glyceryl-2-arachidonate and Neuralgia

Recent years have yielded significant advances in our understanding of microglia, the immune cells of the central nervous system (CNS). Microglia are key players in CNS development, immune surveillance, and the maintenance of proper neuronal function throughout life. In the healthy brain, homeostatic microglia have a unique molecular signature. In neurological diseases, microglia become activated and adopt distinct transcriptomic signatures, including disease-associated microglia (DAM) implicated in neurodegenerative disorders. Homeostatic microglia synthesise the endogenous cannabinoids 2-arachidonoylglycerol and anandamide and express the cannabinoid receptors CB1 and CB2 at constitutively low levels. Upon activation, microglia significantly increase their synthesis of endocannabinoids and upregulate their expression of CB2 receptors, which promote a protective microglial phenotype by enhancing their production of neuroprotective factors and reducing their production of pro-inflammatory factors. Here, we summarise the effects of the microglial cannabinoid system in the CNS demyelinating disease multiple sclerosis, the neurodegenerative diseases Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis, chronic inflammatory and neuropathic pain, and psychiatric disorders including depression, anxiety and schizophrenia. We discuss the therapeutic potential of cannabinoids in regulating microglial activity and highlight the need to further investigate their specific microglia-dependent immunomodulatory effects.

Topics: Alzheimer Disease; Amyotrophic Lateral Sclerosis; Anxiety Disorders; Arachidonic Acids; Chronic Pain; Depressive Disorder; Endocannabinoids; Glycerides; Humans; Mental Disorders; Microglia; Multiple Sclerosis; Neuralgia; Neurodegenerative Diseases; Parkinson Disease; Polyunsaturated Alkamides; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Schizophrenia

The endocannabinoid system was revealed following the understanding of the mechanism of action of marijuana's major psychotropic principle, Δ(9)-tetrahydrocannabinol, and includes two G-protein-coupled receptors (GPCRs; the cannabinoid CB1 and CB2 receptors), their endogenous ligands (the endocannabinoids, the best studied of which are anandamide and 2-arachidonoylglycerol (2-AG)), and the proteins that regulate the levels and activity of these receptors and ligands. However, other minor lipid metabolites different from, but chemically similar to, anandamide and 2-AG have also been suggested to act as endocannabinoids. Thus, unlike most other GPCRs, cannabinoid receptors appear to have more than one endogenous agonist, and it has been often wondered what could be the physiological meaning of this peculiarity. In 1999, it was proposed that anandamide might also activate other targets, and in particular the transient receptor potential of vanilloid type-1 (TRPV1) channels. Over the last decade, this interaction has been shown to occur both in peripheral tissues and brain, during both physiological and pathological conditions. TRPV1 channels can be activated also by another less abundant endocannabinoid, N-arachidonoyldopamine, but not by 2-AG, and have been proposed by some authors to act as ionotropic endocannabinoid receptors. This article will discuss the latest discoveries on this subject, and discuss, among others, how anandamide and 2-AG differential actions at TRPV1 and cannabinoid receptors contribute to making this signalling system a versatile tool available to organisms to fine-tune homeostasis.

Topics: Animals; Arachidonic Acids; Brain; Cannabinoid Receptor Agonists; Endocannabinoids; Glycerides; Humans; Ligands; Neuralgia; Pain Perception; Polyunsaturated Alkamides; Receptor Cross-Talk; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Signal Transduction; Synaptic Transmission; TRPV Cation Channels

Neuropathic pain refers to chronic pain that results from injury to the nervous system. The mechanisms involved in neuropathic pain are complex and involve both peripheral and central phenomena. Although numerous pharmacological agents are available for the treatment of neuropathic pain, definitive drug therapy has remained elusive. Recent drug discovery efforts have identified an original neurobiological approach to the pathophysiology of neuropathic pain. The development of innovative pharmacological strategies has led to the identification of new promising pharmacological targets, including glutamate antagonists, microglia inhibitors and, interestingly, endogenous ligands of cannabinoids and the transient receptor potential vanilloid type 1 (TRPV1). Endocannabinoids (ECs), endovanilloids and the enzymes that regulate their metabolism represent promising pharmacological targets for the development of a successful pain treatment. This review is an update of the relationship between cannabinoid receptors (CB1) and TRPV1 channels and their possible implications for neuropathic pain. The data are focused on endogenous spinal mechanisms of pain control by anandamide, and the current and emerging pharmacotherapeutic approaches that benefit from the pharmacological modulation of spinal EC and/or endovanilloid systems under chronic pain conditions will be discussed.

Topics: Amides; Amidohydrolases; Animals; Arachidonic Acid; Arachidonic Acids; Behavior; Benzamides; Carbamates; Endocannabinoids; Enzyme Inhibitors; Ethanolamines; Glycerides; Humans; Microglia; Neuralgia; Palmitic Acids; Peripheral Nerve Injuries; Polyunsaturated Alkamides; Receptor, Cannabinoid, CB1; Spinal Cord Injuries; TRPV Cation Channels

The analgesic effects of cannabinoid ligands, mediated by CB1 receptors are well established. However, the side-effect profile of CB1 receptor ligands has necessitated the search for alternative cannabinoid-based approaches to analgesia. Herein, we review the current literature describing the impact of chronic pain states on the key components of the endocannabinoid receptor system, in terms of regionally restricted changes in receptor expression and levels of key metabolic enzymes that influence the local levels of the endocannabinoids. The evidence that spinal CB2 receptors have a novel role in the modulation of nociceptive processing in models of neuropathic pain, as well as in models of cancer pain and arthritis is discussed. Recent advances in our understanding of the spinal location of the key enzymes that regulate the levels of the endocannabinoid 2-AG are discussed alongside the outcomes of recent studies of the effects of inhibiting the catabolism of 2-AG in models of pain. The complexities of the enzymes capable of metabolizing both anandamide (AEA) and 2-AG have become increasingly apparent. More recently, it has come to light that some of the metabolites of AEA and 2-AG generated by cyclooxygenase-2, lipoxygenases and cytochrome P450 are biologically active and can either exacerbate or inhibit nociceptive signalling.

Topics: Analgesics; Animals; Arachidonic Acids; Arthritis; Cannabinoid Receptor Agonists; Chronic Pain; Disease Models, Animal; Endocannabinoids; Glycerides; Humans; Neoplasms; Neuralgia; Polyunsaturated Alkamides; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2

Modulation of the endocannabinoid system has been shown to alleviate neuropathic pain. The aim of this study was to evaluate if treatment with paclitaxel, a chemotherapeutic agent that induces neuropathic pain, affects endocannabinoid levels at a time when mice develop paclitaxel-induced mechanical allodynia. We also evaluated the peripheral antiallodynic activity of the endocannabinoid 2-arachidonoyl glycerol (2-AG) and an inhibitor of monoacylglycerol lipase (MAGL), an enzyme responsible for 2-AG hydrolysis.. Female BALB/c mice were treated intraperitoneally with paclitaxel to induce mechanical allodynia. Levels of the endocannabinoids, N-arachidonoylethanolamine (anandamide, AEA), 2-AG, and the N-acylethanolamines (NAEs), N-palmitoylethanolamide (PEA) and N-oleoylethanolamide (OEA), which are structurally-related to AEA, in the brain, spinal cord and paw skin were measured using LC-MS/MS. Protein expression of MAGL in the paw skin was measured using Wes™. The effects of subcutaneous (s.c.) injection of 2-AG and JZL184 (a MAGL inhibitor) into the right hind paw of mice with paclitaxel-induced mechanical allodynia were assessed using the dynamic plantar aesthesiometer. The effects of pretreatment, s.c., into the right hind paw, with cannabinoid type 1 (CB. The levels of 2-AG were reduced only in the paw skin of paclitaxel-treated mice, whilst the levels of AEA, PEA and OEA were not significantly altered. There was no change in the expression of MAGL in the paw skin. Administration of 2-AG and JZL184 produced antiallodynic effects against paclitaxel-induced mechanical allodynia in the injected right paw, but did not affect the uninjected left paw. The antiallodynic activity of 2-AG was antagonized by both AM251 and AM630.. These results indicate that during paclitaxel-induced mechanical allodynia there is a deficiency of 2-AG in the periphery, but not in the CNS. Increasing 2-AG in the paw by local administration of 2-AG or a MAGL inhibitor, alleviates mechanical allodynia in a CB

Topics: Analgesics; Animals; Arachidonic Acids; Benzodioxoles; Cannabinoid Receptor Agonists; Disease Models, Animal; Endocannabinoids; Enzyme Inhibitors; Female; Glycerides; Hyperalgesia; Mice, Inbred BALB C; Monoacylglycerol Lipases; Neuralgia; Paclitaxel; Piperidines; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Skin

Neuropathic pain is often associated with depression. Enhancing endocannabinoids by fatty acid amide hydrolase (FAAH) inhibitors relieves neuropathic pain and stress-induced depressive-like behaviors in animal models. However, it is unclear whether FAAH inhibitor can relieve neuropathic pain-induced depression by or not by its antinociceptive effects.. Adult male Wistar rats with chronic constriction injury (CCI) to the sciatic nerve were treated with the systemic FAAH inhibitor URB597 (5.8 mg·kg·day, intraperitoneally) or peripherally acting FAAH inhibitor URB937 (1.6 mg·kg·d, intraperitoneally; n = 11-12). The treatment was applied from the 15th day after surgery and continued for 15 days. Mechanical withdrawal threshold was examined by Von Frey test before surgery and on the 28th day after CCI. Depressive-like behaviors were evaluated by forced swimming test (FST) and novelty-suppressed feeding (NSF) after 15-day treatment. The levels of anandamide and 2-arachidonoylglycerol in hippocampus were examined by liquid chromatography and mass spectrometry. Hippocampal neurogenesis including proliferation, differentiation, and survival of newborn cells was assessed by immunohistochemistry.. After CCI injury, the rats developed significantly nociceptive and depressive-like behaviors, indicated by persistent mechanical hypersensitivity in Von Frey test, significantly prolonged immobility time in FST (sham: 84.2 ± 13.4 seconds versus CCI: 137.9 ± 18.8 seconds; P < .001), and protracted latency to feed in NSF (sham: 133.4 ± 19.4 seconds versus CCI: 234.9 ± 33.5 seconds; P < .001). For the CCI rats receiving treatment, compared to vehicle placebo group, pain threshold was increased by both URB597 (3.1 ± 1.0 vs 11.2 ± 1.2 g; P < .001) and URB937 (3.1 ± 1.0 vs 12.1 ± 1.3 g; P < .001). Immobility time of FST was reduced by URB597 (135.8 ± 16.6 vs 85.3 ± 17.2 seconds; P < .001) but not by URB937 (135.8 ± 16.6 vs 129.6 ± 17.8 seconds; P = .78). Latency to feed in NSF was also reduced by URB597 (235.9 ± 30.5 vs 131.8 ± 19.8 seconds; P < .001) but not by URB937 (235.9 ± 30.5 vs 232.2 ± 33.2 seconds; P = .72). Meanwhile, CCI decreased the number of proliferating cells and reduced survival of new mature neurons in hippocampus. URB597 but not URB937 treatment improved these cellular deficits.. Inhibition of FAAH can improve depressive-like behaviors induced by neuropathic pain independent of its peripheral antinociceptive action. Enhanced neurogenesis in hippocampus might contribute to the antidepressive effects of URB597.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Behavior, Animal; Benzamides; Carbamates; Depression; Disease Models, Animal; Endocannabinoids; Enzyme Inhibitors; Feeding Behavior; Glycerides; Hippocampus; Locomotion; Male; Neuralgia; Neurogenesis; Pain Threshold; Polyunsaturated Alkamides; Rats, Wistar; Receptor, Cannabinoid, CB1; Signal Transduction; Swimming

Current therapeutics are not effective for orofacial neuropathic pain, and better options are needed. The present study used inferior orbital nerve (ION)-injured mice to investigate the effect of inhibiting monoacylglycerol lipase (MAGL), an enzyme that degrades the major endocannabinoid 2-arachydonoylgycerol (2-AG) in orofacial neuropathic pain. The head-withdrawal threshold to mechanical stimulation of the whisker pad was reduced on days 3, 5, and 7 after ION injury. Injection of JZL184, a selective inhibitor of MAGL, on day 7 after ION injury attenuated the reduction in head-withdrawal threshold at 2 h after administration. Moreover, the numbers of MAGL-immunoreactive neurons in the trigeminal subnucleus caudalis (Vc) and upper cervical spinal cord (C1-C2) were significantly greater in ION-injured mice than in sham-operated mice but were reduced after administration of JZL184. The increase in MAGL immunoreactivity suggests that increased 2-AG production is followed by rapid enzymatic degradation of 2-AG. JZL184 inhibited this degradation and thus increased 2-AG concentration in the brain, particularly in the Vc and C1-C2 regions, thus attenuating pain. Our findings suggest that inhibition of 2-AG degradation by MAGL inhibitors is a promising therapeutic option for treatment of orofacial neuropathic pain.

Topics: Animals; Arachidonic Acids; Behavior, Animal; Benzodioxoles; Endocannabinoids; Enzyme Inhibitors; Facial Pain; Glycerides; Male; Mice, Inbred C57BL; Monoacylglycerol Lipases; Neuralgia; Piperidines; Trigeminal Nerve Injuries

Although opioids are highly efficacious analgesics, their abuse potential and other untoward side effects diminish their therapeutic utility. The addition of non-opioid analgesics offers a promising strategy to reduce required antinociceptive opioid doses that concomitantly reduce opioid-related side effects. Inhibitors of the primary endocannabinoid catabolic enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) show opioid-sparing effects in preclinical models of pain. As simultaneous inhibition of these enzymes elicits enhanced antinociceptive effects compared with single enzyme inhibition, the present study tested whether the dual FAAH-MAGL inhibitor SA-57 [4-[2-(4-chlorophenyl)ethyl]-1-piperidinecarboxylic acid 2-(methylamino)-2-oxoethyl ester] produces morphine-sparing antinociceptive effects, without major side effects associated with either drug class. SA-57 dose-dependently reversed mechanical allodynia in the constriction injury (CCI) of the sciatic nerve model of neuropathic pain and carrageenan inflammatory pain model. As previously reported, SA-57 was considerably more potent in elevating anandamide (AEA) than 2-arachidonyl glycerol (2-AG) in brain. Its anti-allodynic effects required cannabinoid (CB)

Topics: Acetamides; Analgesics; Analgesics, Opioid; Animals; Arachidonic Acid; Arachidonic Acids; Carbamates; Carrageenan; Dose-Response Relationship, Drug; Drug-Seeking Behavior; Endocannabinoids; Glycerides; Heroin; Hydrolysis; Hyperalgesia; Inflammation; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Morphine; Neuralgia; Polyunsaturated Alkamides; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Sciatic Nerve; Self Administration

Serious clinical liabilities associated with the prescription of opiates for pain control include constipation, respiratory depression, pruritus, tolerance, abuse, and addiction. A recognized strategy to circumvent these side effects is to combine opioids with other antinociceptive agents. The combination of opiates with the primary active constituent of cannabis (Δ(9)-tetrahydrocannabinol) produces enhanced antinociceptive actions, suggesting that cannabinoid receptor agonists can be opioid sparing. Here, we tested whether elevating the endogenous cannabinoid 2-arachidonoylglycerol through the inhibition of its primary hydrolytic enzyme monoacylglycerol lipase (MAGL), will produce opioid-sparing effects in the mouse chronic constriction injury (CCI) of the sciatic nerve model of neuropathic pain. The dose-response relationships of i.p. administration of morphine and the selective MAGL inhibitor 2,5-dioxopyrrolidin-1-yl 4-(bis(4-chlorophenyl)methyl)piperazine-1-carboxylate (MJN110) were tested alone and in combination at equieffective doses for reversal of CCI-induced mechanical allodynia and thermal hyperalgesia. The respective ED50 doses (95% confidence interval) of morphine and MJN110 were 2.4 (1.9-3.0) mg/kg and 0.43 (0.23-0.79) mg/kg. Isobolographic analysis of these drugs in combination revealed synergistic antiallodynic effects. Acute antinociceptive effects of the combination of morphine and MJN110 required μ-opioid, CB1, and CB2 receptors. This combination did not reduce gastric motility or produce subjective cannabimimetic effects in the drug discrimination assay. Importantly, combinations of MJN110 and morphine given repeatedly (i.e., twice a day for 6 days) continued to produce antiallodynic effects with no evidence of tolerance. Taken together, these findings suggest that MAGL inhibition produces opiate-sparing events with diminished tolerance, constipation, and cannabimimetic side effects.

Topics: Analgesics, Opioid; Animals; Arachidonic Acids; Behavior, Animal; Carbamates; Constriction, Pathologic; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Synergism; Endocannabinoids; Enzyme Inhibitors; Glycerides; Hyperalgesia; Male; Mice; Mice, Inbred C57BL; Monoacylglycerol Lipases; Morphine; Neuralgia; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Receptors, Opioid, mu; Succinimides

We report the discovery of compound 4a, a potent β-lactam-based monoacylglycerol lipase (MGL) inhibitor characterized by an irreversible and stereoselective mechanism of action, high membrane permeability, high brain penetration evaluated using a human in vitro blood-brain barrier model, high selectivity in binding and affinity-based proteomic profiling assays, and low in vitro toxicity. Mode-of-action studies demonstrate that 4a, by blocking MGL, increases 2-arachidonoylglycerol and behaves as a cannabinoid (CB1/CB2) receptor indirect agonist. Administration of 4a in mice suffering from experimental autoimmune encephalitis ameliorates the severity of the clinical symptoms in a CB1/CB2-dependent manner. Moreover, 4a produced analgesic effects in a rodent model of acute inflammatory pain, which was antagonized by CB1 and CB2 receptor antagonists/inverse agonists. 4a also relieves the neuropathic hypersensitivity induced by oxaliplatin. Given these evidence, 4a, as MGL selective inhibitor, could represent a valuable lead for the future development of therapeutic options for multiple sclerosis and chronic pain.

Topics: Animals; Arachidonic Acids; Blood-Brain Barrier; Brain; Cell Membrane; Drug Design; Encephalomyelitis, Autoimmune, Experimental; Endocannabinoids; Glycerides; HEK293 Cells; Humans; Mice; Models, Molecular; Monoacylglycerol Lipases; Multiple Sclerosis; Mutagenicity Tests; Neuralgia; Organoplatinum Compounds; Oxaliplatin; Pain; Permeability; Proteomics; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Structure-Activity Relationship

There is considerable evidence to support the role of anandamide (AEA), an endogenous ligand of cannabinoid receptors, in neuropathic pain modulation. AEA also produces effects mediated by other biological targets, of which the transient receptor potential vanilloid type 1 (TRPV1) has been the most investigated. Both, inhibition of AEA breakdown by fatty acid amide hydrolase (FAAH) and blockage of TRPV1 have been shown to produce anti-nociceptive effects. Recent research suggests the usefulness of dual-action compounds, which may afford greater anti-allodynic efficacy. Therefore, in the present study, we examined the effect of N-arachidonoyl-serotonin (AA-5-HT), a blocker of FAAH and TRPV1, in a rat model of neuropathic pain after intrathecal administration. We found that treatment with AA-5-HT increased the pain threshold to mechanical and thermal stimuli, with highest effect at the dose of 500nM, which was most strongly attenuated by AM-630, CB2 antagonist, administration. The single action blockers PF-3845 (1000nM, for FAAH) and I-RTX (1nM, for TRPV1) showed lower efficacy than AA-5-HT. Moreover AA-5-HT (500nM) elevated AEA and palmitoylethanolamide (PEA) levels. Among the possible targets of these mediators, only the mRNA levels of CB2, GPR18 and GPR55, which are believed to be novel cannabinoid receptors, were upregulated in the spinal cord and/or DRG of CCI rats. It was previously reported that AA-5-HT acts in CB1 and TRPV1-dependent manner after systemic administration, but here for the first time we show that AA-5-HT action at the spinal level involves CB2, with potential contributions from GRP18 and/or GPR55 receptors.

Topics: Amidohydrolases; Analgesics; Animals; Arachidonic Acids; Cannabinoid Receptor Antagonists; Disease Models, Animal; Dose-Response Relationship, Drug; Endocannabinoids; Ganglia, Spinal; Glycerides; Injections, Spinal; Male; Neuralgia; Nociception; Pain Threshold; Polyunsaturated Alkamides; Rats, Wistar; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Receptors, Cannabinoid; Receptors, G-Protein-Coupled; Serotonin; Signal Transduction; Spinal Cord; Time Factors; TRPV Cation Channels

Chronic stress alters the hypothalamic-pituitary-adrenal (HPA) axis and enhances visceral and somatosensory pain perception. It is unresolved whether chronic stress has distinct effects on visceral and somatosensory pain regulatory pathways. Previous studies reported that stress-induced visceral hyperalgesia is associated with reciprocal alterations of endovanilloid and endocannabinoid pain pathways in DRG neurons innervating the pelvic viscera. In this study, we compared somatosensory and visceral hyperalgesia with respect to differential responses of peripheral pain regulatory pathways in a rat model of chronic, intermittent stress. We found that chronic stress induced reciprocal changes in the endocannabinoid 2-AG (increased) and endocannabinoid degradation enzymes COX-2 and FAAH (decreased), associated with down-regulation of CB1 and up-regulation of TRPV1 receptors in L6-S2 DRG but not L4-L5 DRG neurons. In contrast, sodium channels Nav1.7 and Nav1.8 were up-regulated in L4-L5 but not L6-S2 DRGs in stressed rats, which was reproduced in control DRGs treated with corticosterone in vitro. The reciprocal changes of CB1, TRPV1 and sodium channels were cell-specific and observed in the sub-population of nociceptive neurons. Behavioral assessment showed that visceral hyperalgesia persisted, whereas somatosensory hyperalgesia and enhanced expression of Nav1.7 and Nav1.8 sodium channels in L4-L5 DRGs normalized 3 days after completion of the stress phase. These data indicate that chronic stress induces visceral and somatosensory hyperalgesia that involves differential changes in endovanilloid and endocannabinoid pathways, and sodium channels in DRGs innervating the pelvic viscera and lower extremities. These results suggest that chronic stress-induced visceral and lower extremity somatosensory hyperalgesia can be treated selectively at different levels of the spinal cord.

Topics: Afferent Pathways; Animals; Arachidonic Acids; Corticosterone; Cyclooxygenase 2; Disease Models, Animal; Endocannabinoids; Evoked Potentials, Motor; Ganglia, Spinal; Gene Expression Regulation; Glycerides; Hyperalgesia; Intestine, Large; Male; Neuralgia; Pain Threshold; Physical Stimulation; Rats; Rats, Sprague-Dawley; Reaction Time; Receptor, Cannabinoid, CB1; Spinal Cord; Stress, Psychological; TRPV Cation Channels; Visceral Pain

The two most studied endocannabinoids are anandamide (AEA), principally catalyzed by fatty-acid amide hydrolase (FAAH), and 2-arachidonoyl glycerol (2-AG), mainly hydrolyzed by monoacylglycerol lipase (MGL). Inhibitors targeting these two enzymes have been described, including URB597 and URB602, respectively. Several recent studies examining the contribution of CB₁ and/or CB₂ receptors on the peripheral antinociceptive effects of AEA, 2-AG, URB597 and URB602 in neuropathic pain conditions using either pharmacological tools or transgenic mice separately have been reported, but the exact mechanism is still uncertain. Mechanical allodynia and thermal hyperalgesia were evaluated in 436 male C57BL/6, cnr1KO and cnr2KO mice in the presence or absence of cannabinoid CB₁ (AM251) or CB₂ (AM630) receptor antagonists in a mouse model of neuropathic pain. Peripheral subcutaneous injections of AEA, 2-AG, WIN55,212-2 (WIN; a CB₁/CB₂ synthetic agonist), URB597 and URB602 significantly decreased mechanical allodynia and thermal hyperalgesia. These effects were inhibited by both cannabinoid antagonists AM251 and AM630 for treatments with 2-AG, WIN and URB602 but only by AM251 for treatments with AEA and URB597 in C57BL/6 mice. Furthermore, the antinociceptive effects for AEA and URB597 were observed in cnr2KO mice but absent in cnr1KO mice, whereas the effects of 2-AG, WIN and URB602 were altered in both of these transgenic mice. Complementary genetic and pharmacological approaches revealed that the anti-hyperalgesic effects of 2-AG and URB602 required both CB₁ and CB₂ receptors, but only CB₂ receptors mediated its anti-allodynic actions. The antinociceptive properties of AEA and URB597 were mediated only by CB₁ receptors.

Topics: Animals; Arachidonic Acids; Behavior, Animal; Endocannabinoids; Glycerides; Hyperalgesia; Male; Mice; Mice, Knockout; Neuralgia; Pain Measurement; Pain Threshold; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2

Monoacylglycerol lipase (MGL) is the main enzyme responsible for degradation of the endocannabinoid 2-arachidonoylglycerol (2-AG). Selective inhibitors of MGL have antinociceptive effects upon acute administration and, therefore, hold promise as analgesics. To gain insight into the possible consequences of their prolonged administration, genetically modified mice with the knocked-out MGL gene were tested in several models of acute (phasic, tonic) and chronic (inflammatory, neuropathic) pain. MGL knockout mice showed normal acute phasic pain perception (pain thresholds) and no alleviation of pain perception in models of inflammatory and neuropathic pain. However, compared with wild-type controls, they showed significantly augmented nociceptive behavior in models of acute somatic and visceral tonic pain (formalin and acetic acid tests). The observed proalgesic changes in perception of tonic pain in MGL knockouts could have resulted from desensitization of cannabinoid receptors (known to occur after genetic inactivation of MGL). Supporting this notion, chronic pretreatment with the selective CB1 receptor antagonist AM 251 (employed to re-sensitize cannabinoid receptors in MGL knockouts) resulted in normalization of their tonic pain-related behaviors. Similar augmentation of tonic pain-related behaviors was replicated in C57BL/6N mice pretreated chronically with the selective MGL inhibitor JZL 184 (employed to pharmacologically desensitize CB1 receptors). These findings imply that prolonged use of MGL inhibitors, at doses causing close to complete inhibition of MGL enzymatic activity, not only have no beneficial analgesic effects, they may lead to exacerbation of some types of pain (particularly those with a tonic component).

Topics: Animals; Arachidonic Acids; Benzodioxoles; Cannabinoid Receptor Modulators; Endocannabinoids; Glycerides; Inflammation; Male; Mice; Mice, Inbred C57BL; Mice, Inbred CBA; Mice, Knockout; Neuralgia; Pain; Pain Threshold; Phosphotransferases (Alcohol Group Acceptor); Piperidines; Pyrazoles; Receptor, Cannabinoid, CB1

Cisplatin has been used effectively to treat a variety of cancers but its use is limited by the development of painful peripheral neuropathy. Because the endocannabinoid 2-arachidonoyl-sn-glycerol (2-AG) is anti-hyperalgesic in several preclinical models of chronic pain, the anti-hyperalgesic effect of JZL184, an inhibitor of 2-AG hydrolysis, was tested in a murine model of cisplatin-induced hyperalgesia. Systemic injection of cisplatin (1mg/kg) produced mechanical hyperalgesia when administered daily for 7 days. Daily peripheral administration of a low dose of JZL184 in conjunction with cisplatin blocked the expression of mechanical hyperalgesia. Acute injection of a cannabinoid (CB)-1 but not a CB2 receptor antagonist reversed the anti-hyperalgesic effect of JZL184 indicating that downstream activation of CB1 receptors suppressed the expression of mechanical hyperalgesia. Components of endocannabinoid signaling in plantar hind paw skin and lumbar dorsal root ganglia (DRGs) were altered by treatments with cisplatin and JZL184. Treatment with cisplatin alone reduced levels of 2-AG and AEA in skin and DRGs as well as CB2 receptor protein in skin. Combining treatment of JZL184 with cisplatin increased 2-AG in DRGs compared to cisplatin alone but had no effect on the amount of 2-AG in skin. Evidence that JZL184 decreased the uptake of [(3)H]AEA into primary cultures of DRGs at a concentration that also inhibited the enzyme fatty acid amide hydrolase, in conjunction with data that 2-AG mimicked the effect of JZL184 on [(3)H]AEA uptake support the conclusion that AEA most likely mediates the anti-hyperalgesic effect of JZL184 in this model.

Topics: Amides; Analgesics; Animals; Antineoplastic Agents; Arachidonic Acids; Benzodioxoles; Cells, Cultured; Cisplatin; Disease Models, Animal; Endocannabinoids; Ethanolamines; Ganglia, Spinal; Glycerides; Hyperalgesia; Indoles; Male; Mesencephalon; Mice; Mice, Inbred C3H; Monoacylglycerol Lipases; Morpholines; Neuralgia; Palmitic Acids; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Skin; Spinal Cord

Neuropathic pain elevates spinal anandamide (AEA) levels in a way further increased when URB597, an inhibitor of AEA hydrolysis by fatty acid amide hydrolase (FAAH), is injected intrathecally. Spinal AEA reduces neuropathic pain by acting at both cannabinoid CB1 receptors and transient receptor potential vanilloid-1 (TRPV1) channels. Yet, intrathecal URB597 is only partially effective at counteracting neuropathic pain. We investigated the effect of high doses of intrathecal URB597 on allodynia and hyperalgesia in rats with chronic constriction injury (CCI) of the sciatic nerve. Among those tested, the 200 µg/rat dose of URB597 was the only one that elevated the levels of the FAAH non-endocannabinoid and anti-inflammatory substrates, oleoylethanolamide (OEA) and palmitoylethanolamide (PEA), and of the endocannabinoid FAAH substrate, 2-arachidonoylglycerol, and fully inhibited thermal and tactile nociception, although in a manner blocked almost uniquely by TRPV1 antagonism. Surprisingly, this dose of URB597 decreased spinal AEA levels. RT-qPCR and western blot analyses demonstrated altered spinal expression of lipoxygenases (LOX), and baicalein, an inhibitor of 12/15-LOX, significantly reduced URB597 analgesic effects, suggesting the occurrence of alternative pathways of AEA metabolism. Using immunofluorescence techniques, FAAH, 15-LOX and TRPV1 were found to co-localize in dorsal spinal horn neurons of CCI rats. Finally, 15-hydroxy-AEA, a 15-LOX derivative of AEA, potently and efficaciously activated the rat recombinant TRPV1 channel. We suggest that intrathecally injected URB597 at full analgesic efficacy unmasks a secondary route of AEA metabolism via 15-LOX with possible formation of 15-hydroxy-AEA, which, together with OEA and PEA, may contribute at producing TRPV1-mediated analgesia in CCI rats.

Topics: Amides; Amidohydrolases; Analgesia; Animals; Arachidonate 15-Lipoxygenase; Arachidonic Acids; Benzamides; Calcium Signaling; Carbamates; Diterpenes; Endocannabinoids; Ethanolamines; Flavanones; Glycerides; HEK293 Cells; Humans; Hyperalgesia; Injections, Spinal; Lipoxygenase Inhibitors; Male; Neuralgia; Oleic Acids; Palmitic Acids; Polyunsaturated Alkamides; Posterior Horn Cells; Rats; Rats, Wistar; Sciatic Nerve; Spinal Cord; TRPV Cation Channels

The role of spinal cannabinoid systems in neuropathic pain of streptozotocin (STZ)-induced diabetic mice was studied. In normal mice, injection of the cannabinoid receptor agonist WIN-55,212-2 (1 and 3μg, i.t.) dose-dependently prolonged the tail-flick latency, whereas there were no changes with the injection of either cannabinoid CB1 (AM 251, 1 μg, i.t.) or CB2 (AM 630, 4 μg, i.t.) receptor antagonists. AM 251 (1 μg, i.t.), but not AM 630 (4 μg, i.t.), significantly inhibited the prolongation of the tail-flick latency induced by WIN-55,212-2 (3 μg, i.t.). In STZ-induced diabetic mice, the tail-flick latency was significantly shorter than that in normal mice. A low dose of WIN-55,212-2 (1 μg, i.t.) significantly recovered the tail-flick latency in STZ-induced diabetic mice. The effect of WIN-55,212-2 (1 μg, i.t.) in STZ-induced diabetic mice was significantly inhibited by AM 630 (4 μg, i.t.), but not AM 251 (1 μg). The selective cannabinoid CB2 receptor agonist L-759,656 (19 and 38 μg, i.t.) also dose-dependently recovered the tail-flick latency in STZ-induced diabetic mice, and this recovery was inhibited by AM 630 (4 μg, i.t.). The protein levels of cannabinoid CB1 receptors, CB2 receptors and diacylglycerol lipase α (DGL-α), the enzyme that synthesizes endocannabinoid 2-arachidonoylglycerol, in the spinal cord were examined using Western blotting. The protein levels of both cannabinoid CB1 and CB2 receptors were increased in STZ-induced diabetic mice, whereas the protein level of DGL-α was significantly decreased. These results indicate that spinal cannabinoid systems are changed in diabetic mice and suggest that cannabinoid CB2 receptor agonists might have an ability to recover diabetic neuropathic pain.

Topics: Animals; Arachidonic Acids; Benzoxazines; Blotting, Western; Chromans; Diabetes Mellitus, Experimental; Endocannabinoids; Glycerides; Injections, Spinal; Lipoprotein Lipase; Male; Mice; Mice, Inbred ICR; Morpholines; Naphthalenes; Neuralgia; Pain Measurement; Pain Threshold; Reaction Time; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Spinal Cord

Recurrent myelitis is one of the predominant characteristics in patients with neuromyelitis optica (NMO). While paresis, visual loss, sensory deficits, and bladder dysfunction are well known symptoms in NMO patients, pain has been recognized only recently as another key symptom of the disease. Although spinal cord inflammation is a defining aspect of neuromyelitis, there is an almost complete lack of data on altered somatosensory function, including pain. Therefore, eleven consecutive patients with NMO were investigated regarding the presence and clinical characteristics of pain. All patients were examined clinically as well as by Quantitative Sensory Testing (QST) following the protocol of the German Research Network on Neuropathic Pain (DFNS). Additionally, plasma endocannabinoid levels and signs of chronic stress and depression were determined. Almost all patients (10/11) suffered from NMO-associated neuropathic pain for the last three months, and 8 out of 11 patients indicated relevant pain at the time of examination. Symptoms of neuropathic pain were reported in the vast majority of patients with NMO. Psychological testing revealed signs of marked depression. Compared to age and gender-matched healthy controls, QST revealed pronounced mechanical and thermal sensory loss, strongly correlated to ongoing pain suggesting the presence of deafferentation-induced neuropathic pain. Thermal hyperalgesia correlated to MRI-verified signs of spinal cord lesion. Heat hyperalgesia was highly correlated to the time since last relapse of NMO. Patients with NMO exhibited significant mechanical and thermal dysesthesia, namely dynamic mechanical allodynia and paradoxical heat sensation. Moreover, they presented frequently with either abnormal mechanical hypoalgesia or hyperalgesia, which depended significantly on plasma levels of the endogenous cannabinoid 2-arachidonoylglycerole (2-AG). These data emphasize the high prevalence of neuropathic pain and hyperalgesia in patients with NMO. The degree of mechanical hyperalgesia reflecting central sensitization of nociceptive pathways seems to be controlled by the major brain endocannabinoid 2-AG.

Topics: Adult; Aged; Arachidonic Acids; Brain; Case-Control Studies; Depression; Endocannabinoids; Female; Glycerides; Humans; Hyperalgesia; Hypesthesia; Male; Middle Aged; Neuralgia; Neuromyelitis Optica; Optic Nerve; Pain Measurement; Pain Threshold; Psychological Tests; Spinal Cord

During neuropathic pain, caspases are activated in the limbic cortex. We investigated the role of TRPV1 channels and glial caspases in the mouse prelimbic and infralimbic (PL-IL) cortex after spared nerve injury (SNI). Reverse transcriptase-polymerase chain reaction, western blots, and immunfluorescence showed overexpression of several caspases in the PL-IL cortex 7 days postinjury. Caspase-3 release and upregulation of AMPA receptors in microglia, caspase-1 and IL-1β release in astrocytes, and upregulation of Il-1 receptor-1, TRPV1, and VGluT1 in glutamatergic neurons, were also observed. Of these alterations, only those in astrocytes persisted in SNI Trpv1(-/-) mice. A pan-caspase inhibitor, injected into the PL-IL cortex, reduced mechanical allodynia, this effect being reduced but not abolished in Trpv1(-/-) mice. Single-unit extracellular recordings in vivo following electrical stimulation of basolateral amygdala or application of pressure on the hind paw, showed increased excitatory pyramidal neuron activity in the SNI PL-IL cortex, which also contained higher levels of the endocannabinoid 2-arachidonoylglycerol. Intra-PL-IL cortex injection of mGluR5 and NMDA receptor antagonists and AMPA exacerbated, whereas TRPV1 and AMPA receptor antagonists and a CB(1) agonist inhibited, allodynia. We suggest that SNI triggers both TRPV1-dependent and independent glutamate- and caspase-mediated cross-talk among IL-PL cortex neurons and glia, which either participates or counteracts pain.

Topics: Animals; Arachidonic Acids; Behavior, Animal; Blotting, Western; Caspases; Cerebral Cortex; Endocannabinoids; Ethanolamines; Evoked Potentials; Extracellular Space; Glycerides; Immunohistochemistry; Limbic System; Male; Mice; Mice, Inbred C57BL; Neuralgia; Neuroglia; Pain Perception; Postural Balance; Real-Time Polymerase Chain Reaction; Receptors, Glutamate; RNA; Sciatic Neuropathy; TRPV Cation Channels

The effect of the enol carbamate 1-biphenyl-4-ylethenyl piperidine-1-carboxylate (ST4070), a novel reversible inhibitor of fatty acid amide hydrolase (FAAH), was investigated for acute pain sensitivity and neuropathic pain in rats and mice. Brain enzymatic activity of FAAH and the endogenous levels of its substrates, anandamide (AEA; N-arachidonoylethanolamine), 2-arachidonoylglycerol (2-AG), and N-palmitoylethanolamine (PEA), were measured in control and ST4070-treated mice. ST4070 (10, 30, and 100 mg/kg) was orally administered to assess mechanical nociceptive thresholds and allodynia by using the Randall-Selitto and von Frey tests, respectively. Neuropathy was induced in rats by either the chemotherapeutic agent vincristine or streptozotocin-induced diabetes, whereas the chronic constriction injury (CCI) model was chosen to evaluate neuropathy in mice. ST4070 produced a significant increase of nociceptive threshold in rats and counteracted the decrease of nociceptive threshold in the three distinct models of neuropathic pain. In diabetic mice, ST4070 inhibited FAAH activity and increased the brain levels of AEA and PEA, without affecting that of 2-AG. The administration of ST4070 generated long-lasting pain relief compared with pregabalin and the FAAH inhibitors 1-oxo-1[5-(2-pyridyl)-2-yl]-7-phenylheptane (OL135) and cyclohexylcarbamic acid 3'-carbamoylbiphenyl-3-ylester (URB597) in CCI neuropathic mice. The antiallodynic effects of ST4070 were prevented by pretreatment with cannabinoid type 1 and cannabinoid type 2 receptor antagonists and by the selective peroxisome proliferator-activated receptor α antagonist [(2S)-2-[[(1Z)-1-methyl-3-oxo-3-[4-(trifluoromethyl)phenyl]-1-propenyl]amino]-3-[4-[2-(5-methyl-2-phenyl-4-oxazolyl)ethoxy]phenyl]propyl]-carbamic acid ethyl ester (GW6471). The administration of ST4070 generated long-lasting neuropathic pain relief compared with pregabalin and the FAAH inhibitors OL135 and URB597. Taken together, the reversible FAAH inhibitor ST4070 seems to be a promising novel therapeutic agent for the management of neuropathic pain.

Topics: Acute Pain; Amidohydrolases; Analgesics; Animals; Arachidonic Acids; Brain; Endocannabinoids; Glycerides; Male; Mice; Mice, Inbred NOD; Neuralgia; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley

Activation of spinal microglia contributes to aberrant pain responses associated with neuropathic pain states. Endocannabinoids (ECs) are present in the spinal cord, and inhibit nociceptive processing; levels of ECs may be altered by microglia which modulate the turnover of endocannabinoids in vitro. Here, we investigate the effect of minocycline, an inhibitor of activated microglia, on levels of the endocannabinoids anandamide and 2-arachidonoylglycerol (2-AG), and the related compound N-palmitoylethanolamine (PEA), in neuropathic spinal cord. Selective spinal nerve ligation (SNL) in rats resulted in mechanical allodynia and the presence of activated microglia in the ipsilateral spinal cord. Chronic daily treatment with minocycline (30 mg/kg, ip for 14 days) significantly reduced the development of mechanical allodynia at days 5, 10 and 14 post-SNL surgery, compared to vehicle-treated SNL rats (P < 0.001). Minocycline treatment also significantly attenuated OX-42 immunoreactivity, a marker of activated microglia, in the ipsilateral (P < 0.001) and contralateral (P < 0.01) spinal cord of SNL rats, compared to vehicle controls. Minocycline treatment significantly (P < 0.01) decreased levels of 2-AG and significantly (P < 0.01) increased levels of PEA in the ipsilateral spinal cord of SNL rats, compared to the contralateral spinal cord. Thus, activation of microglia affects spinal levels of endocannabinoids and related compounds in neuropathic pain states.

Topics: Amides; Animals; Arachidonic Acids; Cannabinoid Receptor Modulators; Cell Proliferation; Disease Models, Animal; Endocannabinoids; Ethanolamines; Glycerides; Microglia; Minocycline; Neuralgia; Palmitic Acids; Polyunsaturated Alkamides; Rats; Spinal Cord