and the arachidonic acid cascade through the CB1 receptor using a behavioral paradigm. Therefore, in this study, we clarified the mechanism of cannabinoid-induced suppression of lever pressing in rats, focusing on the arachidonic acid cascade as a novel second messenger of CB1 receptor. Δ 8-THC and the potent synthetic CB1 receptor agonist HU-210 dose-dependently inhibited lever-pressing performance. The Δ 8-THC-induced suppression was significantly antagonized by the cyclooxygenase (COX) inhibitors diclofenac (32 mg/kg, i.p.), aspirin (10 mg/kg, i.p.) and indomethacin (10 mg/kg, i.p.). The suppressive effect of HU-210 was also significantly antagonized by 32 mg/kg diclofenac. Prostaglandin E 2 (3.2 μg/rat, i.c.v.), the final product of the arachidonic acid cascade, significantly inhibited lever pressing similar to Δ 8-THC and HU-210. In conclusion, we found that suppression of lever-pressing behavior induced by cannabinoids was mediated through activation of the arachidonic acid cascade via the CB1 receptor. Therefore, it is possible that the psychoactive effects of cannabinoid are due to an increase in the formation of PGE 2 in the brain.

Levels, metabolism, and pharmacological activity of anandamide in CB(1) cannabinoid receptor knockout mice: evidence for non-CB(1), non-CB(2) receptor-mediated actions of anandamide in mouse brain.

Anandamide [arachidonylethanolamide (AEA)] appears to be an endogenous agonist of brain cannabinoid receptors (CB(1)), yet some of the neurobehavioral effects of this compound in mice are unaffected by a selective CB(1) antagonist. We studied the levels, pharmacological actions, and degradation of AEA in transgenic mice lacking the CB(1) gene. We quantified AEA and the other endocannabinoid, 2-arachidonoyl glycerol, in six brain regions and the spinal cord by isotope-dilution liquid chromatography-mass spectrometry. The distribution of endocannabinoids and their inactivating enzyme, fatty acid amide hydrolase, were found to overlap with CB(1) distribution only in part. In CB(1) knockout homozygotes (CB(1)-/-), the hippocampus and, to a lesser extent, the striatum exhibited lower AEA levels as compared with wild-type (CB(1)+/+) controls. These data suggest a ligand/receptor relationship between AEA and CB(1) in these two brain regions, where tonic activation of the receptor may tightly regulate the biosynthesis of its endogenous ligand. 2-Arachidonoyl glycerol levels and fatty acid amide hydrolase activity were unchanged in CB(1)-/- with respect to CB(1)+/+ mice in all regions. AEA and Delta(9)-tetrahydrocannabinol (THC) were tested in CB(1)-/- mice for their capability of inducing analgesia and catalepsy and decreasing spontaneous activity. The effects of AEA, unlike THC, were not decreased in CB(1)-/- mice. AEA, but not THC, stimulated GTPgammaS binding in brain membranes from CB(1)-/- mice, and this stimulation was insensitive to CB(1) and CB(2) antagonists. We suggest that non-CB(1), non-CB(2) G protein-coupled receptors might mediate in mice some of the neuro-behavioral actions of AEA.

Localization of the CB 1 cannabinoid receptor in the rat brain. An immunohistochemical study☆

The presence of central cannabinoid receptor (CB 1), involving the N-terminal 14 amino acid peptide, was demonstrated in the rat brain by immunohistochemistry. Intensely stained neurons were observed in the principal neurons of the hippocampus, striatum, substantia nigra, cerebellar cortex, including the Purkinje cells. Moderate CB 1-IR cell bodies and fibers were present in the olfactory bulb, cingulate, entorhinal and piriform cortical areas, amygdala and nucleus accumbens. The perivascular glial fibers have shown moderate to high density CB 1-IR in olfactoric and limbic structures. Low density was detected in the thalamus and hypothalamus and area postrema. The CB 1 receptor was widely distributed in the forebrain and sparsely in the hindbrain. These new data support the view that the endogenous cannabinoids play an important role in different neuronal functions as neuromodulators or neurotransmitters.

Biodistribution of [ 18f] SR144385 and [ 18f] SR147963: selective radioligands for positron emission tomographic studies of brain cannabinoid receptors

ABSTRACT. [ 18F] SR144385 and [ 18F] SR147963 were synthesized in a multistep reaction in which fluorine-18 was introduced by nucleophilic halogen displacement on a bromo precursor. The fluorine-18-labeled intermediate was deprotected and coupled with the appropriate alkyl amine to give the final products. Both radioligands had appropriate regional brain distribution for cannabinoid receptors with a target to nontarget ratio of 1.7 for [ 18F] SR147963 and 2.5 for [ 18F] SR144385 at 60 and 90 min postinjection, respectively. The uptake of both tracers was blocked with a 1 mg/kg dose of SR141716A.

Enhancement of anandamide formation in the limbic forebrain and reduction of endocannabinoid contents in the striatum of delta9-tetrahydrocannabinol-tolerant rats.

Recent studies have shown that the pharmacological tolerance observed after prolonged exposure to synthetic or plant-derived cannabinoids in adult rats is accompanied by down-regulation/desensitization of brain cannabinoid receptors. However, no evidence exists on possible changes in the contents of the endogenous ligands of cannabinoid receptors in the brain of cannabinoid-tolerant rats. The present study was designed to elucidate this possibility by measuring, by means of isotope dilution gas chromatography/mass spectrometry, the contents of both anandamide (arachidonoylethanolamide; AEA) and its biosynthetic precursor, N-arachidonoylphosphatidylethanolamine (NArPE), and 2-arachidonoylglycerol (2-AG) in several brain regions of adult male rats treated daily with delta9-tetrahydrocannabinol (delta9-THC) for a period of 8 days. The areas analyzed included cerebellum, striatum, limbic forebrain, hippocampus, cerebral cortex, and brainstem. The same regions were also analyzed for cannabinoid receptor binding and WIN-55,212-2-stimulated guanylyl-5'-O-(gamma-[35S]thio)-triphosphate ([35S]GTPgammaS) binding to test the development of the well known down-regulation/desensitization phenomenon. Results were as follows: As expected, cannabinoid receptor binding and WIN-55,212-2-stimulated [35S]GTPgammaS binding decreased in most of the brain areas of delta9-THC-tolerant rats. The only region exhibiting no changes in both parameters was the limbic forebrain. This same region exhibited a marked (almost fourfold) increase in the content of AEA after 8 days of delta9-THC treatment. By contrast, the striatum exhibited a decrease in AEA contents, whereas no changes were found in the brainstem, hippocampus, cerebellum, or cerebral cortex. The increase in AEA contents observed in the limbic forebrain was accompanied by a tendency of NArPE levels to decrease, whereas in the striatum, no significant change in NArPE contents was found. The contents of 2-AG were unchanged in brain regions from delta9-THC-tolerant rats, except for the striatum where they dropped significantly. In summary, the present results show that prolonged activation of cannabinoid receptors leads to decreased endocannabinoid contents and signaling in the striatum and to increased AEA formation in the limbic forebrain. The pathophysiological implications of these findings are discussed in view of the proposed roles of endocannabinoids in the control of motor behavior and emotional states.

Locomotor activity and occupancy of brain cannabinoid CB1 receptors by the antagonist/inverse agonist AM281.

The goals of this study were to examine the relationship between intravenous doses of the cannabinoid CB1 receptor antagonist AM281 (N-(morpholin-4-yl)-5-(4-iodophenyl)-1-(2, 4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide) and the degree of occupancy of this receptor, and to relate occupancy to the ability of this compound to antagonize the sedative effects of the cannabinoid receptor agonist WIN 55,212-2. Occupancy was determined by measuring the ability of intravenous doses of AM281 to inhibit in vivo binding of [(131)I]AM281 in brain areas, and locomotor activity was assessed by measuring the rate of beam crossings in a photocell apparatus. As previously documented, WIN 55,212-2 (1 mg/kg, i.v.) significantly reduced locomotor activity at early times after administration. Co-injection of AM281 (0.3 mg/kg i/v) and WIN 55, 212-2 restored the rate of beam crossings to that seen on injection of vehicle. In addition, AM281 (0.3 mg/kg i/v) approximately doubled locomotor activity between 60-120 min when injected alone. The IC(50) value for displacement of [(131)I]AM281 by AM281 was 0.45 mg/kg. These observations confirm earlier indications that AM281 is a CB1 receptor antagonist or inverse agonist and suggest the existence of an endogenous cannabinoid tone that moderates exploratory locomotor activity.

Effects of SR141716A on Diazepam Substitution for Δ 9-Tetrahydrocannabinol in Rat Drug Discrimination

Interaction of cannabinoids with GABAergic systems has been noted in a number of previous studies. In the present study, this interaction was examined in a drug-discrimination paradigm. Rats were trained to discriminate either Δ 9-tetrahydrocannabinol (Δ 9-THC; 3 mg/kg) or diazepam (2.5 mg/kg) from vehicle in two-lever drug discrimination procedures for food reinforcement. As in previous studies, diazepam partially substituted for Δ 9-THC, but only at high doses that also decreased response rates. In contrast, Δ 9-THC did not substitute for diazepam in any of the rats. Hence, cross-generalization of these two drugs was asymmetrical. When tested in combination with diazepam, the brain cannabinoid (CB1) receptor antagonist SR141716A did not block the partial substitution of diazepam for Δ 9-THC, nor did it antagonize the discriminative stimulus effects of diazepam in diazepam-trained rats. These results suggest that the partial overlap in the discriminative stimulus effects of Δ 9-THC and diazepam is not mediated by diazepam action at CB1 receptors. However, the fact that diazepam produced partial substitution for Δ 9-THC is consistent with a GABAergic component to cannabinoid drug discrimination.

Cannabis: Discrimination of “Internal Bliss”?

The recent discovery of arachidonylethanolamide (anandamide), an endogenous ligand for cannabinoid receptors, and the synthesis of SR141716A, a cannabinoid antagonist selective for brain cannabinoid (CB1) receptors, have provided new tools to explore the mechanisms underlying cannabis abuse and dependence. Drug discrimination is the animal model with the most predictive validity and specificity for investigation of the psychoactive effects of cannabinoids related to their abuse potential, because, unlike many other drugs of abuse, Δ 9-tetrahydrocannabinol (Δ 9-THC), the major psychoactive ingredient of marijuana, is not self-administered by animals. Results of Δ 9-THC discrimination studies have revealed that the subjective effects of cannabis intoxication are pharmacologically selective for centrally active cannabinoid compounds, and that cannabis action at CB1 receptors is involved in medication of these effects. Less clear is the role of endogenous cannabinoid system(s) in cannabis intoxication. Anandamide, named for a Sanskrit word for “internal bliss,” unreliably substitutes for Δ 9-THC. Further, substitution, when it is observed, occurs only at doses that also significantly decrease response rates. In contrast, Δ 9-THC and other structurally diverse cannabinoids fully substitute for Δ 9-THC at doses that do not substantially affect response rates. Attempts to train animals to discriminate anandamide (or SR141716A) have so far been unsuccessful. Preliminary evidence from drug discrimination studies with more metabolically stable anandamide analogs have suggested that these differences in the discriminative stimulus effects of Δ 9- THC and anandamide-like cannabinoids are not entirely due to pharmacokinetic factors, but the exact role of “internal bliss” in cannabis intoxication and dependence is still not completely understood.

A light and electron microscopic study of the CB1 cannabinoid receptor in primate brain

The immunohistochemical distribution and subcellular localization of the cannabinoid CB1 receptor was determined in the adult monkey using a polyclonal antiserum raised against the amino terminus of the rat CB1 receptor. At the level of light microscopy, our results generally parallel earlier studies investigating CB1 distribution in rodent brain with a few differences. In particular, high levels of receptor were found in the cortex, hippocampus, amygdala, cerebellum. However significant differences were also noted. The most striking differences were high levels of CB1 receptor in the monkey substantia nigra pars compacta, cerebellar Purkinje cells, and the principal cells of the hippocampus, while few receptors were found in the globus pallidus or substantia nigra pars reticulata. In contrast, in a previous study investigating the rat, using the same antibody, the opposite staining pattern was observed. At the electron microscopic level CB1 receptor was restricted to neurons. Here it was found both pre- and postsynaptically, particularly on dendritic spines and axon terminals. The CB1 receptor is widely distributed in higher brain regions in the monkey. While its distribution is similar to that in the rat, there are major differences, some of which may be significant when extrapolating the behavioral effects of cannabinoids observed in rodents to primates (e.g., humans). The ultrastructural localization of the CB1 receptor suggests that it modulates neuronal excitability by both pre- and postsynaptic mechanisms.

Synthesis of [18F] SR144385: a selective radioligand for positron emission tomographic studies of brain cannabinoid receptors

A cannabinoid receptor antagonist, SR144385, has been labeled with fluorine-18. [18F] SR144385 was synthesized in a multi-step reaction in which fluorine-18 was introduced by nucleophilic halogen displacement on a bromo precursor. The fluorine-18 labeled intermediate was deprotected and coupled with 1-aminopiperidine to give [18F]SR144385. The time for radiosynthesis, HPLC purification, and formulation was 2 hours from end-of-bombardment. [18F] SR144385 of high radiochemical purity was obtained at end-of-synthesis with an average (n=11) specific radioactivity of 1852 mCi/μmol and an average isolated, non-decay corrected radiochemical yield of 4% from potassium [18F] fluoride. Copyright © 1999 John Wiley & Sons, Ltd.

Synthesis of [18F] SR144385: a selective radioligand for positron emission tomographic studies of brain cannabinoid receptors

Synthesis of [18F] SR144385: a selective radioligand for positron emission tomographic studies of brain cannabinoid receptors

Substrate specificity and stereoselectivity of rat brain microsomal anandamide amidohydrolase.

Anandamide amidohydrolase (AAH) catalyzes the hydrolysis of arachidonylethanolamide (anandamide), an endogenous cannabinoid receptor ligand. To delineate the structural requirements of AAH substrates, rat brain microsomal AAH hydrolysis of a series of anandamide congeners was studied using two reverse-phase high-performance liquid chromatography (RP-HPLC) assays developed in our laboratory. Arachidonamide (1) was found to be the best substrate with an apparent Km of 2.34 mM and a Vmax of 2.89 nmol/min/mg of protein. Although anandamide (2) has a similar Km value, its Vmax is approximately one-half that of arachidonamide. N, N-Bis(2-hydroxyethyl)arachidonamide (3) was not hydrolyzed, suggesting specificity for unsubstituted or mono-N-substituted arachidonamides. Analogues with a methyl group at the 1'-position of the ethanolamido headgroup were also found to have greater resistance to enzymatic turnover and therefore increased metabolic stability. The enzyme exhibited high stereoselectivity as the rate of hydrolysis of (R)-alpha-methanandamide (2.4%) (anandamide = 100%) was about 10-fold lower than that of its (S)-enantiomer (23%). In contrast, (R)-beta-methanandamide was 6-times more susceptible (121%) than the (S)-beta-enantiomer (21%). Interestingly, an inverse correlation was shown between AAH stereoselectivity and the brain cannabinoid receptor affinity as the enantiomers with high receptor affinity displayed low susceptibility to hydrolysis by AAH. Metabolic stability is also imparted to analogues with a short hydrocarbon headgroup as well as to those possessing 2-monomethyl or 2,2-dimethyl substituents. 2-Arachidonylglycerol and racemic 1-arachidonylglycerol were shown to be excellent AAH substrates. To identify AAH inhibitors, hydrolysis of anandamide was also studied in the presence of a select group of cannabimimetics. Of these, (-)-Delta8-THC and SR141716A, a biarylpyrazole CB1 antagonist, were found to inhibit enzymatic activity. These newly defined enzyme recognition parameters should provide a foundation for the rational development of stable, therapeutically useful anandamide analogues with high receptor affinity.

Structure-activity relationships of pyrazole derivatives as cannabinoid receptor antagonists.

As a potent, specific antagonist for the brain cannabinoid receptor (CB1), the biarylpyrazole N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2, 4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141716A; 1) was the lead compound for initiating studies designed to examine the structure-activity relationships of related compounds and to search for more selective and potent cannabimimetic ligands. A series of pyrazole derivatives was designed and synthesized to aid in the characterization of the cannabinoid receptor binding sites and also to serve as potentially useful pharmacological probes. Therapeutically, such compounds may have the ability to antagonize harmful side effects of cannabinoids and cannabimimetic agents. Structural requirements for potent and selective brain cannabinoid CB1 receptor antagonistic activity included (a) a para-substituted phenyl ring at the 5-position, (b) a carboxamido group at the 3-position, and (c) a 2,4-dichlorophenyl substituent at the 1-position of the pyrazole ring. The most potent compound of this series contained a p-iodophenyl group at the 5-position, a piperidinyl carboxamide at the 3-position, and a 2,4-dichlorophenyl group at the 1-position of the pyrazole ring. The iodinated nature of this compound offers additional utility as a gamma-enriching SPECT (single photon emission computed tomography) ligand that may be useful in characterizing brain CB1 receptor binding in vivo.

Investigation of structural analogs of prostaglandin amides for binding to and activation of CB1 and CB2 cannabinoid receptors in rat brain and human tonsils.

Arachidonyl ethanolamide (anandamide, AEA) is an endogenously produced lipid amide which binds to and activates the CB1 and CB2 cannabinoid receptors. Further, administration of arachidonyl ethanolamide to animals produces many of the same physiological effects as classical and non-classical cannabinoids1,2. The CB1 cannabinoid receptors are found in the brain and central nervous system; the CB2 cannabinoid receptors are found in immune cells and tissue. Because arachidonyl ethanolamide is an arachidonic acid derivative, it is possible that it may be metabolized to a prostaglandin derivative. The carboxylic acid moiety is not necessary for binding to cyclooyxgenase, the enzyme which begins the arachidonic acid cascade3,4, suggesting that an ethanol amide derivative might be possible as a substrate for this enzyme. This lends potential physiological relevance to a prostaglandin amide.

A light and electron microscopic study of the CB1 cannabinoid receptor in the primate spinal cord.

The distribution of cannabinoid receptors was studied in the monkey spinal cord by immunocytochemistry and electron microscopy, using an antibody to the CB1 brain cannabinoid receptor. Large numbers of labelled neurons were observed in all portions of the grey matter of the spinal cord. These included small diameter 9-16 microm neurons in the dorsal horn, larger (40-60 microm) neurons in the intermediate grey, and very large (60-100 microm), motor neurons in the ventral horn. Reaction product was observed in dendrites postsynaptic to unlabelled axon terminals. Since cannabinoid receptor activation decreases neuronal excitability by several mechanisms, including inhibition of voltage dependent calcium channels, the dense staining of CB1 in dorsal horn neurons suggests that CB1 could reduce calcium influx through such channels in these neurons. This, in turn, could decrease calcium-dependent changes in synaptic transmission and decrease sensitisation to nociceptive stimuli in these neurons. Similarly, the dense staining of CB1 in ventral horn cells suggests that cannabinoid receptors could limit calcium influx through voltage dependent calcium channels in these neurons, and could be significant in terms of neuroprotection to these neurons.