Tetrahydrolipstatin analogues as modulators of endocannabinoid 2-arachidonoylglycerol metabolism

A series of 21 analogues of tetrahydrolipstatin (THL, 1) were synthesized and tested as inhibitors of the formation or hydrolysis of the endocannabinoid 2-arachidonoylglycerol (2-AG). Three of the novel compounds, i.e., 11, 13, and 15, inhibited 2-AG formation via the diacylglycerol lipase alpha (DAGLalpha) with IC 50 values lower than 50 nM (IC 50 of THL = 1 microM) and were between 23- and 375-fold selective vs 2-AG hydrolysis by monoacylglycerol lipase (MAGL) as well as vs cannabinoid CB 1 and CB 2 receptors and anandamide hydrolysis by fatty acid amide hydrolase (FAAH). Three other THL analogues, i.e., 14, 16, and 18, were slightly more potent than THL against DAGLalpha and appreciably selective vs MAGL, CB receptors, and FAAH (15-26-fold). One compound, i.e., 8, was a potent inhibitor of MAGL-like activity (IC 50 = 0.41 microM), and relatively ( approximately 7-fold) selective vs the other targets tested.     

Synthesis and pharmacological evaluation of 2,4-dinitroaryldithiocarbamate derivatives as novel monoacylglycerol lipase inhibitors

Monoacylglycerol lipase (MAGL) is responsible for signal termination of 2-arachidonoylglycerol (2-AG), an endocannabinoid neurotransmitter endowed with several physiological effects. Previously, we showed that the arylthioamide scaffold represents a privileged template for designing MAGL inhibitors. A series of 37 compounds resulting from pharmacomodulations around the arylthioamide template were synthesized and tested to evaluate their inhibitory potential on MAGL activity as well as their selectivity over fatty acid amide hydrolase (FAAH), another endocannabinoid-hydrolyzing enzyme. We have identified 2,4-dinitroaryldithiocarbamate derivatives as a novel class of MAGL inhibitors. Among the synthesized compounds, we identified [2,4-dinitrophenyl-4-(4-tert-butylbenzyl)piperazine-1-carbodithioate] (CK37), as the most potent MAGL inhibitor within this series (IC(50) = 154 nM). We have also identified [2,4-dinitrophenyl-4-benzhydrylpiperazine-1-carbodithioate] (CK16) as a selective MAGL inhibitor. These compounds are irreversible MAGL inhibitors that probably act by interacting with Cys208 or Cys242 and Ser122 residues of the enzyme. Moreover, CK37 is able to raise 2-arachidonoylglycerol (2-AG) levels in intact cells.     

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.     

Structural and energetic insights into the selective interactions of monoacylglycerol lipase with its natural substrate and small-molecule inhibitors

The monoacylglycerol lipase (MAGL) regulates 2-arachidonoyl glycerol (2-AG) level in the endocannabinoid system (ECS), which is implicated in a number of severe diseases such as cancer and Alzheimer’s disease. However, most existing MAGL inhibitors also show additional inhibitory effects on fatty acid amide hydrolase (FAAH), another member of the ECS that degrades the 2-AG analog N-arachidonoyl ethanolamine. Understanding of molecular mechanism and biological implication underlying the specific interactions in MAGL–ligand recognition is thus fundamentally important for the rational design of selective MAGL inhibitors. In the current study, the structural basis and energetic property regarding the binding of several MAGL inhibitors as well as its substrate 2-AG to both the MAGL and FAAH are investigated systematically by integrating molecular docking, quantum mechanics/molecular mechanics analysis, and Poisson–Boltzmann/surface area solvent model. In addition, a novel quantitative structure–selectivity relationship method is proposed to help in the explanation and prediction of inhibitor selectivity between MAGL and FAAH. It is suggested that the selectivity is primarily determined by the size, topology, and property of the rear moiety of inhibitor compounds; a bulky, bifurcated rear is the prerequisite for a inhibitor to have high selectivity for MAGL over FAAH, whereas those dual-type MAGL–FAAH inhibitors should possess a small, rear moiety—the ideal choice is a single aromatic branch occupying this position.     

Monoglyceride lipase-like enzymatic activity is responsible for hydrolysis of 2-arachidonoylglycerol in rat cerebellar membranes

2-Arachidonoylglycerol (2-AG) is an endogenous cannabinoid that binds to CB1 and CB2 cannabinoid receptors, inducing cannabimimetic effects. However, the cannabimimetic effects of 2-AG are weak in vivo due to its rapid enzymatic hydrolysis. The enzymatic hydrolysis of 2-AG has been proposed to mainly occur by monoglyceride lipase (monoacylglycerol lipase). Fatty acid amide hydrolase (FAAH), the enzyme responsible for the hydrolysis of N-arachidonoylethanolamide (AEA), is also able to hydrolyse 2-AG. In the present study, we investigated the hydrolysis of endocannabinoids in rat cerebellar membranes and observed that enzymatic activity towards 2-AG was 50-fold higher than that towards AEA. Furthermore, various inhibitors for 2-AG hydrolase activity were studied in rat cerebellar membranes. 2-AG hydrolysis was inhibited by methyl arachidonylfluorophosphonate, hexadecylsulphonyl fluoride and phenylmethylsulphonyl fluoride with ic(50) values of 2.2 nM, 241 nM and 155 microM, respectively. Potent FAAH inhibitors, such as OL-53 and URB597, did not inhibit the hydrolysis of 2-AG, suggesting that 2-AG is inactivated in rat cerebellar membranes by an enzyme distinct of FAAH. The observation that the hydrolysis of 1(3)-AG and 2-AG occurred at equal rates supports the role of MGL in 2-AG inactivation. This enzyme assay provides a useful method for future inhibition studies of 2-AG degrading enzyme(s) in brain membrane preparation having considerably higher MGL-like activity when compared to FAAH activity.     

Overview of the chemical families of fatty acid amide hydrolase and monoacylglycerol lipase inhibitors

The family of the endogenous agonists of the cannabinoid receptors--i.e., the endocannabinoids--includes several polyunsaturated fatty acid amides and esters. Arachidonoylethanolamide (anandamide, AEA) and 2-arachidon-oylglycerol (2-AG) are, respectively, the leads of these chemical families. So far, two enzymes responsible for the metabolism of AEA and 2-AG have been described: fatty acid amide hydrolase (FAAH) which hydrolyzes AEA and in some cells 2-AG, and Monoacylglycerol Lipase (MAGL) which hydrolyzes 2-AG. In spite of the early characterisation of MAGL and the nearly simultaneous clonings of the two enzymes, most of the efforts were dedicated to the study of FAAH and consequentially, the range of FAAH inhibitors available nowadays exceeds the number of compounds active upon MAGL. FAAH inhibitors can be divided in two major groups, the first one includes the inhibitors inspired by the chemical structures of FAAH substrates, which carry an arachidonoyl-, oleoyl- or palmitoyl-carbon chain that mimic the fatty acid chains of anandamide, oleamide and palmitoylethanolamide. The second group involves compounds that do not share similarities with the endocannabinoids, such as the carbamates, oxazolopyridins, 2-thioxoimidazolidin-4-ones, imidazolidine-2,4-diones and the non-steroidal anti-inflammatory drugs. However, the family of MAGL inhibitors contains few members and most of them exhibit a lack of selectivity. The purpose of this review is to give an overview of the families of synthetic inhibitors of FAAH and MAGL. The synthetic pathways, the chemical features, potencies, selectivities and modes of inhibition are listed and discussed in order to facilitate their comparison.     

New insights into endocannabinoid degradation and its therapeutic potential

Endocannabinoids are amides, esters and ethers of long chain polyunsaturated fatty acids, which act as new lipidic mediators. Anandamide (N-arachidonoylethanolamine; AEA) and 2-arachidonoylglycerol (2-AG) are the main endogenous agonists of cannabinoid receptors, able to mimic several pharmacological effects of (-)-Delta9-tetrahydrocannabinol (THC), the active principle of Cannabis sativa preparations like hashish and marijuana. The activity of AEA and 2-AG at their receptors is limited by cellular uptake through an anandamide membrane transporter (AMT), followed by intracellular degradation. A fatty acid amide hydrolase (FAAH) is the main AEA hydrolase, whereas a monoacylglycerol lipase (MAGL) is critical in degrading 2-AG. Here, we will review growing evidence that demonstrates that these hydrolases are pivotal regulators of the endogenous levels of AEA and 2-AG in vivo, overall suggesting that specific inhibitors of AMT, FAAH or MAGL may serve as attractive therapeutic targets for the treatment of human disorders. Recently, the N-acylphosphatidylethanolamine-specific phospholipase D (NAPE-PLD), which synthesizes AEA from N-arachidonoylphosphatidylethanolamine (NArPE), and the diacylglycerol lipase (DAGL), which generates 2-AG from diacylglycerol (DAG) substrates, have been characterized. The role of these synthetic routes in maintaining the endocannabinoid tone in vivo will be discussed. Finally, the effects of inhibitors of endocannabinoid degradation in animal models of human disease will be reviewed, with an emphasis on their ongoing applications in anxiety, cancer and neurodegenerative disorders.     

Inhibitors of Endocannabinoid-Metabolizing Enzymes Reduce Precipitated Withdrawal Responses in THC-Dependent Mice

Abstinence symptoms in cannabis-dependent individuals are believed to contribute to the maintenance of regular marijuana use. However, there are currently no medications approved by the FDA to treat cannabis-related disorders. The only treatment currently shown consistently to alleviate cannabinoid withdrawal in both animals and humans is substitution therapy using the psychoactive constituent of marijuana, Δ⁹-tetrahydrocannabinol (THC). However, new genetic and pharmacological tools are available to increase endocannabinoid levels by targeting fatty acid amide hydrolase (FAAH) or monoacylglycerol lipase (MAGL), the enzymes responsible for the degradation of the endogenous cannabinoid ligands anandamide and 2-arachidonoylglycerol, respectively. In the present study, we investigated whether increasing endogenous cannabinoids levels, through the use of FAAH (−/−) mice as well as the FAAH inhibitor URB597 or the MAGL inhibitor JZL184, would reduce the intensity of withdrawal signs precipitated by the CB₁ receptor antagonist rimonabant in THC-dependent mice. Strikingly, acute administration of either URB597 or JZL184 significantly attenuated rimonabant-precipitated withdrawal signs in THC-dependent mice. In contrast, FAAH (−/−) mice showed identical withdrawal responses as wild-type mice under a variety of conditions, suggesting that the absence of this enzyme across the development of dependence and during rimonabant challenge does not affect withdrawal responses. Of importance, subchronic administration of URB597 did not lead to cannabinoid dependence and neither URB597 nor JZL184 impaired rotarod motor coordination. These results support the concept of targeting endocannabinoid metabolizing enzymes as a promising treatment for cannabis withdrawal.Key words: anandamide, cannabis dependence, fatty acid amide hydrolase (FAAH), monoacylglycerol lipase (MAGL), 2-arachindonoylglycerol (2-AG)     

Kavalactones and the endocannabinoid system: the plant-derived yangonin is a novel CB? receptor ligand

To investigate the possible interactions between kavalactone-based molecules and proteins of the endocannabinoid system and provide novel and synthetically accessible structural scaffolds for the design of cannabinoid receptor ligands sharing pharmacological properties with kavapyrones, a preliminary SAR analysis was performed on five commercially available natural kavalactones and nine kavalactone-analogues properly synthesized. These compounds were investigated for assessing their cannabinoid receptor binding affinity and capability of inhibiting the activity of the two major metabolic enzymes of the endocannabinoid system, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL). Among the molecules tested, only yangonin exhibited affinity for the human recombinant CB? receptor with a K(i)=0.72 μM and selectivity vs. the CB? receptor (K(i)>10 μM). None of the compounds exhibited strong inhibitory effects on the two enzymes analyzed. The CB? receptor affinity of yangonin suggests that the endocannabinoid system might contribute to the complex human psychopharmacology of the traditional kava drink and the anxiolytic preparations obtained from the kava plant.     

Fatty acid amide hydrolase inhibitors from virtual screening of the endocannabinoid system

The endocannabinoid system consists of two cannabinoid receptors (CB1 and CB2), endogenous ligands (endocannabinoids), and the enzymes involved in the metabolism of the endocannabinoids, including fatty acid amide hydrolase (FAAH) and monoglyceride lipase (MGL). In the present study, virtual screening of MGL inhibitors was performed by utilizing a comparative model of the human MGL enzyme. All hit molecules were tested for their potential MGL inhibitory activity, but no compounds were found capable of inhibiting MGL-like enzymatic activity in rat cerebellar membranes. However, these compounds were also tested for their potential FAAH inhibitory activity and five compounds (2-6) inhibiting FAAH were found with IC50 values between 4 and 44 microM. In addition, the hit molecules from the virtual screening of CB2 receptor ligands (reported previously in Salo et al. J. Med. Chem. 2005, 48, 7166) were also tested in our FAAH assay, and four active compounds (7-10) were found with IC50 values between 0.52 and 22 microM. Additionally, compound 7 inhibited MGL-like enzymatic activity with an IC50 value of 31 microM.     

Focus on the three key enzymes hydrolysing endocannabinoids as new drug targets

The family of endocannabinoids (i.e., the endogenous agonists of cannabinoid receptors) contains several polyunsaturated fatty acid amides such as anandamide (AEA) and oleamide but also esters such as 2-arachidonoylglycerol (2-AG). These compounds are the subject of growing interest in pharmacology for their multiple therapeutic potentials. Unfortunately, they are rapidly inactivated by enzymatic hydrolysis, which prevents their effective medical use. Inhibitors of endocannabinoid degradation seem to be necessary tools for the development of endocannabinoid therapeutics. But hitting this target is inconceivable without good knowledge of the enzymes. Fatty acid amide hydrolase (FAAH) is the oldest and the best characterised enzyme involved in the degradation of endocannabinoids. Cloning, distribution in the body and crystal structure of FAAH have been described. A large number of FAAH inhibitors have also been synthesised and tested. For a long time, FAAH was considered as the only key enzyme hydrolysing endocannabinoids. But recent findings indicate that at least two other enzymes have critical role in the endocannabinoids degradation. Monoglyceride lipase participates in 2-AG degradation and some data indicate that it is the primary mechanism for 2-AG inactivation in intact neurons. N-palmitoylethanolamine-selective acid amidase (NPAA) is a second fatty acid amide hydrolase more active with N-palmitoylethanolamine, an anti-inflammatory substance. The purpose of this review is to collect and compare the catalytic properties of these 3 key enzymes hydrolysing endocannabinoids.     

Endocannabinoids at the spinal level regulate, but do not mediate, nonopioid stress-induced analgesia

Recent work in our laboratories has demonstrated that an opioid-independent form of stress-induced analgesia (SIA) is mediated by endogenous cannabinoids [Hohmann et al., 2005. Nature 435, 1108]. Non-opioid SIA, induced by a 3-min continuous foot shock, is characterized by the mobilization of two endocannabinoid lipids--2-arachidonoylglycerol (2-AG) and anandamide--in the midbrain periaqueductal gray (PAG). The present studies were conducted to examine the contributions of spinal endocannabinoids to nonopioid SIA. Time-dependent increases in levels of 2-AG, but not anandamide, were observed in lumbar spinal cord extracts derived from shocked relative to non-shocked rats. Notably, 2-AG accumulation was of smaller magnitude than that observed previously in the dorsal midbrain following foot shock. 2-AG is preferentially degraded by monoacylglycerol lipase (MGL), whereas anandamide is hydrolyzed primarily by fatty-acid amide hydrolase (FAAH). This metabolic segregation enabled us to manipulate endocannabinoid tone at the spinal level to further evaluate the roles of 2-AG and anandamide in nonopioid SIA. Intrathecal administration of the competitive CB1 antagonist SR141716A (rimonabant) failed to suppress nonopioid SIA, suggesting that supraspinal rather than spinal CB1 receptor activation plays a pivotal role in endocannabinoid-mediated SIA. By contrast, spinal inhibition of MGL using URB602, which selectively inhibits 2-AG hydrolysis in the PAG, enhanced SIA through a CB1-selective mechanism. Spinal inhibition of FAAH, with either URB597 or arachidonoyl serotonin (AA-5-HT), also enhanced SIA through a CB1-mediated mechanism, presumably by increasing accumulation of tonically released anandamide. Our results suggest that endocannabinoids in the spinal cord regulate, but do not mediate, nonopioid SIA.     

Inhibition of endocannabinoid catabolic enzymes elicits anxiolytic-like effects in the marble burying assay

Cannabinoids have long been shown to have a range of potential therapeutic effects, including antiemetic actions, analgesia, and anxiolysis. However, psychomimetic and memory disruptive side effects, as well as the potential for abuse and dependence, have restricted their clinical development. Endogenous cannabinoids (i.e., endocannabinoids; eCBs), such as anandamide (AEA) and 2-arachidonoylglycerol (2-AG), are produced throughout the limbic system and other brain regions associated with emotionality and are believed to modulate behavioral responses to stress-related conditions. AEA and 2-AG are rapidly metabolized by the respective enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL). Accordingly, inhibition of each enzyme increases brain levels of the appropriate eCB. Although FAAH inhibition has been established to decrease anxiety-like behavior, the role of 2-AG has been difficult to ascertain until the recent synthesis of JZL184, a potent and selective MAGL inhibitor. In the present study, we investigated the effects of inhibiting FAAH or MAGL on anxiety-like behavior in marble burying, a model of repetitive, compulsive behaviors germane to anxiety disorders such as obsessive-compulsive disorder. The FAAH inhibitor PF-3845, the MAGL inhibitor JZL184, and the benzodiazepine diazepam decreased marble burying at doses that did not affect locomotor activity. In contrast, Δ⁹-tetrahydrocannabinol (THC), the primary psychoactive constituent of marijuana, did not consistently reduce marble burying without also eliciting profound decreases in locomotor behavior. The CB₁ cannabinoid receptor antagonist rimonabant blocked the reduction in marble burying caused by FAAH and MAGL inhibitors, but not by diazepam, indicating a CB₁ receptor mechanism of action. These data indicate that elevation of AEA or 2-AG reduces marble burying behavior and suggest that their catabolic enzymes represent potential targets for the development of new classes of pharmacotherapeutics to treat anxiety-related disorders.     

Therapeutic potential of endocannabinoid-hydrolysing enzyme inhibitors

The specific protein target of delta9-tetrahydrocannabinol (delta9-THC), the main active ingredient of Cannabis sativa L., was characterized from rat brain nearly 20 years ago, and several endogenous compounds and proteins comprising the endocannabinoid (eCB) system have since been discovered. It has become evident that the eCB system consists of at least two cannabinoid receptors (i.e. the CB1 and CB2 receptors), in addition to their endogenous ligands (the eCBs) and several enzymes involved in the biosynthesis and catabolism of the eCBs. The two well-established eCBs, N-arachidonoylethanolamide (AEA) and 2-arachidonoylglycerol (2-AG), are produced by neurons on demand, act near their sites of synthesis and are effectively metabolized by fatty acid amide hydrolase (FAAH) and monoglyceride lipase (MGL), respectively. Inhibitors specifically targeting these enzymes could offer novel therapeutic approaches (e.g. for the treatment of pain and movement disorders). This MiniReview summarizes the literature concerning the potential therapeutic potential of FAAH and MGL inhibitors.     

Inhibitors of Fatty Acid Amide Hydrolase and Monoacylglycerol Lipase: New Targets for Future Antidepressants

Cannabis and analogs of Δ⁹-tetrahydrocannabinol have been used for therapeutic purposes, but their therapeutic use remains limited because of various adverse effects. Endogenous cannabinoids have been discovered, and dysregulation of endocannabinoid signaling is implicated in the pathophysiology of major depressive disorder (MDD). Recently, endocannabinoid hydrolytic enzymes such as fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) have become new therapeutic targets in the treatment of MDD. Several FAAH or MAGL inhibitors are reported to have no cannabimimetic side effects and, therefore, are new potential therapeutic options for patients with MDD who are resistant to first-line antidepressants (selective serotonin and serotonin-norepinephrine reuptake inhibitors). In this review, we focus on the possible relationships between MDD and the endocannabinoid system as well as the inhibitors’ therapeutic potential. MAGL inhibitors may reduce inflammatory responses through activation of cannabinoid receptor type 2. In the hypothalamic–pituitary–adrenal axis, repeated FAAH inhibitor administration may be beneficial for reducing circulating glucocorticoid levels. Both FAAH and MAGL inhibitors may contribute to dopaminergic system regulation. Recently, several new inhibitors have been developed with strong potency and selectivity. FAAH inhibitor, MAGL inhibitor, or dual blocker use would be promising new treatments for MDD. Further pre-clinical studies and clinical trials using these inhibitors are warranted.     

Endocannabinoid overload

The signaling capacity of endogenous cannabinoids ("endocannabinoids") is tightly regulated by degradative enzymes. This Perspective highlights a research article in this issue (p. 996) in which the authors show that genetic disruption of monoacylglycerol lipase (MAGL), the principal degradative enzyme for the endocannabinoid 2-arachidonoylglycerol (2-AG), causes marked elevations in 2-AG levels that lead to desensitization of brain cannabinoid receptors. These findings highlight the central role that MAGL plays in endocannabinoid metabolism in vivo and reveal that excessive 2-AG signaling can lead to functional antagonism of the brain cannabinoid system.     

Dual inhibition of MAGL and type II topoisomerase by N-phenylmaleimides as a potential strategy to reduce neuroblastoma cell growth

The endocannabinoid system is implicated in numerous physiopathological processes while more and more pieces of evidence wave the link between this complex machinery and cancer related phenomenon. In these lines, we confirmed the effects of 2-arachidonoylglycerol (2-AG), the main endocannabinoid, on neuroblastoma cells proliferation in vitro, and proved that some N-phenylmaleimide compounds that were previously shown as MAGL inhibitors can also inhibit type 2 topoisomerase. We also shed light on their antiproliferative effects on a neuroblastoma cell line. In order to establish a link between MAGL inhibition, topoisomerase inhibition and the effects on N1E-115 cells, we tested combinations of maleimides or known endocannabinoid metabolism inhibitors and 2-AG, the major MAGL substrate, on N1E-115 cells. However, none of the inhibitors tested, except the carbamate CAY10499, managed to increase 2-AG's effects. Even the MAGL reference inhibitor JZL184 failed to induce a stronger inhibition of proliferation.     

Cannabinoid signaling in rat cerebellar granule cells: G-protein activation, inhibition of glutamate release and endogenous cannabinoids

Previous studies have indicated that cannabinoids inhibit presynaptic neurotransmitter release in brain through CB1 receptors. To examine this issue in a primary neuronal culture system, rat cerebellar granule cells (CGCs) were prepared. [35S]GTPgammaS binding assays in saponin-permeabilized CGCs showed that G-protein activation by the CB1 agonist, WIN55212-2, and adenosine A1 agonist, phenylisopropyladenosine, was maximal during the second week in culture. Delta9-tetrahydrocannabinol stimulated [35S]GTPgammaS binding to a lesser degree than WIN55212-2, and the antagonists SR141716A and AM281 acted as inverse agonists in intact CGCs, but not in CGC membrane preparations. Ten micromolar WIN55212-2 and Delta9-tetrahydrocannabinol decreased depolarization-evoked efflux of [3H]-D-aspartate from CGCs by 32% and 13%, respectively. SR141716A and AM281 increased [3H]-D-aspartate release by 28%. The fatty acid amide hydrolase (FAAH) inhibitor phenylmethylsulfonyl fluoride (PMSF) and the anandamide uptake inhibitor AM404 inhibited transmitter release, implying that the antagonist effects were mediated by blockade of endocannabinoid activity. Levels of endocannabinoids (both anandamide and 2-arachidonyl glycerol [2-AG]) in extracts of the cells and cell incubation buffer were increased by PMSF pre-treatment. Depolarization with KCl significantly decreased the amount of anandamide and 2-AG in PMSF-treated CGCs. These results suggest that endogenous cannabinoids inhibit neurotransmitter release in CGCs, which may also release endocannabioids upon neural stimulation.     

Effect of repeated systemic administration of selective inhibitors of endocannabinoid inactivation on rat brain endocannabinoid levels

Several selective inhibitors of endocannabinoid inactivation via either the fatty acid amide hydrolase (FAAH) or the putative endocannabinoid transporter have been developed so far. Here, we have studied the effect in rats of a subchronic intraperitoneal treatment with three recently developed selective inhibitors of endocannabinoid uptake (VDM-11, UCM-707 and OMDM-2) or with a selective FAAH inhibitor (N-arachidonoyl-serotonin, AA-5-HT), on the brain levels of anandamide and 2-arachidonoylglycerol (2-AG) measured by means of isotope dilution LC-MS 1, 5 and 12 h after the last treatment. OMDM-2 was the most efficacious compound at enhancing the levels of anandamide at all time points, with a maximal effect (1.9-fold enhancement) after 5h. This compound also enhanced 2-AG levels by approximately 1.3-fold, but only 5 and 12h from administration. VDM-11 slightly, albeit significantly, enhanced anandamide levels (1.3-fold) only at 1h from administration and 2-AG levels (1.3-fold) only after 5h. Finally, UCM-707 only affected 2-AG levels (by two-fold) at only 1h from administration. FAAH inhibition by AA-5-HT significantly enhanced the levels of both anandamide (between 1.3- and 1.5-fold, maximal effect after 1 h) and 2-AG (between 1.3- and 1.6-fold, maximal effect after 12 h) at all time points. Brains from rats treated with AA-5-HT did never exhibit enhanced levels of serotonin, thus pointing to the metabolic stability of this FAAH inhibitor. These data indicate that: (1) the pharmacological effects reported so far for the four compounds under study in animal models of diseases may be due to enhancement of both anandamide and 2-AG levels; (2) 2-AG seems to need a longer time after the last administration in order to be augmented; (3) OMDM-2 and AA-5-HT should be regarded as enhancers of endocannabinoid levels suitable for use in vivo.     

Monoacylglycerol lipase inhibition by organophosphorus compounds leads to elevation of brain 2-arachidonoylglycerol and the associated hypomotility in mice

Three components of the cannabinoid system are sensitive to selected organophosphorus (OP) compounds: monoacylglycerol (MAG) lipase that hydrolyzes the major endogenous agonist 2-arachidonoylglycerol (2-AG); fatty acid amide hydrolase (FAAH) that cleaves the agonist anandamide present in smaller amounts; the CB1 receptor itself. This investigation considers which component of the cannabinoid system is the most likely contributor to OP-induced hypomotility in mice. Structure-activity studies by our laboratory and others rule against major involvement of a direct toxicant-CB1 receptor interaction for selected OPs. Attention was therefore focused on the OP sensitivities of MAG lipase and FAAH, assaying 19 structurally diverse OP chemicals (pesticides, their metabolites and designer compounds) for in vitro inhibition of both enzymes. Remarkably high potency and low selectivity is observed with three O-alkyl (C1, C2, C3) alkylphosphonofluoridates (C8, C12) (IC50 0.60-3.0 nM), five S-alkyl (C5, C7, C9) and alkyl (C10, C12) benzodioxaphosphorin oxides (IC50 0.15-5.7 nM) and one OP insecticide metabolite (chlorpyrifos oxon, IC50 34-40 nM). In ip-treated mice, the OPs at 1-30 mg/kg more potently inhibit brain FAAH than MAG lipase, but FAAH inhibition is not correlated with hypomotility. However, the alkylphosphonofluoridate-treated mice show dose-dependent increases in severity of hypomotility, inhibition of MAG lipase activity and elevation of 2-AG. Moderate to severe hypomotility is accompanied by 64 to 86% MAG lipase inhibition and about 6-fold elevation of brain 2-AG level. It therefore appears that OP-induced MAG lipase inhibition leads to elevated 2-AG and the associated hypomotility.