anandamide 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

Peripheral neuropathy (PN) is a condition of peripheral nerve damage leading to severe pain. The first line therapies are associated with adverse psychotropic effects (PSE) and second line therapies are not efficient enough to relieve pain. There is an unmet drug need for relieving pain effectively without PSE in PN. Anandamide, an endocannabinoid activates cannabinoid receptors to relieve the pain due to peripheral neuropathy (PN). Anandamide has a very short biological half-life as they are extensively metabolized by fatty acid amide hydrolase enzyme (FAAH). Regional delivery of safe FAAH inhibitor (FI) with anandamide would be beneficial for PN without PSE. The objective of the study is to identify a safe FI and deliver the anandamide in combination with the FI topically for the management of PN. The FAAH inhibition potential of silymarin constituents was evaluated by molecular docking and in vitro studies. The topical gel formulation was developed to deliver anandamide and FI. The formulation was assessed in chemotherapeutic agent-induced PN rat models to relieve mechanical-allodynia and thermal-hyperalgesia. The molecular docking studies demonstrated that the Prime MM-GBSA free energy of silymarin constituents were in the order of silybin > isosilybin > silychristin > taxifolin > silydianin. In in vitro studies, silybin 20 µM inhibited > 61.8% of FAAH activity and increased the half-life of anandamide. The developed formulation increased permeation of anandamide and silybin across the porcine skin. Furthermore, on the application of anandamide and anandamide-silybin gel to rat paws, there was a significant increase in the pain threshold for allodynic and hyperalgesic stimulus up to 1 h and 4 h, respectively. The topical anandamide with silybin delivery approach could serve to alleviate PN efficiently and thus could minimize unwanted CNS side effects of synthetic or natural cannabinoids in patients.

Topics: Animals; Endocannabinoids; Hyperalgesia; Molecular Docking Simulation; Neuralgia; Polyunsaturated Alkamides; Rats; Silybin

The antiallodynic effect of PhAR-DBH-Me was evaluated on two models of neuropathic pain, and the potential roles of CB1, CB2, and TRPV1 receptors as molecular targets of PhAR-DBH-Me were studied. Female Wistar rats were submitted to L5/L6 spinal nerve ligation (SNL) or repeated doses of cisplatin (0.1 mg/kg, i.p.) to induce experimental neuropathy. Then, tactile allodynia was determined, and animals were treated with logarithmic doses of PhAR-DBH-Me (3.2-100 mg/kg, i.p.). To evaluate the mechanism of action of PhAR-DBH-Me, in silico studies using crystallized structures of CB1, CB2, and TRPV1 receptors were performed. To corroborate the computational insights, animals were intraperitoneally administrated with antagonists for CB1 (AM-251, 3 mg/kg), CB2 (AM-630, 1 mg/kg), and TRPV1 receptors (capsazepine, 3 mg/kg), 15 min before to PhAR-DBH-Me (100 mg/kg) administration. Vagal stimulation evoked on striated muscle contraction in esophagus, was used to elicited pharmacological response of PhAR-DBH-ME on nervous tissue. Systemic administration of PhAR-DBH-Me reduced the SNL- and cisplatin-induced allodynia. Docking studies suggested that PhAR-DBH-Me acts as an agonist for CB1, CB2, and TRPV1 receptors, with similar affinity to the endogenous ligand anandamide. Moreover antiallodynic effect of PhAR-DBH-Me was partially prevented by administration of AM-251 and AM-630, and completely prevented by capsazepine. Finally, PhAR-DBH-Me decreased the vagally evoked electrical response in esophagus rat. Taken together, results indicate that PhAR-DBH-Me induces an antiallodynic effect through partial activation of CB1 and CB2 receptors, as well as desensitization of TRPV1 receptors. Data also shed light on the novel vanilloid nature of the synthetic compound PhAR-DBH-Me.

Topics: Animals; Antineoplastic Agents; Arachidonic Acids; Azabicyclo Compounds; Cannabinoid Receptor Antagonists; Capsaicin; Cisplatin; Endocannabinoids; Female; Hyperalgesia; Injections, Intraperitoneal; Ligation; Models, Animal; Neuralgia; Oleic Acids; Polyunsaturated Alkamides; Rats; Rats, Wistar; Spinal Nerves; TRPV Cation Channels; Vagus Nerve Stimulation

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

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

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

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

The endocannabinoid anandamide (AEA) is an antinociceptive lipid that is inactivated through cellular uptake and subsequent catabolism by fatty acid amide hydrolase (FAAH). Fatty acid binding proteins (FABPs) are intracellular carriers that deliver AEA and related N-acylethanolamines (NAEs) to FAAH for hydrolysis. The mammalian brain expresses three FABP subtypes: FABP3, FABP5, and FABP7. Recent work from our group has revealed that pharmacological inhibition of FABPs reduces inflammatory pain in mice. The goal of the current work was to explore the effects of FABP inhibition upon nociception in diverse models of pain. We developed inhibitors with differential affinities for FABPs to elucidate the subtype(s) that contributes to the antinociceptive effects of FABP inhibitors. Inhibition of FABPs reduced nociception associated with inflammatory, visceral, and neuropathic pain. The antinociceptive effects of FABP inhibitors mirrored their affinities for FABP5, while binding to FABP3 and FABP7 was not a predictor of in vivo efficacy. The antinociceptive effects of FABP inhibitors were mediated by cannabinoid receptor 1 (CB1) and peroxisome proliferator-activated receptor alpha (PPARα) and FABP inhibition elevated brain levels of AEA, providing the first direct evidence that FABPs regulate brain endocannabinoid tone. These results highlight FABPs as novel targets for the development of analgesic and anti-inflammatory therapeutics.

Topics: Analgesia; Analgesics; Animals; Arachidonic Acids; Brain; Disease Models, Animal; Endocannabinoids; Fatty Acid-Binding Proteins; Hyperalgesia; Male; Mice; Models, Molecular; Molecular Conformation; Molecular Docking Simulation; Neuralgia; Polyunsaturated Alkamides; PPAR alpha; Protein Binding; Rats; Receptor, Cannabinoid, CB1

Amelioration of neuropathic spinal cord injury (SCI) pain is a clinical challenge. Increasing the endocannabinoid anandamide and other fatty acid amides (FAA) by blocking fatty acid amide hydrolase (FAAH) has been shown to be antinociceptive in a number of animal models of chronic pain. However, an antinociceptive effect of blocking FAAH has yet to be demonstrated in a rat model of neuropathic SCI pain. Four weeks following a SCI, rats developed significantly decreased hind paw withdrawal thresholds, indicative of below-level cutaneous hypersensitivity. A group of SCI rats were systemically treated (i.p.) with either the selective FAAH inhibitor URB597 or vehicle twice daily for seven days. A separate group of SCI rats received a single dose (p.o.) of either the selective FAAH inhibitor PF-3845 or vehicle. Following behavioral testing, levels of the FAA N-arachidonoylethanolamide, N-oleoyl ethanolamide and N-palmitoyl ethanolamide were quantified in brain and spinal cord from SCI rats. Four weeks following SCI, FAA levels were markedly reduced in spinal cord tissue. Although systemic treatment with URB597 significantly increased CNS FAA levels, no antinociceptive effect was observed. A significant elevation of CNS FAA levels was also observed following oral PF-3845 treatment, but only a modest antinociceptive effect was observed. Increasing CNS FAA levels alone does not lead to robust amelioration of below-level neuropathic SCI pain. Perhaps utilizing FAAH inhibition in conjunction with other analgesic mechanisms could be an effective analgesic therapy.

Topics: Amidohydrolases; Analgesics; Animals; Arachidonic Acids; Benzamides; Benzoxazines; Brain; Carbamates; Endocannabinoids; Enzyme Inhibitors; Hindlimb; Male; Morpholines; Naphthalenes; Neuralgia; Pain Measurement; Pain Threshold; Piperidines; Polyunsaturated Alkamides; Pyridines; Rats, Sprague-Dawley; Spinal Cord; Spinal Cord Injuries; Treatment Outcome

Endocannabinoids (EC), particularly anandamide (AEA), released constitutively in pain pathways might be accountable for the inhibitory effect on nociceptors. Pathogenesis of neuropathic pain may reflect complex remodeling of the dorsal root ganglia (DRGs) and spinal cord EC system. Multiple pathways involved both in the biosynthesis and degradation of AEA have been suggested. We investigated the local synthesis and degradation features of AEA in DRGs and spinal cord during the development and maintenance of pain in a model of chronic constriction injury (CCI). All AEA synthesis and degradation enzymes are present on the mRNA level in DRGs and lumbar spinal cord of intact as well as CCI-treated animals. Deregulation of EC system components was consistent with development of pain phenotype at days 3, 7, and 14 after CCI. The expression levels of enzymes involved in AEA degradation was significantly upregulated ipsilateral in DRGs and spinal cord at different time points. Expression of enzymes of the alternative, sPLA2-dependent and PLC-dependent, AEA synthesis pathways was elevated in both of the analyzed structures at all time points. Our data have shown an alteration of alternative AEA synthesis and degradation pathways, which might contribute to the variation of AEA levels and neuropathic pain development.

Topics: Animals; Arachidonic Acids; Chronic Disease; Constriction, Pathologic; Endocannabinoids; Ganglia, Spinal; Gene Expression Regulation, Enzymologic; Hyperalgesia; Ligation; Lumbar Vertebrae; Male; Neuralgia; Pain Threshold; Phosphatidylethanolamines; Phospholipase D; Polyunsaturated Alkamides; Rats, Wistar; Receptor, Cannabinoid, CB2; RNA, Messenger; Sciatic Nerve; TRPV Cation Channels; Up-Regulation

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

Damaging of peripheral nerves may result in chronic neuropathic pain for which the likelihood is increased in the elderly. We assessed in mice if age-dependent alterations of endocannabinoids contributed to the heightened vulnerability to neuropathic pain at old age. We assessed nociception, endocannabinoids and the therapeutic efficacy of R-flurbiprofen in young and aged mice in the spared nerve injury model of neuropathic pain. R-flurbiprofen was used because it is able to reduce neuropathic pain in young mice in part by increasing anandamide. Aged mice developed stronger nociceptive hypersensitivity after sciatic nerve injury than young mice. This was associated with low anandamide levels in the dorsal root ganglia, spinal cord, thalamus and cortex, which further decreased after nerve injury. In aged mice, R-flurbiprofen had only weak antinociceptive efficacy and it failed to restore normal anandamide levels after nerve injury. In terms of the mechanisms, we found that fatty acid amide hydrolase (FAAH) which degrades anandamide, was upregulated after nerve injury at both ages, so that this upregulation likely did not account for the age-dependent differences. However, enzymes contributing to oxidative metabolism of anandamide, namely cyclooxygenase-1 and Cyp2D6, were increased in the brain of aged mice, possibly enhancing the oxidative breakdown of anandamide. This may overwhelm the capacity of R-flurbiprofen to restore anandamide homeostasis and may contribute to the heightened risk for neuropathic pain at old age.

Topics: Aging; Amidohydrolases; Animals; Arachidonic Acids; Behavior, Animal; Brain; Cyclooxygenase 1; Cyclooxygenase Inhibitors; Cytochrome P-450 CYP2D6; Disease Models, Animal; Endocannabinoids; Enzyme Induction; Flurbiprofen; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Nerve Tissue Proteins; Neuralgia; Peripheral Nerves; Polyunsaturated Alkamides; Spinal Cord; Stereoisomerism

The endocannabinoid anandamide (AEA) activates also transient receptor potential vanilloid-1 (TRPV1) channels. However, no data exist on the potential role of spinal TRPV1 activation by AEA in neuropathic pain. We tested the effect of: 1) AEA (5-100 μg), alone or in the presence of an inhibitor of its hydrolysis, and 2) elevated levels of endogenous AEA (following inhibition of AEA hydrolysis), in CCI rats, and the involvement of TRPV1 or cannabinoid CB(1) receptors in the observed effects. Levels of AEA in the spinal cord of CCI rats were measured following all treatments. AEA (50 μg) displayed anti-allodynic and anti-hyperalgesic effects which were abolished by previous antagonism of TRPV1, but not CB(1), receptors. Depending on the administered dose, the selective inhibitor of AEA enzymatic hydrolysis, URB597 (10-100 μg), reduced thermal and tactile nociception via CB(1) or CB(1)/TRPV1 receptors. The anti-nociceptive effects of co-administered per se ineffective doses of AEA (5 μg) and URB597 (5 μg) was abolished by antagonism of CB(1), but not TRPV1, receptors. Spinal AEA levels were increased after CCI, slightly increased further by URB597, 10 μg i.t., and strongly elevated by URB597, 100 μg. Injection of AEA (50 μg) into the lumbar spinal cord led to its dramatic elevation in this tissue, whereas, when a lower dose was used (5 μg) AEA endogenous levels were elevated only in the presence of URB597 (5 μg). We suggest that spinal AEA reduces neuropathic pain via CB(1) or TRPV1, depending on its local concentration.

Topics: Analgesia; Animals; Arachidonic Acids; Cannabinoid Receptor Modulators; Endocannabinoids; Hyperalgesia; Male; Neuralgia; Pain Measurement; Polyunsaturated Alkamides; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Sciatic Nerve; 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

Fatty acid amide hydrolase (FAAH) is an integral membrane enzyme within the amidase-signature family. It catalyzes the hydrolysis of several endogenous biologically active lipids, including anandamide (arachidonoyl ethanolamide), oleoyl ethanolamide, and palmitoyl ethanolamide. These endogenous FAAH substrates have been shown to be involved in a variety of physiological and pathological processes, including synaptic regulation, regulation of sleep and feeding, locomotor activity, pain and inflammation. Here we describe the biochemical and biological properties of a potent and selective FAAH inhibitor, 4-(3-phenyl-[1,2,4]thiadiazol-5-yl)-piperazine-1-carboxylic acid phenylamide (JNJ-1661010). The time-dependence of apparent IC(50) values at rat and human recombinant FAAH, dialysis and mass spectrometry data indicate that the acyl piperazinyl fragment of JNJ-1661010 forms a covalent bond with the enzyme. This bond is slowly hydrolyzed, with release of the piperazinyl fragment and recovery of enzyme activity. The lack of inhibition observed in a rat liver esterase assay suggests that JNJ-1661010 is not a general esterase inhibitor. JNJ-1661010 is >100-fold preferentially selective for FAAH-1 when compared to FAAH-2. JNJ-1661010 dose-dependently increases arachidonoyl ethanolamide, oleoyl ethanolamide, and palmitoyl ethanolamide in the rat brain. The compound attenuates tactile allodynia in the rat mild thermal injury model of acute tissue damage and in the rat spinal nerve ligation (Chung) model of neuropathic pain. JNJ-1661010 also diminishes thermal hyperalgesia in the inflammatory rat carrageenan paw model. These data suggest that FAAH inhibitors with modes of action similar to JNJ-1661010 may be useful clinically as broad-spectrum analgesics.

Topics: Amides; Amidohydrolases; Analgesics; Animals; Arachidonic Acids; Brain; Carrageenan; Disease Models, Animal; Dose-Response Relationship, Drug; Endocannabinoids; Enzyme Inhibitors; Ethanolamines; Hot Temperature; Humans; Hydrolysis; Isoenzymes; Kinetics; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Neuralgia; Oleic Acids; Pain; Pain Measurement; Pain Threshold; Palmitic Acids; Piperazines; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley; Reaction Time; Recombinant Proteins; Thiadiazoles

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