The endocannabinoid system: physiology and pharmacology

The endogenous cannabinoid system is an ubiquitous lipid signalling system that appeared early in evolution and which has important regulatory functions throughout the body in all vertebrates. The main endocannabinoids (endogenous cannabis-like substances) are small molecules derived from arachidonic acid, anandamide (arachidonoylethanolamide) and 2-arachidonoylglycerol. They bind to a family of G-protein-coupled receptors, of which the cannabinoid CB(1) receptor is densely distributed in areas of the brain related to motor control, cognition, emotional responses, motivated behaviour and homeostasis. Outside the brain, the endocannabinoid system is one of the crucial modulators of the autonomic nervous system, the immune system and microcirculation. Endocannabinoids are released upon demand from lipid precursors in a receptor-dependent manner and serve as retrograde signalling messengers in GABAergic and glutamatergic synapses, as well as modulators of postsynaptic transmission, interacting with other neurotransmitters, including dopamine. Endocannabinoids are transported into cells by a specific uptake system and degraded by two well-characterized enzymes, the fatty acid amide hydrolase and the monoacylglycerol lipase. Recent pharmacological advances have led to the synthesis of cannabinoid receptor agonists and antagonists, anandamide uptake blockers and potent, selective inhibitors of endocannabinoid degradation. These new tools have enabled the study of the physiological roles played by the endocannabinoids and have opened up new strategies in the treatment of pain, obesity, neurological diseases including multiple sclerosis, emotional disturbances such as anxiety and other psychiatric disorders including drug addiction. Recent advances have specifically linked the endogenous cannabinoid system to alcoholism, and cannabinoid receptor antagonism now emerges as a promising therapeutic alternative for alcohol dependence and relapse.     

Cardiovascular effects of cannabinoids

The psychoactive properties of cannabinoids, the biologically active constituents of the marijuana plant, have long been recognized. Recent research has revealed that cannabinoids elicit not only neurobehavioral, and immunological, but also profound cardiovascular effects. Similar effects can be elicited by the endogenous ligand arachidonyl ethanolamine (anandamide) and 2-arachidonoyl-glycerol. The biological effects of cannabinoids are mediated by specific receptors. Two cannabinoid receptors have been identified so far: CB1-receptors are expressed by different cells of the brain and in peripheral tissues, while CB2-receptors were found almost exclusively in immune cells. Through the use of a selective CB1 receptor antagonist and CB1 receptor-knockout mice the hypotensive and bradycardic effects of cannabinoids in rodents could be attributed to activation of peripheral CB1 receptors. In hemodynamic studies using the radioactive microsphere technique in anesthetized rats, cannabinoids were found to be potent CB1-receptor dependent vasodilators in the coronary and cerebrovascular beds. Recent findings implicate the endogenous cannabinoid system in the pathomechanism of haemorrhagic, endotoxic and cardiogenic shock. Finally, there is evidence that the extreme mesenteric vasodilation, portal hypertension and systemic hypotension present in advanced liver cirrhosis are also mediated by the endocannabinoid system. These exciting, recent research developments indicate that the endogenous cannabinoid system plays an important role in cardiovascular regulation, and pharmacological manipulation of this system may offer novel therapeutic approaches in a variety of pathological conditions.     

Carbamoyl tetrazoles as inhibitors of endocannabinoid inactivation: a critical revisitation

We have synthesized a series of 18 1,5- and 2,5-disubstituted carbamoyl tetrazoles, including LY2183240 (1) and LY2318912 (7), two compounds previously described as potent inhibitors of the cellular uptake of the endocannabinoid anandamide, and their regioisomers 2 and 8. We confirm that compound 1 is a potent inhibitor of both the cellular uptake and, like the other new compounds synthesized here, the enzymatic hydrolysis of anandamide. With the exception of 9, 12, 15, and the 2,5-regioisomer of LY2183240 2, the other compounds were all found to be weakly active or inactive on anandamide uptake. Several compounds also inhibited the enzymatic hydrolysis of the other main endocannabinoid, 2-arachidonoylglycerol, as well as its enzymatic release from sn-1-oleoyl-2-arachidonoyl-glycerol, at submicromolar concentrations. Four of the novel compounds, i.e. 3, 4, 17, and 18, inhibited anandamide hydrolysis potently (IC50=2.1-5.4nM) and selectively over all the other targets tested (IC50 >or= 10microM), thus representing new potentially useful tools for the inhibition of fatty acid amide hydrolase.     

Expression of endocannabinoid synthetic enzyme mRnas is correlated with cannabinoid 1 receptor mRNA in the mouse brain

The two principal endocannabinoids, N-arachidonoyl-ethanolamine (anandamide) and 2-arachidonoyl-glycerol (2-AG), are synthesized from arachidonic acid (AA) and AA is released as they are degraded. Therefore, the function of endocannabinoids is closely linked to AA, but the exact relationships have not been clarified, especially with respect to endocannabinoid metabolism. In the present study, oil rich in AA was administered (0, 100, 200 and 300 microl) orally to male mice for 7 days. Phospholipase D (PLD), fatty acid amide hydrolase (FAAH), diacyl-glycerol lipase (DAGL), monoacyl-glycerol lipase (MAGL) and cannabinoid 1 (CB1) receptor mRNA expressions were determined in the whole brain. No changes in the expression of any gene investigated were detected following AA treatment. However, it was demonstrated that the expression of the CB1 receptor was positively correlated with PLD, FAAH and DAGL expression. This suggests that expression of the CB1 receptor is closely coordinated with that of the enzymes which synthesize its ligands.     

Expression of endocannabinoid synthetic enzyme mRNAs is correlated with cannabinoid 1 receptor mRNA in the mouse brain

The two principal endocannabinoids, N-arachidonoyl-ethanolamine (anandamide) and 2-arachidonoyl-glycerol (2-AG), are synthesized from arachidonic acid (AA) and AA is released as they are degraded. Therefore, the function of endocannabinoids is closely linked to AA, but the exact relationships have not been clarified, especially with respect to endocannabinoid metabolism. In the present study, oil rich in AA was administered (0, 100, 200 and 300 m l) orally to male mice for 7 days. Phospholipase D (PLD), fatty acid amide hydrolase (FAAH), diacyl-glycerol lipase (DAGL), monoacyl-glycerol lipase (MAGL) and cannabinoid 1 (CB1) receptor mRNA expressions were determined in the whole brain. No changes in the expression of any gene investigated were detected following AA treatment. However, it was demonstrated that the expression of the CB1 receptor was positively correlated with PLD, FAAH and DAGL expression. This suggests that expression of the CB1 receptor is closely coordinated with that of the enzymes which synthesize its ligands.     

Role of the endocannabinoid system in the development of tolerance to alcohol

The present review evaluates the evidence that the endocannabinoid system plays in the development of tolerance to alcohol. The identification of a G-protein-coupled receptor, namely, the cannabinoid receptor (CB(1) receptor), which was activated by Delta(9)-tetrahydrocannabinol (Delta(9)-THC), the major psychoactive component of marijuana, led to the discovery of endogenous cannabinoid agonists. Until now, four fatty acid derivatives identified to be arachidonylethanolamide (AEA), 2-arachidonylglycerol (2-AG), 2-arachidonylglycerol ether (noladin ether) and virodhamine have been isolated from both nervous and peripheral tissues. Both AEA and 2-AG have been shown to mimic the pharmacological and behavioural effects of Delta(9)-THC. The role of the endocannabinoid system in the development of tolerance to alcohol was not known until recently. Recent studies from our laboratory have implicated for the first time a role for the endocannabinoid system in development of tolerance to alcohol. Chronic alcohol treatment has been shown to down-regulate CB(1) receptors and its signal transduction. The observed downregulation of CB(1) receptor function results from the persistent stimulation of the receptors by AEA and 2-AG, the synthesis of which has been shown to be increased by chronic alcohol treatment. The enhanced formation of endocannabinoids may subsequently influence the release of neurotransmitters. It was found that the DBA/2 mice, known to avoid alcohol intake, have significantly reduced CB(1) receptor function in the brain, consistent with other studies in which the CB(1) receptor antagonist SR 141716A has been shown to block voluntary alcohol intake in rodents. Similarly, activation of the CB(1) receptor system promoted alcohol craving, suggesting a role for the CB(1) receptor gene in excessive alcohol drinking behaviour and development of alcoholism. Ongoing investigations may lead to a better understanding of the mechanisms underlying the development of tolerance to alcohol and to develop therapeutic strategies to treat alcoholism.     

Brain endocannabinoid system is involved in fluoxetine-induced anorexia

In order to describe the effects of chronic fluoxetine administration on the brain endocannabinoid system in lean and obese Zucker rats, brain immunostaining for the CB1 and CB1-phosphorylated cannabinoid receptors was carried out. Obese Zucker rats showed significantly increased the numbers of neural cells positively immunostained for the CB1-phosphorylated receptor in the striatum, compared to their lean litter-mates. Chronic fluoxetine administration decreased the number of neural cells immunostained for CB1-phosphorylated receptor in several striatal and hippocampal regions of obese Zucker rats, compared to controls treated with saline. In contrast, no change in CB1-phosphorylated receptor immunostaining was observed in fluoxetine-treated lean rats, with respect to controls. Taken together, these results suggest the involvement of the hippocampal and striatal endocannabinoid receptor system in fluoxetine-induced anorexia in lean and obese Zucker rats.     

Brain endocannabinoid system is involved in fluoxetine-induced anorexia

Abstract

In order to describe the effects of chronic fluoxetine administration on the brain endocannabinoid system in lean and obese Zucker rats, brain immunostaining for the CB1 and CB1-phosphorylated cannabinoid receptors was carried out. Obese Zucker rats showed significantly increased the numbers of neural cells positively immunostained for the CB1-phosphorylated receptor in the striatum, compared to their lean litter-mates. Chronic fluoxetine administration decreased the number of neural cells immunostained for CB1-phosphorylated receptor in several striatal and hippocampal regions of obese Zucker rats, compared to controls treated with saline. In contrast, no change in CB1-phosphorylated receptor immunostaining was observed in fluoxetine-treated lean rats, with respect to controls. Taken together, these results suggest the involvement of the hippocampal and striatal endocannabinoid receptor system in fluoxetine-induced anorexia in lean and obese Zucker rats.     

Are anandamide and cannabinoid receptors involved in ethanol tolerance? A review of the evidence

There have been significant developments towards the elucidation of molecular and cellular changes in neuronal second messenger pathways involved in the development of tolerance to and dependence on ethanol (EtOH). The long-term exposure to EtOH has been shown to affect several aspects of neuronal signal transduction as well as ligand-gated ion channels and receptor systems, including the receptors that are coupled to the superfamily of GTP binding regulatory proteins (G-proteins). The recent identification of a G-protein coupled receptor that was activated by delta-9-tetrahydrocannabinol (THC), the major psychoactive component of marijuana, led to the discovery of endogenous agonists. One such agonist found to exist in mammalian brain was characterized to be an arachidonic acid (AA) metabolite and was named anandamide (AnNH). AnNH has been shown to bind specifically to the cannabinoid receptor (CB(1)) and mimic many of the pharmacological and behavioural effects of THC including tolerance development. The role of endocannabinoids and the CB(1) receptor signal transduction system in tolerance development to drugs of abuse has not been explored until recently. The findings presented in this review provide evidence for the first time that some of the pharmacological actions of EtOH including tolerance development may be mediated through participation of the endocannabinoid-CB(1) receptor signal transduction system. Recent studies have shown that chronic EtOH exposure produces downregulation of CB(1) receptors and an inhibition of CB(1) receptor agonist-stimulated GTPgammaS binding in mouse brain synaptic plasma membranes (SPM). The observed receptor downregulation results from the persistent stimulation of the receptors by the endogenous CB(1) receptor agonist AnNH, the synthesis of which is increased by chronic EtOH exposure. Further, the CB(1) receptor antagonist SR-141716A has been shown to block voluntary EtOH intake in rats and mice. Based on these studies, a hypothesis is presented to explain the possible involvement of the endocannabinoid system in the pharmacological and behavioural effects of EtOH.     

New N-methylpiperazinyl derivatives of bicyclic antiprotozoal compounds

The 4-methylpiperazinyl group was inserted as substituent at the bridgehead of bicyclic compounds or as terminal group of their aminoacyl and aminoalkyl side chains. The new compounds were tested in?vitro for their activities against the multidrug-resistant K(1) strain of Plasmodium falciparum and Trypanosoma brucei rhodesiense (STIB 900). The results were compared to those of formerly prepared analogues and of drugs in use. A couple of bicyclo-octyl ω-(4-piperazin-1-yl)alkanoates showed high antitrypanosomal (IC(50)≤0.087μM) and antiplasmodial activity (IC(50)≤0.06μM). The most active ω-(4-methylpiperazin-1-yl)alkyl-2-azabicyclo-nonane possessed higher antiplasmodial activity (IC(50)≤0.023μM) and selectivity (S.I.=IC(50) (Cytotox.)/IC(50) (P.?falciparum)=2188) than the antimalarial drug chloroquine (IC(50)=0.15μM, S.I.=1257).     

Nuclear magnetic resonance for studying recognition processes between anandamide and cannabinoid receptors

The understanding of the molecular basis of cannabinoid activity has greatly improved since the discovery of CB1 and CB2 receptors. In this paper, the ligand binding processes between the endogenous cannabimimetic ligand, anandamide (AEA), and the cannabinoid receptors from different parts of rat brain were studied by nuclear magnetic resonance spectroscopy. The NMR approach is based on the comparison of selective (R1(SE)) and non-selective (R1(NS)) proton spin-lattice relaxation rates of the ligand in the presence and absence of macromolecular receptors, as well as R1(NS) and R1(SE) temperature dependency analysis. From these studies, the ligand-receptor binding strength was evaluated on the basis of the calculation of the "affinity index". The derivation of the "affinity index" from chemical equilibrium kinetics for all systems allowed the comparison of the ability of anandamide to interact with cannabinoid receptors present in different brain sectors.     

Structural-activity relationship study on C-4 carbon atom of the CB1 antagonist SR141716: synthesis and pharmacological evaluation of 1,2,4-triazole-3-carboxamides

A series of 1,2,4-triazole-3-carboxamides has been prepared from alkyl-1,2,4-triazole-3-carboxylates under mild conditions. The ability of these triazoles to displace [3H]-CP55940 from CB1 cannabinoid receptor was measured. However, they showed only poor to moderate binding affinities, indicating that substitution of the C-4 pyrazole atom of the CB1 reference compound SR141716 by a nitrogen atom results in loss of affinity. Further investigations for functionality indicated that the compound 6a exhibited significant cannabinoid antagonistic properties in the mouse vas deferens functional assay. This leads us to the conclusion that 6a binds at a different CB1 binding site or at a new cannabinoid receptor subtype.     

Endocannabinoid system in cancer cachexia

PURPOSE OF REVIEW: More than 60% of advanced cancer patients suffer from anorexia and cachexia. This review focuses on the possible mechanisms by which the endocannabinoid system antagonizes cachexia-anorexia processes in cancer patients and how it can be tapped for therapeutic applications. RECENT FINDINGS: Cannabinoids stimulate appetite and food intake. Hepatocytes express functional cannabinoid type 1 receptors, activation of which increases the expression of lipogenic genes (e.g those encoding sterol regulatory element binding protein 1c, acetyl-coenzyme A carboxylase-1, and fatty acid synthase in the liver and hypothalamus) and increase de-novo fatty acid synthesis, which contributes to development of diet-induced obesity. Both ghrelin and cannabinoids stimulate AMP-activated protein kinase in the hypothalamus, whereas they inhibit it in the liver and adipose tissues. Both anandamide and synthetic cannabinoid type 1 receptor agonists such as HU210 and the plant-derived cannabinoid tetrahydro-cannabinol also significantly inhibit tumor necrosis factor-alpha. SUMMARY: Cannabinoid type 1 receptor activation stimulates appetite and promotes lipogenesis and energy storage. Further study of cancer-cachexia pathophysiology and the role of endocannabinoids will help us to develop cannabinoids without psychotropic properties, which will help cancer patients suffering from cachexia and improve outcomes of clinical antitumor therapy.     

Effect of dietary krill oil supplementation on the endocannabinoidome of metabolically relevant tissues from high-fat-fed mice

Background

Omega-3 polyunsaturated fatty acids (ω-3-PUFA) are known to ameliorate several metabolic risk factors for cardiovascular disease, and an association between elevated peripheral levels of endogenous ligands of cannabinoid receptors (endocannabinoids) and the metabolic syndrome has been reported. We investigated the dose-dependent effects of dietary ω-3-PUFA supplementation, given as krill oil (KO), on metabolic parameters in high fat diet (HFD)-fed mice and, in parallel, on the levels, in inguinal and epididymal adipose tissue (AT), liver, gastrocnemius muscle, kidneys and heart, of: 1) the endocannabinoids, anandamide and 2-arachidonoylglycerol (2-AG), 2) two anandamide congeners which activate PPARα but not cannabinoid receptors, N-oleoylethanolamine and N-palmitoylethanolamine, and 3) the direct biosynthetic precursors of these compounds.

Methods

Lipids were identified and quantified using liquid chromatography coupled to atmospheric pressure chemical ionization single quadrupole mass spectrometry (LC-APCI-MS) or high resolution ion trap-time of flight mass spectrometry (LC-IT-ToF-MS).

Results

Eight-week HFD increased endocannabinoid levels in all tissues except the liver and epididymal AT, and KO reduced anandamide and/or 2-AG levels in all tissues but not in the liver, usually in a dose-dependent manner. Levels of endocannabinoid precursors were also generally down-regulated, indicating that KO affects levels of endocannabinoids in part by reducing the availability of their biosynthetic precursors. Usually smaller effects were found of KO on OEA and PEA levels.

Conclusions

Our data suggest that KO may promote therapeutic benefit by reducing endocannabinoid precursor availability and hence endocannabinoid biosynthesis.     

Chronic ethanol consumption regulates cannabinoid CB1 receptor gene expression in selected regions of rat brain

AIMS: The aim of this study was to examine the effects of chronic ethanol consumption in cannabinoid CB(1) receptor gene expression in Wistar rats. METHODS: Rats were exposed to a bottle containing a solution of ethanol (10% v/v) and saccharin (0.25% w/v) for 52 days. At the end of this period, rats were killed by decapitation and cannabinoid CB(1) receptor gene expression was measured by in situ hybridization histochemistry. RESULTS: Our results indicated that chronic ethanol consumption reduced cannabinoid CB(1) receptor gene expression in caudate-putamen (CPu) (24%), ventromedial nucleus of the hypothalamus (VMN) (43%), CA1 (27%) and CA2 (22%) fields of hippocampus and increased dentate gyrus (DG) (30%). CONCLUSIONS: These results reveal for the first time that prolonged exposure to ethanol produces marked alterations in cannabinoid CB(1) receptor gene expression in selected regions of the rat brain, supporting an interaction between ethanol consumption and the endogenous cannabinoid receptor. Furthermore, these findings suggest that cannabinoid CB(1) receptor may be considered as a new pharmacological target for treating ethanol dependence.     

Three-dimensional quantitative structure-selectivity relationships analysis guided rational design of a highly selective ligand for the cannabinoid receptor 2

This paper describes a three-dimensional quantitative structure-selectivity relationships (3D-QSSR) study for selectivity of a series of ligands for cannabinoid CB1 and CB2 receptors. 3D-QSSR exploration was expected to provide design information for drugs with high selectivity toward the CB2 receptor. The proposed 3D computational model was performed by Phase and generated taking into account a number of structurally diverse compounds characterized by a wide range of selectivity index values. The model proved to be predictive, with r² of 0.95 and Q² of 0.63. In order to get prospective experimental validation, the selectivity of an external data set of 39 compounds reported in the literature was predicted. The correlation coefficient (r² = 0.56) obtained on this unrelated test set provided evidence that the correlation shown by the model was not a chance result. Subsequently, we essayed the ability of our approach to help the design of new CB2-selective ligands. Accordingly, based on our interest in studying the cannabinergic properties of quinolones, the N-(adamantan-1-yl)-4-oxo-8-methyl-1-pentyl-1,4-dihydroquinoline-3-carboxamide (65) was considered as a potential synthetic target. The log(SI) value predicted by using our model was indicative of high CB2 selectivity for such a compound, thus spurring us to synthesize it and to evaluate its CB1 and CB2 receptor affinity. Compound 65 was found to be an extremely selective CB2 ligand as it displayed high CB2 affinity (Ki = 4.9 nM), while being devoid of CB1 affinity (Ki > 10,000 nM). The identification of a new selective CB2 receptor ligand lends support for the practicability of quantitative ligand-based selectivity models for cannabinoid receptors. These drug discovery tools might represent a valuable complementary approach to docking studies performed on homology models of the receptors.     

Non-nucleoside HIV-1 reverse transcriptase inhibitors. Part 11: structural modulations of diaryltriazines with potent anti-HIV activity

A series of novel 6-naphthyloxy substituted DATA analogues bearing different substituents on the C-6 position of triazine ring were synthesized and evaluated for their in vitro anti-HIV activity in MT-4 cells. The results demonstrated that most of the compounds in this series are potent activity against HIV-1 with moderate to high selectivity. Among these analogues, two compounds exhibited excellent effect in inhibiting HIV-1 replication at nanomolar concentration (for compound 9h: IC(50)=9.3 nM, SI=15,385; for compound 9i: IC(50)=9.4 nM, SI=14,094), which are about 15-fold more active than nevirapine. In addition, several compounds are active against both HIV-1 and HIV-2, whose mechanism may be different from typical NNRTIs.     

Inhibition of PDGFR tyrosine kinase activity by a series of novel N-(3-(4-(pyridin-3-yl)-1H-imidazol-2-ylamino)phenyl)amides: a SAR study on the bioisosterism of pyrimidine and imidazole

A series of N-(3-(4-(pyridin-3-yl)-1H-imidazol-2-ylamino)phenyl)amides were synthesized and tested for inhibition of PDGFR and FLT3 autophosphorylation. The novel N-(3-(4-(pyridin-3-yl)-1H-imidazol-2-ylamino)phenyl)amides, obtained by replacement of the pyrimidine system in Imatinib (1) with an imidazole ring, exhibit potent inhibitory activity on PDGFR, similar to the parent compound (IC(50) (9e)=0.2 microM; IC(50) Imatinib (1)=0.3 microM). Selectivity hereby seems to be conserved, as shown by the lack of activity on FLT3, a closely related class III receptor tyrosine kinase, which is not affected by the parent compound Imatinib.