Monoacylglycerol lipase activity is a critical modulator of the tone and integrity of the endocannabinoid system.

Endocannabinoids are lipid molecules that serve as natural ligands for the cannabinoid receptors CB1 and CB2. They modulate a diverse set of physiological processes such as pain, cognition, appetite, and emotional states, and their levels and functions are tightly regulated by enzymatic biosynthesis and degradation. 2-Arachidonoylglycerol (2-AG) is the most abundant endocannabinoid in the brain and is believed to be hydrolyzed primarily by the serine hydrolase monoacylglycerol lipase (MAGL). Although 2-AG binds and activates cannabinoid receptors in vitro, when administered in vivo, it induces only transient cannabimimetic effects as a result of its rapid catabolism. Here we show using a mouse model with a targeted disruption of the MAGL gene that MAGL is the major modulator of 2-AG hydrolysis in vivo. Mice lacking MAGL exhibit dramatically reduced 2-AG hydrolase activity and highly elevated 2-AG levels in the nervous system. A lack of MAGL activity and subsequent long-term elevation of 2-AG levels lead to desensitization of brain CB1 receptors with a significant reduction of cannabimimetic effects of CB1 agonists. Also consistent with CB1 desensitization, MAGL-deficient mice do not show alterations in neuropathic and inflammatory pain sensitivity. These findings provide the first genetic in vivo evidence that MAGL is the major regulator of 2-AG levels and signaling and reveal a pivotal role for 2-AG in modulating CB1 receptor sensitization and endocannabinoid tone.

Depression-like phenotype following chronic CB1 receptor antagonism.

Rimonabant was the first clinically marketed cannabinoid (CB)(1) receptor antagonist developed to treat obesity. Unfortunately, CB(1) receptor antagonism produced adverse psychiatric events in patients. To determine whether this occurs pre-clinically, we investigated the effects of rimonabant in rodent models of mood disorders. Chronic treatment with rimonabant increased immobility time in the rat forced swim test and reduced the consumption of sucrose-sweetened water in an assay postulated to model anhedonia. These responses were similar to the effects elicited by chronic mild stress in these behavioral models, which, taken together, are indicative of a depression-like phenotype. Additionally, chronic treatment with rimonabant produced decreases in frontal cortex serotonin levels, marked reductions in hippocampal cell proliferation, survival, and BDNF levels, and elevations in the concentrations of pro-inflammatory cytokines including interferon gamma and TNF alpha. These preclinical findings mimic clinical reports and implicate possible mechanisms responsible for the unfavorable psychiatric events reported following chronic rimonabant use.

Loss of retrograde endocannabinoid signaling and reduced adult neurogenesis in diacylglycerol lipase knock-out mice.

Endocannabinoids (eCBs) function as retrograde signaling molecules at synapses throughout the brain, regulate axonal growth and guidance during development, and drive adult neurogenesis. There remains a lack of genetic evidence as to the identity of the enzyme(s) responsible for the synthesis of eCBs in the brain. Diacylglycerol lipase-alpha (DAGLalpha) and -beta (DAGLbeta) synthesize 2-arachidonoyl-glycerol (2-AG), the most abundant eCB in the brain. However, their respective contribution to this and to eCB signaling has not been tested. In the present study, we show approximately 80% reductions in 2-AG levels in the brain and spinal cord in DAGLalpha(-/-) mice and a 50% reduction in the brain in DAGLbeta(-/-) mice. In contrast, DAGLbeta plays a more important role than DAGLalpha in regulating 2-AG levels in the liver, with a 90% reduction seen in DAGLbeta(-/-) mice. Levels of arachidonic acid decrease in parallel with 2-AG, suggesting that DAGL activity controls the steady-state levels of both lipids. In the hippocampus, the postsynaptic release of an eCB results in the transient suppression of GABA-mediated transmission at inhibitory synapses; we now show that this form of synaptic plasticity is completely lost in DAGLalpha(-/-) animals and relatively unaffected in DAGLbeta(-/-) animals. Finally, we show that the control of adult neurogenesis in the hippocampus and subventricular zone is compromised in the DAGLalpha(-/-) and/or DAGLbeta(-/-) mice. These findings provide the first evidence that DAGLalpha is the major biosynthetic enzyme for 2-AG in the nervous system and reveal an essential role for this enzyme in regulating retrograde synaptic plasticity and adult neurogenesis.

Abnormal gait, due to inflammation but not nerve injury, reflects enhanced nociception in preclinical pain models.

Validation of gait analysis has the potential to bridge the gap between data from animal pain models and clinical observations. The goal of these studies was to compare alterations in gait due to inflammation or nerve injury to traditional pain measurements in animals. Pharmacological experiments determined whether gait alterations were related to enhanced nociception, edema, or motor nerve dysfunction. Gait was analyzed using an automated system (DigiGait) after injection of an inflammatory agent (carrageenan; CARR or FCA; Freund's complete adjuvant) or nerve injury (axotomy; AXO, partial sciatic nerve ligation; PSNL, spinal nerve ligation; SNL or chronic constriction injury; CCI). All models caused significant alterations in gait and thermal (inflammatory) or mechanical (nerve injury) hyperalgesia. Both indomethacin and morphine were able to block or reverse thermal hyperalgesia and normalize gait in the CARR model. Indomethacin partially blocked and did not reverse paw edema, suggesting that gait alterations must be primarily driven by enhanced nociception. In nerve injury models, AXO, PSNL, CCI, and SNL caused changes to the largest number of gait indices with the rank order being AXO>PSNL=CCI >> SNL. Gabapentin and duloxetine reversed mechanical hyperalgesia but did not normalize gait in any nerve injury model. Collectively, these data suggest that pain is the primary driver of abnormal gait in models of inflammatory but not nerve injury-related pain and suggests that, in the latter, disruption in gait is due to perturbation to the motor system. Gait may therefore constitute an alternative and potentially clinically relevant measure of pain due to inflammation.

Pharmacological characterization of the muscarinic agonist (3R,4R)-3-(3-hexylsulfanyl-pyrazin-2-yloxy)-1-aza-bicyclo[2.2.1]heptane (WAY-132983) in in vitro and in vivo models of chronic pain.

Here, we have investigated the in vitro pharmacology of a muscarinic agonist, (3R,4R)-3-(3-hexylsulfanyl-pyrazin-2-yloxy)-1-aza-bicyclo[2.2.1]heptane (WAY-132983), and we demonstrated its activity in several models of pain. WAY-132983 had a similar affinity for the five muscarinic receptors (9.4-29.0 nM); however, in calcium mobilization studies it demonstrated moderate selectivity for M(1) (IC(50) = 6.6 nM; E(max) = 65% of 10 muM carbachol-stimulation) over the M(3) (IC(50) = 23 nM; E(max) = 41%) and M(5) receptors (IC(50) = 300 nM; E(max) = 18%). WAY-132983 also activated the M(4) receptor, fully inhibiting forskolin-induced increase in cAMP levels (IC(50) = 10.5 nM); at the M(2) receptor its potency was reduced by 5-fold (IC(50) = 49.8 nM). In vivo, WAY-132983 demonstrated good systemic bioavailability and high brain penetration (>20-fold over plasma levels). In addition, WAY-1329823 produced potent and efficacious antihyperalgesic and antiallodynic effects in rodent models of chemical irritant, chronic inflammatory, neuropathic, and incisional pain. It is noteworthy that efficacy in these models was observed at doses that did not produce analgesia or ataxia. Furthermore, a series of antagonist studies demonstrated that the in vivo activity of WAY-132983 is mediated through activation of muscarinic receptors primarily through the M(4) receptor. The data presented herein suggest that muscarinic agonists, such as WAY-132983, may have a broad therapeutic efficacy for the treatment of pain.

WAY-163909 [(7bR,10aR)-1,2,3,4,8,9,10,10a-octahydro-7bH-cyclopenta-[b][1,4]diazepino[6,7,1hi]indole]: A novel 5-hydroxytryptamine 2C receptor-selective agonist with preclinical antipsychotic-like activity.

Serotonin-2C (5-HT2C) receptor antagonists and agonists have been shown to affect dopamine (DA) neurotransmission, with agonists selectively decreasing mesolimbic DA. As antipsychotic efficacy is proposed to be associated with decreased mesolimbic DA neurotransmission by virtue of DA D2 receptor antagonism, the 5-HT2C-selective receptor agonist, WAY-163909 [(7bR,10aR)-1,2, 3,4,8,9,10,10a-octahydro-7bH-cyclopenta-[b][1,4]diazepino[6,7, 1hi]indole], was evaluated in animal models of schizophrenia and in vivo microdialysis and electrophysiology to determine the effects on mesolimbic and nigrostriatal DA neurotransmission. Similar to clozapine, WAY-163909 (1.7-30 mg/kg i.p.) decreased apomorphine-induced climbing with little effect on stereotypy and no significant induction of catalepsy. WAY-163909 (0.3-3 mg/kg s.c.) more potently reduced phencyclidine-induced locomotor activity compared with d-amphetamine with no effect on spontaneous activity. WAY-163909 (1.7-17 mg/kg i.p.) reversed MK-801 (5H-dibenzo[a,d]cyclohepten-5,10-imine (dizocilpine maleate)- and DOI [1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane]-disrupted prepulse inhibition of startle (PPI) and improved PPI in DBA/2N mice. In conditioned avoidance responding, WAY-163909 (0.3-3 mg/kg i.p.; 1-17 mg/kg p.o.) reduced avoidance responding, an effect blocked by the 5-HT(2B/2C) receptor antagonist SB 206553 [5-methyl-1-(3-pyridylcarbamoyl)-1,2,3,5-tetrahydropyrrolo[2,3-f]indole]. WAY-163909 (10 mg/kg s.c.) selectively decreased extracellular levels of DA in the nucleus accumbens without affecting the striatum. Likewise, in vivo electrophysiological recordings showed a decrease in the number of spontaneously firing DA neurons in the ventral tegmental area but not in the substantia nigra with both acute and chronic (21-day) administration of WAY-163909 (1-10 mg/kg i.p.). Thus, the profile of the 5-HT2C selective receptor agonist WAY-163909 is similar to that of an atypical antipsychotic and additionally may have rapid onset properties.

An estrogen receptor-beta agonist is active in models of inflammatory and chemical-induced pain.

ERB-041 (2-(3-Fluoro-4-hydroxyphenyl)-7-vinyl-1,3 benzoxazol-5-ol) is a selective estrogen receptor-beta agonist with activity in rodent models of rheumatoid arthritis and endometriosis. Clinical trials for these diseases are underway: however, the role of estrogen receptor-beta in modulating pain associated with inflammation remains unknown. These studies demonstrate that acutely administered ERB-041 is anti-hyperalgesic in preclinical models of chemical-induced and acute inflammatory pain, thus suggesting that ERB-041 may be useful for modulating pain associated with some types of inflammation.