Control of experimental spasticity by targeting the degradation of endocannabinoids using selective fatty acid amide hydrolase inhibitors.

BACKGROUND: It has been previously shown that CB1 cannabinoid receptor agonism using cannabis extracts alleviates spasticity in both a mouse experimental autoimmune encephalomyelitis (EAE) model and multiple sclerosis (MS) in humans. However, this action can be associated with dose-limiting side effects. OBJECTIVE: We hypothesised that blockade of anandamide (endocannabinoid) degradation would inhibit spasticity, whilst avoiding overt cannabimimetic effects. METHODS: Spasticity eventually developed following the induction of EAE in either wild-type or congenic fatty acid amide hydrolase (FAAH)-deficient Biozzi ABH mice. These animals were treated with a variety of different FAAH inhibitors and the effect on the degree of limb stiffness was assessed using a strain gauge. RESULTS: Control of spasticity was achieved using FAAH inhibitors CAY100400, CAY100402 and URB597, which was sustained following repeated administrations. Therapeutic activity occurred in the absence of overt cannabimimetic effects. Importantly, the therapeutic value of the target could be definitively validated as the treatment activity was lost in FAAH-deficient mice. Spasticity was also controlled by a selective monoacyl glycerol lipase inhibitor, JZL184. CONCLUSIONS: This study demonstrates definitively that FAAH inhibitors provide a new class of anti-spastic agents that may have utility in treating spasticity in MS and avoid the dose-limiting side effects associated with cannabis use.

Protective role of palmitoylethanolamide in contact allergic dermatitis.

BACKGROUND: Palmitoylethanolamide (PEA) is an anti-inflammatory mediator that enhances the activation by anandamide (AEA) of cannabinoid receptors and transient receptor potential vanilloid type-1 (TRPV1) channels, and directly activates peroxisome proliferator-activated receptor-alpha (PPAR-alpha). In mice, 2,4-dinitrofluorobenzene (DNFB)-induced contact allergic dermatitis (CAD) in inflamed ears is partly mediated by the chemokine Monocyte Chemotactic Protein-2 (MCP-2) and accompanied by elevation of AEA levels. No datum is available on PEA regulation and role in CAD. OBJECTIVE: We examined whether PEA is produced during DNFB-induced CAD, and if it has any direct protective action in keratinocytes in vitro. METHODS: Eight- to ten-week-old female C57BL/6J wild-type and CB(1)/CB(2) double knock-out mice were used to measure PEA levels and the expression of TRPV1, PPAR-alpha receptors and enzymes responsible for PEA biosynthesis and degradation. Human keratinocytes (HaCaT) cells were stimulated with polyinosinic polycytidylic acid [poly-(I:C)], and the expression and release of MCP-2 were measured in the presence of PEA and antagonists of its proposed receptors. RESULTS: 2,4-Dinitrofluorobenzene increased ear skin PEA levels and up-regulated TRPV1, PPAR-alpha and a PEA-biosynthesizing enzyme in ear keratinocytes. In HaCaT cells, stimulation with poly-(I:C) elevated the levels of both PEA and AEA, and exogenous PEA (10 microM) inhibited poly-(I:C)-induced expression and release of MCP-2 in a way reversed by antagonism at TRPV1, but not PPAR-alpha. PEA (5-10 mg/kg, intraperitoneal) also inhibited DNFB-induced ear inflammation in mice in vivo, in a way attenuated by TRPV1 antagonism. CONCLUSIONS: We suggest that PEA is an endogenous protective agent against DNFB-induced keratinocyte inflammation and could be considered for therapeutic use against CAD.

2-arachidonoylglycerol levels are increased in leukocytospermia and correlate with seminal macrophages.

Evidence has been produced that macrophages can actively generate endocannabinoids (eCBs) in response to inflammatory stimuli. As eCBs are involved in the control of several physiological processes, including reproduction, here, we explored whether seminal levels of the eCBs, N-arachidonoylethanolamine (AEA), and 2-arachidonoylglycerol (2-AG), were higher in the presence of leukocytospermia, and were correlated with semen concentration of macrophages. The content of AEA and 2-AG was measured by high-performance liquid chromatography/mass spectrometry in seminal plasma of ejaculates from 18 leukocytospermic patients (>1 × 106 leukocytes/mL) and 21 normozoospermic controls. In the same ejaculates, round cells were phenotyped by flow-cytometry as leukocytes (CD45+), macrophages (CD14+), and activated macrophages (CD14+, HLA-DR+). The levels of 2-AG, but not of AEA, were significantly higher in ejaculates from leukocytospermic patients than in controls and exhibited a significant correlation with semen concentration of macrophages and activated macrophages. Significant associations of 2-AG with macrophages and activated macrophages persisted after adjustment for semen volume and sperm concentration. In conclusion, here we provide evidence that seminal plasma levels of 2-AG are higher in the presence of leukocytospermia, as a marker of macrophages activation. Further studies are warranted to elucidate possible clinical implications.

Endocannabinoids: endogenous cannabinoid receptor ligands with neuromodulatory action.

The existence of an endogenous cannabinoid system was demonstrated conclusively with the discovery of endogenous brain constituents capable of activating the cannabinoid receptors functionally. These compounds are synthesized by neuronal cells and inactivated through re-uptake and enzymatic hydrolysis by both neurons and astrocytes. In analogy with the endorphins they can be referred to as endocannabinoids. Apart from the identification of their metabolic pathways, research carried out in the past six years has focused on the possible cellular and molecular targets for the actions of endocannabinoids. These studies have confirmed a similarity between the endocannabinoids and the psychoactive substance in marijuana, delta9(-)-tetrahydrocannabinol, and have suggested a role for endocannabinoids in the modulation of neurotransmitter action and release.

In vitro and in vivo pharmacology of synthetic olivetol- or resorcinol-derived cannabinoid receptor ligands.

BACKGROUND AND PURPOSE: We have previously reported the development of CB-25 and CB-52, two ligands of CB1 and CB2 cannabinoid receptors. We assessed here their functional activity. EXPERIMENTAL APPROACH: The effect of the two compounds on forskolin-induced cAMP formation in intact cells or GTP-gamma-S binding to cell membranes, and their action on nociception in vivo was determined. KEY RESULTS: CB-25 enhanced forskolin-induced cAMP formation in N18TG2 cells (EC50 approximately 20 nM, max. stimulation = 48%), behaving as an inverse CB1 agonist, but it stimulated GTP-gamma-S binding to mouse brain membranes, behaving as a partial CB1 agonist (EC50 =100 nM, max. stimulation = 48%). At human CB1 receptors, CB-25 inhibited cAMP formation in hCB1-CHO cells (EC50 = 1600 nM, max. inhibition = 68% of CP-55,940 effect). CB-52 inhibited forskolin-induced cAMP formation by N18TG2 cells (IC50 = 450 nM, max. inhibition = 40%) and hCB1-CHO cells (EC50 = 2600 nM, max. inhibition = 62% of CP-55,940 effect), and stimulated GTP-gamma-S binding to mouse brain membranes (EC50 = 11 nM, max. stimulation approximately 16%). Both CB-25 and CB-52 showed no activity in all assays of CB2-coupled functional activity and antagonized CP55940-induced stimulation of GTP-gamma-S binding to hCB2-CHO cell membranes. In vivo, both compounds, administered i.p., produced dose-dependent nociception in the plantar test carried out in healthy rats, and antagonised the anti-nociceptive effect of i.p. WIN55,212-2. In the formalin test in mice, however, the compounds counteracted both phases of formalin-induced nociception. CONCLUSIONS AND IMPLICATIONS: CB-25 and CB-52 behave in vitro mostly as CB1 partial agonists and CB2 neutral antagonists, whereas their activity in vivo might depend on the tonic activity of cannabinoid receptors.

The bright side of psychoactive substances: cannabinoid-based drugs in motor diseases.

INTRODUCTION: Psychoactive substances are associated with the idea of drugs with high addictive liability, affecting mental states, cognition, emotion and motor behavior. However these substances can modify synaptic transmission and help to disclose some mechanisms underlying alterations in brain processing and pathophysiology of motor disease. Hence, the 'bright side' of e cannabinoid-based drugs must be thoroughly examined to be identified within the latter framework. Areas covered: We will analyze the preclinical and clinical evidence of cannabinoid-based drugs, discussing their therapeutic value in basal ganglia motor disorders such as Parkinson's disease and Huntington disease. Expert commentary: Despite the knowledge acquired in the last years, the therapeutic potential of cannabinoid-based drugs should be further tested by novel routes of investigation. This should be focused on the role of cannabinoid signaling system in mitochondrial function as well as on the physical and functional interaction with other key receptorial targets belonging to this network.

Exogenous anandamide protects rat brain against acute neuronal injury in vivo.

The endocannabinoid anandamide [N-arachidonoylethanolamine (AEA)] is thought to function as an endogenous protective factor of the brain against acute neuronal damage. However, this has never been tested in an in vivo model of acute brain injury. Here, we show in a longitudinal pharmacological magnetic resonance imaging study that exogenously administered AEA dose-dependently reduced neuronal damage in neonatal rats injected intracerebrally with the Na(+)/K(+)-ATPase inhibitor ouabain. At 15 min after injury, AEA (10 mg/kg) administered 30 min before ouabain injection reduced the volume of cytotoxic edema by 43 +/- 15% in a manner insensitive to the cannabinoid CB(1) receptor antagonist SR141716A. At 7 d after ouabain treatment, 64 +/- 24% less neuronal damage was observed in AEA-treated (10 mg/kg) rats compared with control animals. Coadministration of SR141716A prevented the neuroprotective actions of AEA at this end point. In addition, (1) no increase in AEA and 2-arachidonoylglycerol levels was detected at 2, 8, or 24 hr after ouabain injection; (2) application of SR141716A alone did not increase the lesion volume at days 0 and 7; and (3) the AEA-uptake inhibitor, VDM11, did not affect the lesion volume. These data indicate that there was no endogenous endocannabinoid tone controlling the acute neuronal damage induced by ouabain. Although our data seem to question a possible role of the endogenous cannabinoid system in establishing a brain defense system in our model, AEA may be used as a structural template to develop neuroprotective agents.

Occurrence and metabolism of anandamide and related acyl-ethanolamides in ovaries of the sea urchin Paracentrotus lividus.

Cannabinoid receptors have been described in sea urchin sperm and shown to mediate inhibition of sperm acrosome reaction. Anandamide (arachidonoyl-ethanolamide), the mammalian physiological ligand at the cannabinoid CB1 receptor, has been subsequently found to effect this inhibition. Here we present data showing that ovaries from the sea urchin Paracentrotus lividus contain anandamide and two related acyl-ethanolamides, as well as enzymatic activities potentially responsible for their biosynthesis and degradation. Pilot experiments carried out with either ovaries or spermatozoa, extracted from both P. lividus and Arbacea lixula and radiolabelled with [14C]ethanolamine, showed that in sexually mature ovaries of both species significant levels of radioactivity were incorporated into a lipid component with the same chromatographic behaviour as anandamide. Lipid extracts from P. lividus ovaries were purified and analysed by gas chromatography/mass spectrometry which showed the presence of low but measurable amounts of anandamide, palmitoyl- and stearoyl-ethanolamides. The extracts were also found to contain lipid components with the same chromatographic behaviour as the N-acyl-phosphatidyl-ethanolamines, the phospholipid precursors of acyl-ethanolamides in mammalian tissues, and capable of releasing anandamide, palmitoyl- and stearoyl-ethanolamides upon digestion with S. chromofuscus phospholipase D. Accordingly, whole homogenates from P. lividus contained an enzymatic activity capable of converting synthetic [3H]N-arachidonoyl-phosphatidyl-ethanolamine into [3H]anandamide. Finally, mature ovaries of P. lividus were shown also to contain an amidohydrolase activity which catalyses the hydrolysis of anandamide and palmitoyl-ethanolamide to ethanolamine. This enzyme displayed subcellular distribution, pH/temperature dependency profiles and sensitivity to inhibitors similar but not identical to those of the previously described 'anandamide amidohydrolase' from mammalian tissues. These data support the hypothesis, formulated in previous studies, that anandamide or related metabolites may be oocyte-derived cannabimimetic regulators of sea urchin fertility.

Anandamide: some like it hot.

Anandamide was the first endogenous ligand of cannabinoid receptors to be discovered in 1992. Yet, this compound also efficiently activates receptors specific for capsaicin, known as vanilloid type 1 receptors (VR1). Whether anandamide is a physiological VR1 ligand is controversial. However, very recent reports demonstrate that activation of VR1 by anandamide can be significantly enhanced by various regulatory factors, suggesting that this compound might act as an "endovanilloid" under certain conditions.

The biosynthesis, fate and pharmacological properties of endocannabinoids.

The finding of endogenous ligands for cannabinoid receptors, the endocannabinoids, opened a new era in cannabinoid research. It meant that the biological role of cannabinoid signalling could be finally studied by investigating not only the pharmacological actions subsequent to stimulation of cannabinoid receptors by their agonists, but also how the activity of these receptors was regulated under physiological and pathological conditions by varying levels of the endocannabinoids. This in turn meant that the enzymes catalysing endocannabinoid biosynthesis and inactivation had to be identified and characterized, and that selective inhibitors of these enzymes had to be developed to be used as (1) probes to confirm endocannabinoid involvement in health and disease, and (2) templates for the design of new therapeutic drugs. This chapter summarizes the progress achieved in this direction during the 12 years following the discovery of the first endocannabinoid.

Suppression of nerve growth factor Trk receptors and prolactin receptors by endocannabinoids leads to inhibition of human breast and prostate cancer cell proliferation.

Anandamide and 2-arachidonoylglycerol (2-AG), two endogenous ligands of the CB1 and CB2 cannabinoid receptor subtypes, inhibit the proliferation of PRL-responsive human breast cancer cells (HBCCs) through down-regulation of the long form of the PRL receptor (PRLr). Here we report that 1) anandamide and 2-AG inhibit the nerve growth factor (NGF)-induced proliferation of HBCCs through suppression of the levels of NGF Trk receptors; 2) inhibition of PRLr levels results in inhibition of the proliferation of other PRL-responsive cells, the prostate cancer DU-145 cell line; and 3) CB1-like cannabinoid receptors are expressed in HBCCs and DU-145 cells and mediate the inhibition of cell proliferation and Trk/PRLr expression. Beta-NGF-induced HBCC proliferation was potently inhibited (IC50 = 50-600 nM) by the synthetic cannabinoid HU-210, 2-AG, anandamide, and its metabolically stable analogs, but not by the anandamide congener, palmitoylethanolamide, or the selective agonist of CB2 cannabinoid receptors, BML-190. The effect of anandamide was blocked by the CB1 receptor antagonist, SR141716A, but not by the CB2 receptor antagonist, SR144528. Anandamide and HU-210 exerted a strong inhibition of the levels of NGF Trk receptors as detected by Western immunoblotting; this effect was reversed by SR141716A. When induced by exogenous PRL, the proliferation of prostate DU-145 cells was potently inhibited (IC50 = 100-300 nM) by anandamide, 2-AG, and HU-210. Anandamide also down-regulated the levels of PRLr in DU-145 cells. SR141716A attenuated these two effects of anandamide. HBCCs and DU-145 cells were shown to contain 1) transcripts for CB1 and, to a lesser extent, CB2 cannabinoid receptors, 2) specific binding sites for [3H]SR141716A that could be displaced by anandamide, and 3) a CB1 receptor-immunoreactive protein. These findings suggest that endogenous cannabinoids and CB1 receptor agonists are potential negative effectors of PRL- and NGF-induced biological responses, at least in some cancer cells.

Overlap between the ligand recognition properties of the anandamide transporter and the VR1 vanilloid receptor: inhibitors of anandamide uptake with negligible capsaicin-like activity.

Some synthetic agonists of the VR1 vanilloid (capsaicin) receptor also inhibit the facilitated transport into cells of the endogenous cannabinoid anandamide (arachidonoylethanolamide, AEA). Here we tested several AEA derivatives containing various derivatized phenyl groups or different alkyl chains as either inhibitors of the AEA membrane transporter (AMT) in intact cells or functional agonists of the VR1 vanilloid receptor in HEK cells transfected with the human VR1. We found that four known AMT inhibitors, AM404, arvanil, olvanil and linvanil, activate VR1 receptors at concentrations 400-10000-fold lower than those necessary to inhibit the AMT. However, we also found three novel AEA derivatives, named VDM11, VDM12 and VDM13, which inhibit the AMT as potently as AM404 but exhibit little or no agonist activity at hVR1. These compounds are weak inhibitors of AEA enzymatic hydrolysis and poor CB(1)/CB(2) receptor ligands. We show for the first time that, despite the overlap between the chemical moieties of AMT inhibitors and VR1 agonists, selective inhibitors of AEA uptake that do not activate VR1 (e.g. VDM11) can be developed.

Two novel classes of neuroactive fatty acid amides are substrates for mouse neuroblastoma 'anandamide amidohydrolase'.

The endogenous cannabimimetic substance, anandamide (N-arachidonoyl-ethanolamine) and the recently isolated sleep-inducing factor, oleoyl-amide (cis-9,10-octadecenoamide), belong to two neuroactive fatty acid amide classes whose action in mammals has been shown to be controlled by enzymatic amide bond hydrolysis. Here we report the partial characterisation and purification of 'anandamide amidohydrolase' from membrane fractions of N18 neuroblastoma cells, and provide evidence for a further and previously unsuspected role of this enzyme. An enzymatic activity catalysing the hydrolysis of [14C]anandamide was found in both microsomal and 10,000 x g pellet fractions. The latter fractions, which displayed the highest Vmax for anandamide, were used for further characterisation of the enzyme, and were found to catalyse the hydrolysis also of [14C]oleoyl-amide, with an apparent Km of 9.0 +/- 2.2 microM. [14C]anandamide- and [14C]oleoyl-amide-hydrolysing activities: (i) exhibited identical pH- and temperature-dependency profiles; (ii) were inhibited by alkylating agents; (iii) were competitively inhibited by the phospholipase A2 inhibitor arachidonyl-trifluoromethyl-ketone with the same IC50 (3 microM); (iv) were competitively inhibited by both anandamide (or other polyunsaturated fatty acid-ethanolamides) and oleoyl-amide. Proteins solubilised from 10,000 x g pellets were directly analysed by isoelectric focusing, yielding purified fractions capable of catalysing the hydrolysis of both [14C]anandamide and [14C]oleoyl-amide. These data suggest that 'anandamide amidohydrolase' enzymes, such as that characterised in this study, may be used by neuronal cells also to hydrolyse the novel sleep-inducing factor oleoyl-amide.

Interactions between synthetic vanilloids and the endogenous cannabinoid system.

The chemical similarity between some synthetic agonists of vanilloid receptors, such as olvanil (N-vanillyl-cis-9-octadecenoamide), and the 'endocannabinoid' anandamide (arachidonoyl-ethanolamide, AEA), suggests possible interactions between the cannabinoid and vanilloid signalling systems. Here we report that olvanil is a stable and potent inhibitor of AEA facilitated transport into rat basophilic leukemia (RBL-2H3) cells. Olvanil blocked both the uptake and the hydrolysis of [14C]AEA by intact RBL-2H3 cells (IC50 = 9 microM), while capsaicin and pseudocapsaicin (N-vanillyl-nonanamide) were much less active. Olvanil was more potent than previously reported inhibitors of AEA facilitated transport, i.e. phloretin (IC50 = 80 microM), AM404 (12.9% inhibition at 10 microM) or oleoylethanolamide (27.5% inhibition at 10 microM). Olvanil was a poor inhibitor of [14C]AEA hydrolysis by RBL-2H3 and N18TG2 cell membranes, suggesting that the inhibitory effect on [14C]AEA breakdown observed in intact cells was due to inhibition of [14C]AEA uptake. Olvanil was stable to enzymatic hydrolysis, and (i) displaced the binding of high affinity cannabinoid receptor ligands to membrane preparations from N18TG2 cells and guinea pig forebrain (Ki = 1.64-7.08 microM), but not from cells expressing the CB2 cannabinoid receptor subtype; (ii) inhibited forskolin-induced cAMP formation in intact N18TG2 cells (IC50 = 1.60 microM), this effect being reversed by the selective CB1 antagonist SR141716A. Pseudocapsaicin, but not capsaicin, also selectively bound to CB1 receptor-containing membranes. These data suggest that some of the analgesic actions of olvanil may be due to its interactions with the endogenous cannabinoid system, and may lead to the design of a novel class of cannabimimetics with potential therapeutic applications as analgesics.

Cannabimimetic fatty acid derivatives: the anandamide family and other endocannabinoids.

In agreement with the highly lipophilic nature of (-)-delta9-tetrahydrocannabinol, all the endogenous ligands of cannabinoid receptors identified so far are derivatives of long chain fatty acids. N- Arachidonoylethanolamine (anandamide) and some of its polyunsaturated congeners have been found in mammalian brain and shown to activate the CB1 and, with a lower efficacy, CB2 cannabinoid receptor subtypes. More recently, 2-arachidonoylglycerol (2-AG), a widespread intermediate in the metabolism of phosphoglycerides, diacylglycerols and triglycerides, was also found to activate the cannabinoid receptors. The capability of palmitoylethanolamide, an anti-inflammatory metabolite, to activate CB2-like receptors is still being debated. Here we review: 1) the metabolic pathways suggested so far to underlie the biosynthesis and inactivation of anandamide and 2-AG, and 2) the current knowledge of the chemical bases for the interactions of anandamide and 2-AG with proteins of the endogenous cannabinoid system characterized so far, i.e. the CB1 and CB2 receptor subtypes, the membrane anandamide carrier , which facilitates anandamide diffusion into cells, and the enzyme fatty acid amide hydrolase , which catalyzes anandamide and, to a certain extent, 2-AG hydrolysis in vivo.

Endocannabinoids: new targets for drug development.

The possible therapeutic use of marijuana s active principles, the cannabinoids, is currently being debated. It is now known that these substances exert several of their pharmacological actions by activating specific cell membrane receptors, the CB1 and CB2 cannabinoid receptor subtypes. This knowledge led to the design of synthetic cannabinoid agonists and antagonists with high therapeutic potential. The recent discovery of the endocannabinoids, i.e. endogenous metabolites capable of activating the cannabinoid receptors, and the understanding of the molecular mechanisms leading to their biosynthesis and inactivation, opened a new era in research on the pharmaceutical applications of cannabinoids. Ongoing studies on the pathological and physiological conditions regulating the tissue levels of endocannabinoids, and on the pharmacological activity of these compounds and their derivatives, may provide a lead for the development of new drugs for the treatment of nervous and immune disorders, cardiovascular diseases, pain, inflammation and cancer. These studies are reviewed in this article with special emphasis on the chemical features that determine the interaction of endocannabinoids with the proteins mediating their activity and degradation.

Finding of the endocannabinoid signalling system in Hydra, a very primitive organism: possible role in the feeding response.

Hydra (Cnidaria) is the first animal organism to have developed a neural network, which has been proposed to control, inter alia, the "feeding response", i.e. a mechanism through which the coelenterate opens and then closes its mouth in the presence of prey and/or glutathione. Here, we report that Hydra contains: (i) selective cannabinoid binding sites; (ii) the endogenous cannabinoid receptor ligand, anandamide (arachidonoylethanolamide); (iii) a fatty acid amide hydrolase-like activity catalysing anandamide hydrolysis; and (iv) the putative biosynthetic precursor of anandamide, N-arachidonoylphosphatidylethanolamine. We suggest that this "endogenous cannabinoid system" is involved in the modulation of the "feeding response". Anandamide (1 nM-1 microM) potently inhibited (up to 45%) the glutathione-induced "feeding response" by accelerating Hydra vulgaris mouth closure. The effect was maximal at 100 nM anandamide and was reversed by the selective antagonist of the CB1 subtype of mammalian cannabinoid receptors, SR 141716A (50-100 nM). Specific cannabinoid binding sites were detected in membranes from Hydra polyps by using [3H]SR 141716A (Kd= 1.87 nM, Bmax = 26.7 fmol/mg protein), and increasing anandamide concentrations were found to displace the binding of [3H]SR 141716A to these membranes (Ki = .505 nM). Hydra polyps were also found to contain amounts of anandamide (15.6 pmol/g) and N-arachidonoylphosphatidylethanolamine (32.4 pmol/g), as well as the other "endocannabinoid" 2-arachidonoylglycerol (11.2 nmol/g), comparable to those described previously for mammalian brain. Finally, a fatty acid amide hydrolase activity (Vmax = 3.4 nmol/min/mg protein), with subcellular distribution, pH dependency and sensitivity to inhibitors similar to those reported for the mammalian enzyme, but with a lower affinity for anandamide (Km = 400 microM), was also detected in Hydra polyps. These data suggest that the endocannabinoid signalling system plays a physiological role in Hydra that is to control the feeding response. Hydra is the simplest living organism described so far to use this recently discovered regulatory system.

Cannabinoid CB1-receptor mediated regulation of gastrointestinal motility in mice in a model of intestinal inflammation.

1. We have studied the effect of cannabinoid agonists (CP 55,940 and cannabinol) on intestinal motility in a model of intestinal inflammation (induced by oral croton oil in mice) and measured cannabinoid receptor expression, endocannabinoids (anandamide and 2-arachidonylglycerol) and anandamide amidohydrolase activity both in physiological and pathophysiological states. 2. CP 55,940 (0.03 - 10 nmol mouse(-1)) and cannabinol (10 - 3000 nmol mouse(-1)) were more active in delaying intestinal motility in croton oil-treated mice than in control mice. These inhibitory effects were counteracted by the selective cannabinoid CB(1) receptor antagonist SR141716A (16 nmol mouse(-1)). SR141716A (1 - 300 nmol mouse(-1)), administered alone, increased intestinal motility to the same extent in both control and croton oil-treated mice. 3. Croton oil-induced intestinal inflammation was associated with an increased expression of CB(1) receptor, an unprecedented example of up-regulation of cannabinoid receptors during inflammation. 4. High levels of anandamide and 2-arachidonylglycerol were detected in the small intestine, although no differences were observed between control and croton oil-treated mice; by contrast anandamide amidohydrolase activity increased 2 fold in the inflamed small intestine. 5. It is concluded that inflammation of the gut increases the potency of cannabinoid agonists possibly by 'up-regulating' CB(1) receptor expression; in addition, endocannabinoids, whose turnover is increased in inflamed gut, might tonically inhibit intestinal motility.

Molecular targets for cannabidiol and its synthetic analogues: effect on vanilloid VR1 receptors and on the cellular uptake and enzymatic hydrolysis of anandamide.

1. (-)-Cannabidiol (CBD) is a non-psychotropic component of Cannabis with possible therapeutic use as an anti-inflammatory drug. Little is known on the possible molecular targets of this compound. We investigated whether CBD and some of its derivatives interact with vanilloid receptor type 1 (VR1), the receptor for capsaicin, or with proteins that inactivate the endogenous cannabinoid, anandamide (AEA). 2. CBD and its enantiomer, (+)-CBD, together with seven analogues, obtained by exchanging the C-7 methyl group of CBD with a hydroxy-methyl or a carboxyl function and/or the C-5' pentyl group with a di-methyl-heptyl (DMH) group, were tested on: (a) VR1-mediated increase in cytosolic Ca(2+) concentrations in cells over-expressing human VR1; (b) [(14)C]-AEA uptake by RBL-2H3 cells, which is facilitated by a selective membrane transporter; and (c) [(14)C]-AEA hydrolysis by rat brain membranes, which is catalysed by the fatty acid amide hydrolase. 3. Both CBD and (+)-CBD, but not the other analogues, stimulated VR1 with EC(50)=3.2 - 3.5 microM, and with a maximal effect similar in efficacy to that of capsaicin, i.e. 67 - 70% of the effect obtained with ionomycin (4 microM). CBD (10 microM) desensitized VR1 to the action of capsaicin. The effects of maximal doses of the two compounds were not additive. 4. (+)-5'-DMH-CBD and (+)-7-hydroxy-5'-DMH-CBD inhibited [(14)C]-AEA uptake (IC(50)=10.0 and 7.0 microM); the (-)-enantiomers were slightly less active (IC(50)=14.0 and 12.5 microM). 5. CBD and (+)-CBD were also active (IC(50)=22.0 and 17.0 microM). CBD (IC(50)=27.5 microM), (+)-CBD (IC(50)=63.5 microM) and (-)-7-hydroxy-CBD (IC(50)=34 microM), but not the other analogues (IC(50)>100 microM), weakly inhibited [(14)C]-AEA hydrolysis. 6. Only the (+)-isomers exhibited high affinity for CB(1) and/or CB(2) cannabinoid receptors. 7. These findings suggest that VR1 receptors, or increased levels of endogenous AEA, might mediate some of the pharmacological effects of CBD and its analogues. In view of the facile high yield synthesis, and the weak affinity for CB(1) and CB(2) receptors, (-)-5'-DMH-CBD represents a valuable candidate for further investigation as inhibitor of AEA uptake and a possible new therapeutic agent.

Evidence for an endocannabinoid system in the central nervous system of the leech Hirudo medicinalis.

In invertebrates, like Hydra and sea urchins, evidence for a functional cannabinoid system was described. The partial characterization of a putative CB1 cannabinoid receptor in the leech Hirudo medicinalis led us to investigate the presence of a complete endogenous cannabinoid system in this organism. By using gas chromatography-mass spectrometry, we demonstrate the presence of the endocannabinoids anandamide (N-arachidonoylethanolamine, 21.5+/-0.7 pmol/g) and 2-arachidonoyl-glycerol (147.4+/-42.7 pmol/g), and of the biosynthetic precursor of anandamide, N-arachidonylphosphatidyl-ethanolamine (16.5+/-3.3 pmol/g), in the leech central nervous system (CNS). Anandamide-related molecules such as N-palmitoylethanolamine (32.4+/-1.6 pmol/g) and N-linolenoylethanolamine (5.8 pmol/g) were also detected. We also found an anandamide amidase activity in the leech CNS cytosolic fraction with a maximal activity at pH 7 and little sensitivity to typical fatty acid amide hydrolase (FAAH) inhibitors. Using an antiserum directed against the amidase signature sequence, we focused on the identification and the localization of the leech amidase. Firstly, leech nervous system protein extract was subjected to Western blot analysis, which showed three immunoreactive bands at ca. approximately 42, approximately 46 and approximately 66 kDa. The former and latter bands were very faint and were also detected in whole homogenates from the coelenterate Hydra vulgaris, where the presence of CB1-like receptors, endocannabinoids and a FAAH-like activity was reported previously. Secondly, amidase immunocytochemical detection revealed numerous immunoreactive neurons in the CNS of three species of leeches. In addition, we observed that leech amidase-like immunoreactivity matches to a certain extent with CB1-like immunoreactivity. Finally, we also found that stimulation by anandamide of this receptor leads, as in mammals, to inhibition of cAMP formation, although this effect appeared to be occurring through the previously described anandamide-induced and CB1-mediated activation of nitric oxide release. Taken together, these results suggest the existence of a complete and functional cannabinoid system in leeches.