The endocannabinoid system - current implications for drug development.

In this review, the state of the art for compounds affecting the endocannabinoid (eCB) system is described with a focus on the treatment of pain. Amongst directly acting CB receptor ligands, clinical experience with ∆9 -tetrahydracannabinol and medical cannabis in chronic non-cancer pain indicates that there are differences between the benefits perceived by patients and the at best modest effect seen in meta-analyses of randomized controlled trials. The reason for this difference is not known but may involve differences in the type of patients that are recruited, the study conditions that are chosen and the degree to which biases such as reporting bias are operative. Other directly acting CB receptor ligands such as biased agonists and allosteric receptor modulators have not yet reached the clinic. Amongst indirectly acting compounds targeting the enzymes responsible for the synthesis and catabolism of the eCBs anandamide and 2-arachidonoylglycerol, fatty acid amide hydrolase (FAAH) inhibitors have been investigated clinically but were per se not useful for the treatment of pain, although they may be useful for the treatment of post-traumatic stress disorder and cannabis use disorder. Dual-acting compounds targeting this enzyme and other targets such as cyclooxygenase-2 or transient potential vanilloid receptor 1 may be a way forward for the treatment of pain.

Juniperonic Acid Biosynthesis is Essential in Caenorhabditis Elegans Lacking Δ6 Desaturase (fat-3) and Generates New ω-3 Endocannabinoids.

In eukaryotes, the C20:4 polyunsaturated fatty acid arachidonic acid (AA) plays important roles as a phospholipid component, signaling molecule and precursor of the endocannabinoid-prostanoid axis. Accordingly, the absence of AA causes detrimental effects. Here, compensatory mechanisms involved in AA deficiency in Caenorhabditis elegans were investigated. We show that the ω-3 C20:4 polyunsaturated fatty acid juniperonic acid (JuA) is generated in the C. elegansfat-3(wa22) mutant, which lacks Δ6 desaturase activity and cannot generate AA and ω-3 AA. JuA partially rescued the loss of function of AA in growth and development. Additionally, we observed that supplementation of AA and ω-3 AA modulates lifespan of fat-3(wa22) mutants. We described a feasible biosynthetic pathway that leads to the generation of JuA from α-linoleic acid (ALA) via elongases ELO-1/2 and Δ5 desaturase which is rate-limiting. Employing liquid chromatography mass spectrometry (LC-MS/MS), we identified endocannabinoid-like ethanolamine and glycerol derivatives of JuA and ω-3 AA. Like classical endocannabinoids, these lipids exhibited binding interactions with NPR-32, a G protein coupled receptor (GPCR) shown to act as endocannabinoid receptor in C. elegans. Our study suggests that the eicosatetraenoic acids AA, ω-3 AA and JuA share similar biological functions. This biosynthetic plasticity of eicosatetraenoic acids observed in C. elegans uncovers a possible biological role of JuA and associated ω-3 endocannabinoids in Δ6 desaturase deficiencies, highlighting the importance of ALA.

Forsythiaside prevents ß-amyloid-induced hippocampal slice injury by upregulating 2-arachidonoylglycerol via cannabinoid receptor 1-dependent NF-κB pathway.

In the study, the neuroprotectivities of forsythiaside, a main constituent of Forsythia suspensa (Thunb.) Vahl (F. suspensa, Lianqiao in Chinese), were investigated in the hippocampal slices. Forsythiaside suppressed the overexpression of cyclooxygenase-2 (COX-2) and monoacylglycerol lipase (MAGL) proteins induced by ß-amyloid (Aß25-35) to upregulate the levels of 2-arachidonoylglycerol (2-AG), an endogenous endocannabinoids. Then the inhibition of forsythiaside on COX-2 was deeply studied by the molecular docking. Forsythiaside prevented neuroinflammation and apoptosis from Aß25-35 insults, and this action appeared to be mediated via cannabinoid receptor 1 (CB1R)-dependent nuclear factor-κB (NF-κB) signaling pathways. More importantly, forsythiaside functionally improved Aß25-35-induced learning and memory deficits, which was indicated by long term potentiation (LTP). Taken together, forsythiaside may have therapeutic potential for Alzheimer's diseases (AD) by increasing the levels of 2-AG.

Conditioned gaping produced by high dose Δ9-tetrahydracannabinol: Dysregulation of the hypothalamic endocannabinoid system.

Δ9-tetrahydracannabinol (THC) is recognized as an effective treatment for nausea and vomiting via its action on the cannabinoid 1 (CB1) receptor. Paradoxically, there is evidence that THC can also produce nausea and vomiting. Using the conditioned gaping model of nausea in rats, we evaluated the ability of several doses of THC (0.0, 0.5, 5 and 10 mg/kg, i.p.) to produced conditioned gaping reactions. We then investigated the ability of the CB1 receptor antagonist, rimonabant, to block the establishment of THC-induced conditioned gaping. Real-time polymerase chain reaction (RT-PCR) was then used to investigate changes in endocannabinoid related genes in various brain regions in rats chronically treated with vehicle (VEH), 0.5 or 10 mg/kg THC. THC produced dose-dependent gaping, with 5 and 10 mg/kg producing significantly more gaping reactions than VEH or 0.5 mg/kg THC, a dose known to have anti-emetic properties. Pre-treatment with rimonabant reversed this effect, indicating that THC-induced conditioned gaping was CB1 receptor mediated. The RT-PCR analysis revealed an upregulation of genes for the degrading enzyme, monoacylglycerol lipase (MAGL), of the endocannabinoid, 2-arachidolyl glycerol (2-AG), in the hypothalamus of rats treated with 10 mg/kg THC. No changes in the expression of relevant genes were found in nausea (interoceptive insular cortex) or vomiting (dorsal vagal complex) related brain regions. These findings support the hypothesis that THC-induced nausea is a result of a dysregulated hypothalamic-pituitary-adrenal axis leading to an overactive stress response.

Enhanced endocannabinoid tone as a potential target of pharmacotherapy.

The endocannabinoid system is up-regulated in numerous pathophysiological states such as inflammatory, neurodegenerative, gastrointestinal, metabolic and cardiovascular diseases, pain, and cancer. It has been suggested that this phenomenon primarily serves an autoprotective role in inhibiting disease progression and/or diminishing signs and symptoms. Accordingly, enhancement of endogenous endocannabinoid tone by inhibition of endocannabinoid degradation represents a promising therapeutic approach for the treatment of many diseases. Importantly, this allows for the avoidance of unwanted psychotropic side effects that accompany exogenously administered cannabinoids. The effects of endocannabinoid metabolic pathway modulation are complex, as endocannabinoids can exert their actions directly or via numerous metabolites. The two main strategies for blocking endocannabinoid degradation are inhibition of endocannabinoid-degrading enzymes and inhibition of endocannabinoid cellular uptake. To date, the most investigated compounds are inhibitors of fatty acid amide hydrolase (FAAH), an enzyme that degrades the endocannabinoid anandamide. However, application of FAAH inhibitors (and consequently other endocannabinoid degradation inhibitors) in medicine became questionable due to a lack of therapeutic efficacy in clinical trials and serious adverse effects evoked by one specific compound. In this paper, we discuss multiple pathways of endocannabinoid metabolism, changes in endocannabinoid levels across numerous human diseases and corresponding experimental models, pharmacological strategies for enhancing endocannabinoid tone and potential therapeutic applications including multi-target drugs with additional targets outside of the endocannabinoid system (cyclooxygenase-2, cholinesterase, TRPV1, and PGF2α-EA receptors), and currently used medicines or medicinal herbs that additionally enhance endocannabinoid levels. Ultimately, further clinical and preclinical studies are warranted to develop medicines for enhancing endocannabinoid tone.

Endocannabinoids in arthritis: current views and perspective.

Preclinical and clinical studies using cannabis-based therapy have been shown to provide both analgesia and anti-inflammatory effects, with an overall alleviation of clinical symptoms in animal models of arthritis, highlighting its promising therapeutic application for humans. Despite this, the development of cannabis-based therapeutics remains in its infancy, with further investigation into its efficacy and safety profile in patients still required. This synopsis reviews the various components of the endocannabinoid system in health and disease and their potential as therapeutic targets.

Formation of OX-1R/CB1R heteromeric complexes in embryonic mouse hypothalamic cells: Effect on intracellular calcium, 2-arachidonoyl-glycerol biosynthesis and ERK phosphorylation.

Orexin 1 (OX-1R) and cannabinoid receptor (CB1R) belong to the superfamily of G-protein-coupled receptors (GPCRs) and are mostly coupled to Gq and Gi/o proteins, respectively. In vitro studies in host cells over-expressing OX-1R and CB1R revealed a functional interaction between these receptors, through either their ability to form heteromers or the property for OX-1R to trigger the biosynthesis of 2-arachidonoylglycerol (2-AG), an endogenous CB1R ligand. Since: i) OX-1R and CB1R co-espression has been described at postsynaptc sites in hypothalamic circuits involved the regulation of energy homeostasis, and ii) increased orexin-A (OX-A) and 2-AG levels occur in hypothalamic neurons during obesity, we sought here to investigate the OX-1R/CB1R interaction in embryonic mouse hypothalamic NPY/AgRP mHypoE-N41 neurons which express, constitutively, both receptors. Treatment of mHypoE-N41 cells with OX-A (0.1-0.3µM), but not with the selective CB1R agonist, arachidonyl-2-chloroethylamide (ACEA; 0.1-0.3µM), transiently elevated [Ca(2+)]i. Incubation with a subeffective dose of OX-A (0.1µM)+ACEA (0.1µM) led to stronger and longer lasting elevation of [Ca(2+)]i, antagonized by OX-1R or CB1R antagonism with SB-334867 or AM251, respectively. FRET and co-immunoprecipitation experiments showed the formation of OX-1R/CB1R heteromers after incubation with OX-A (0.2µM), or OX-A (0.1µM)+ACEA (0.1µM), but not after ACEA (0.2µM), in a manner antagonized by SB-334867 or AM251. OX-A (0.2µM) or OX-A (0.1µM)+ACEA (0.1µM) also led to 2-AG biosynthesis. Finally, a stronger activation of ERK1/2(Thr202/185) phosphorylation in comparison to basal or each agonist alone (0.1-0.2µM), was induced by incubation with OX-A (0.1µM)+ACEA (0.1µM), again in a manner prevented by OX-1R or CB1R antagonism. We suggest that OX-A, alone at effective concentrations on [Ca(2+)]i, or in combination with ACEA, at subeffective concentrations, triggers intracellular signaling events via the formation of OX-1R/CB1R heteromers and an autocrine loop mediated by 2-AG.

Phytocannabinoids and epilepsy.

WHAT IS KNOWN AND OBJECTIVE: Antiepileptic drugs often produce serious adverse effects, and many patients do not respond to them properly. Phytocannabinoids produce anticonvulsant effects in preclinical and preliminary human studies, and appear to produce fewer adverse effects than available antiepileptic drugs. The present review summarizes studies on the anticonvulsant properties of phytocannabinoids. METHODS: Literature search using the PubMed database to identify studies on phytocannabinoids and epilepsy. RESULTS AND DISCUSSION: Preclinical studies suggest that phytocannabinoids, especially cannabidiol and cannabidivarin, have potent anticonvulsant effects which are mediated by the endocannabinoid system. Human studies are limited in number and quality, but suggest that cannabidiol has anticonvulsant effects in adult and infantile epilepsy and is well tolerated after prolonged administration. WHAT IS NEW AND CONCLUSION: Phytocannabinoids produce anticonvulsant effects through the endocannabinoid system, with few adverse effects. Cannabidiol and cannabidivarin should be tested in randomized, controlled clinical trials, especially in infantile epileptic syndromes.

Sheep cheese naturally enriched in α-linolenic, conjugated linoleic and vaccenic acids improves the lipid profile and reduces anandamide in the plasma of hypercholesterolaemic subjects.

Intake of dairy fat has long been considered as a risk factor for CVD. Pasture and dietary lipid supplementation have been reported to be reliable strategies in ruminant nutrition, in order to increase the content of α-linolenic acid (ALA), conjugated linoleic acid (CLA) and vaccenic acid (VA), and decrease SFA in milk fat. In the present study, we aimed at verifying whether consumption of a sheep cheese, naturally enriched in ALA, CLA and VA, would modify the plasma lipid and endocannabinoid profiles in mildly hypercholesterolaemic subjects. A total of forty-two adult volunteers (nineteen males and twenty-three females) with diagnosed mildly hypercholesterolaemia (total cholesterol 5·68-7·49 mmol/l) were randomly assigned to eat 90 g/d of a control or enriched cheese for 3 weeks, with a cross-over after 3 weeks of washout. Plasma lipids, endocannabinoids, adipokines and inflammatory markers were measured. The intake of enriched cheese significantly increased the plasma concentrations of CLA, VA, the n-3 fatty acids ALA and EPA, and more remarkably decreased that of the endocannabinoid anandamide. LDL-cholesterol decreased significantly (7%). No changes were detected in the levels of inflammatory markers; however, a significant correlation was found between the plasma levels of anandamide and leptin. The control cheese modified none of the parameters measured. The results obtained do not support the view that intake of dairy fat is detrimental to hypercholesterolaemic subjects. Indeed, they show that a naturally enriched cheese possesses beneficial properties, since it ameliorates the plasma lipid profile, and more remarkably reduces endocannabinoid biosynthesis.

DAGLß inhibition perturbs a lipid network involved in macrophage inflammatory responses.

The endocannabinoid 2-arachidonoylglycerol (2-AG) is biosynthesized by diacylglycerol lipases DAGLα and DAGLß. Chemical probes to perturb DAGLs are needed to characterize endocannabinoid function in biological processes. Here we report a series of 1,2,3-triazole urea inhibitors, along with paired negative-control and activity-based probes, for the functional analysis of DAGLß in living systems. Optimized inhibitors showed high selectivity for DAGLß over other serine hydrolases, including DAGLα (∼60-fold selectivity), and the limited off-targets, such as ABHD6, were also inhibited by the negative-control probe. Using these agents and Daglb(-/-) mice, we show that DAGLß inactivation lowers 2-AG, as well as arachidonic acid and eicosanoids, in mouse peritoneal macrophages in a manner that is distinct and complementary to disruption of cytosolic phospholipase-A2. We observed a corresponding reduction in lipopolysaccharide-induced tumor necrosis factor-α release. These findings indicate that DAGLß is a key metabolic hub within a lipid network that regulates proinflammatory responses in macrophages.