anandamide and Pain

It has been established that the endogenous cannabinoid (endocannabinoid) system plays key modulatory roles in a wide variety of pathological conditions. The endocannabinoid system comprises both cannabinoid receptors, their endogenous ligands including 2-arachidonoylglycerol (2-AG), N-arachidonylethanolamine (anandamide, AEA), and enzymes that regulate the synthesis and degradation of endogenous ligands which include diacylglycerol lipase alpha (DAGL-α), diacylglycerol lipase beta (DAGL-β), fatty acid amide hydrolase (FAAH), monoacylglycerol lipase (MAGL), α/β hydrolase domain 6 (ABHD6). As the endocannabinoid system exerts considerable involvement in the regulation of homeostasis and disease, much effort has been made towards understanding endocannabinoid-related mechanisms of action at cellular, physiological, and pathological levels as well as harnessing the various components of the endocannabinoid system to produce novel therapeutics. However, drug discovery efforts within the cannabinoid field have been slower than anticipated to reach satisfactory clinical endpoints and raises an important question into the validity of developing novel ligands that therapeutically target the endocannabinoid system. To answer this, we will first examine evidence that supports the existence of an endocannabinoid system role within inflammatory diseases, neurodegeneration, pain, substance use disorders, mood disorders, as well as metabolic diseases. Next, this review will discuss recent clinical studies, within the last 5 years, of cannabinoid compounds in context to these diseases. We will also address some of the challenges and considerations within the cannabinoid field that may be important in the advancement of therapeutics into the clinic.

Topics: Animals; Arachidonic Acids; Cannabinoid Receptor Agonists; Cannabinoids; Drug Discovery; Endocannabinoids; Glycerides; Humans; Inflammation; Metabolic Diseases; Mood Disorders; Nervous System Diseases; Pain; Polyunsaturated Alkamides; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Substance-Related Disorders

Potassium (K(+)) channels are membrane proteins expressed in most living cells that selectively control the flow of K(+) ions. More than 80 genes encode the K(+) channel subunits in the human genome. The TWIK-related K(+) channel (TREK-1) belongs to the two-pore domain K(+) channels (K2P) and displays various properties including sensitivity to physical (membrane stretch, acidosis, temperature) and chemical stimuli (signaling lipids, volatile anesthetics). The distribution of TREK-1 in the central nervous system, coupled with the physiological consequences of its opening and closing, leads to the emergence of this channel as an attractive therapeutic target. We review the TREK-1 channel, its structural and functional properties, and the pharmacological agents (agonists and antagonists) able to modulate its gating.

Topics: Arrhythmias, Cardiac; Depression; Epilepsy; Humans; Inflammation; Models, Molecular; Molecular Structure; Neuroprotective Agents; Pain; Potassium Channels, Tandem Pore Domain; Structure-Activity Relationship

2-Arachidonoyl-glycerol (2-AG) and arachidonyl-ethanolamide (AEA) are endocannabinoids that have been implicated in many physiologic disorders, including obesity, metabolic syndromes, hepatic diseases, pain, neurologic disorders, and inflammation. Their immunomodulatory effects are numerous and are not always mediated by cannabinoid receptors, reflecting the presence of an arachidonic acid (AA) molecule in their structure, the latter being the precursor of numerous bioactive lipids that are pro- or anti-inflammatory. 2-AG and AEA can thus serve as a source of AA but can also be metabolized by most eicosanoid biosynthetic enzymes, yielding additional lipids. In this regard, enhancing endocannabinoid levels by using endocannabinoid hydrolysis inhibitors is likely to augment the levels of these lipids that could regulate inflammatory cell functions. This review summarizes the metabolic pathways involved in the biosynthesis and metabolism of AEA and 2-AG, as well as the biologic effects of the 2-AG and AEA lipidomes in the regulation of inflammation.

Topics: Animals; Arachidonic Acids; Dendritic Cells; Endocannabinoids; Glycerides; Humans; Inflammation; Lipid Metabolism; Liver Diseases; Lymphocytes; Metabolic Syndrome; Neurodegenerative Diseases; Obesity; Pain; Phosphatidic Acids; Polyunsaturated Alkamides; Receptors, Cannabinoid

Fatty acid amide hydrolase (FAAH) is a key enzyme responsible for the degradation of the endocannabinoid anandamide. FAAH inactivation is emerging as a strategy to treat several CNS and peripheral diseases, including inflammation and pain. The search for effective FAAH inhibitors has thus become a key focus in present drug discovery.. Patents and patent applications published from 2009 to 2014 in which novel chemical classes are claimed to inhibit FAAH.. FAAH is a promising target for treating many disease conditions including pain, inflammation and mood disorders. In the last few years, remarkable efforts have been made to develop new FAAH inhibitors (either reversible and irreversible) characterized by excellent potency and selectivity, to complete the arsenal of tools for modulating FAAH activity. The failure of PF-04457845 in a Phase II study on osteoarthritis pain has not flattened the interest in FAAH inhibitors. New clinical trials on 'classical' FAAH inhibitors are now ongoing, and new strategies based on compounds with peculiar in vivo distribution (e.g., peripheral) or with multiple pharmacological activities (e.g., FAAH and COX) are under investigation and could boost the therapeutic potential of this class in the next future.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Central Nervous System Diseases; Drug Design; Endocannabinoids; Enzyme Inhibitors; Humans; Inflammation; Pain; Patents as Topic; Polyunsaturated Alkamides

The endocannabinoid (EC) system consists of two main receptors: cannabinoid type 1 receptor cannabinoid receptors are found in both the central nervous system (CNS) and periphery, whereas the cannabinoid type 2 receptor cannabinoid receptor is found principally in the immune system and to a lesser extent in the CNS. The EC family consists of two classes of well characterised ligands; the N-acyl ethanolamines, such as N-arachidonoyl ethanolamide or anandamide (AEA), and the monoacylglycerols, such as 2-arachidonoyl glycerol. The various synthetic and catabolic pathways for these enzymes have been (with the exception of AEA synthesis) elucidated. To date, much work has examined the role of EC in nociceptive processing and the potential of targeting the EC system to produce analgesia. Cannabinoid receptors and ligands are found at almost every level of the pain pathway from peripheral sites, such as peripheral nerves and immune cells, to central integration sites such as the spinal cord, and higher brain regions such as the periaqueductal grey and the rostral ventrolateral medulla associated with descending control of pain. EC have been shown to induce analgesia in preclinical models of acute nociception and chronic pain states. The purpose of this review is to critically evaluate the evidence for the role of EC in the pain pathway and the therapeutic potential of EC to produce analgesia. We also review the present clinical work conducted with EC, and examine whether targeting the EC system might offer a novel target for analgesics, and also potentially disease-modifying interventions for pathophysiological pain states.

Topics: Analgesia; Analgesics; Arachidonic Acids; Endocannabinoids; Glycerides; Humans; Nervous System; Pain; Polyunsaturated Alkamides; Receptors, Cannabinoid

The quest for possible targets for the development of novel analgesics has identified the activation of the cannabinoid type 1 (CB1) receptor outside the CNS as a potential means of providing relief from persistent pain, which currently constitutes an unmet medical need. Increasing tissue levels of the CB1 receptor endogenous ligand N-arachidonoylethanolamine (anandamide), by inhibiting anandamide degradation through blocking the anandamide-hydrolysing enzyme fatty acid amide hydrolase, has been suggested to be used to activate the CB1 receptor. However, recent clinical trials revealed that this approach does not deliver the expected relief from pain. Here, we discuss one of the possible reasons, the activation of the transient receptor potential vanilloid type 1 ion channel (TRPV1) on nociceptive primary sensory neurons (PSNs) by anandamide, which may compromise the beneficial effects of increased tissue levels of anandamide. We conclude that better design such as concomitant blocking of anandamide hydrolysis and anandamide uptake into PSNs, to inhibit TRPV1 activation, could overcome these problems.

Topics: Animals; Arachidonic Acids; Cannabinoid Receptor Agonists; Endocannabinoids; Humans; Nociceptors; Pain; Polyunsaturated Alkamides; TRPV Cation Channels

The endocannabinoid signaling system regulates diverse physiologic processes and has attracted considerable attention as a potential pharmaceutical target for treating diseases, such as pain, anxiety/depression, and metabolic disorders. The principal ligands of the endocannabinoid system are the lipid transmitters N-arachidonoylethanolamine (anandamide) and 2-arachidonoylglycerol (2-AG), which activate the two major cannabinoid receptors, CB1 and CB2. Anandamide and 2-AG signaling pathways in the nervous system are terminated by enzymatic hydrolysis mediated primarily by the serine hydrolases fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively. In this review, we will discuss the development of FAAH and MAGL inhibitors and their pharmacological application to investigate the function of anandamide and 2-AG signaling pathways in preclinical models of neurobehavioral processes, such as pain, anxiety, and addiction. We will place emphasis on how these studies are beginning to discern the different roles played by anandamide and 2-AG in the nervous system and the resulting implications for advancing endocannabinoid hydrolase inhibitors as next-generation therapeutics.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Depression; Disease Models, Animal; Endocannabinoids; Enzyme Inhibitors; Glycerides; Humans; Ligands; Molecular Structure; Monoacylglycerol Lipases; Pain; Polyunsaturated Alkamides; Signal Transduction; Substance-Related Disorders

In the late 1990's, a series of experiments carried out independently in two laboratories led to establish an important connection between the function of the endocannabinoids, which, as exemplified in this special issue, is per se very complex and ubiquitous in animals, and that of the transient receptor potential (TRP) channels, a large family of plasma membrane cation channels involved in several mammalian and non-mammalian physiological and pathological conditions, overlapping only in part with those in which the cannabinoid receptors participate. These experiments were initially based on the observation that the endocannabinoid anandamide and the xenobiotic ligand of TRP channels of V1 type (TRPV1), capsaicin, are somehow chemically similar, both compounds being fatty acid amides, as are also synthetic activators of these channels and inhibitors of anandamide cellular re-uptake. As discussed in this article, the same type of "chemical thoughts" led to the discovery of N-arachidonoyl-dopamine, an endogenous ligand of TRPV1 channels that behaves also an endocannabinoid. The overlap between the ligand recognition properties of some TRP channels and proteins of the endocannabinoid system, namely the cannabinoid receptors and the proteins and enzymes catalyzing anandamide cellular re-uptake and hydrolysis, is being actively explored through the rational design and synthesis of new endocannabinoid-based drugs with multiple mechanisms of action. These aspects are discussed in this review article, together with the possible functional and pharmacological consequences of endocannabinoid-TRP channel interactions.

Topics: Animals; Arachidonic Acids; Cannabinoid Receptor Modulators; Endocannabinoids; Humans; Pain; Polyunsaturated Alkamides; Transient Receptor Potential Channels

The endogenous cannabinoid N-arachidonoyl ethanolamine (anandamide; AEA) produces most of its pharmacological effects by binding and activating CB(1) and CB(2) cannabinoid receptors within the CNS and periphery. However, the actions of AEA are short lived because of its rapid catabolism by fatty acid amide hydrolase (FAAH). Indeed, FAAH knockout mice as well as animals treated with FAAH inhibitors are severely impaired in their ability to hydrolyze AEA as well as a variety of noncannabinoid lipid signaling molecules and consequently possess greatly elevated levels of these endogenous ligands. In this mini review, we describe recent research that has investigated the functional consequences of inhibiting this enzyme in a wide range of animal models of inflammatory and neuropathic pain states. FAAH-compromised animals reliably display antinociceptive and anti-inflammatory phenotypes with a similar efficacy as direct-acting cannabinoid receptor agonists, such as Delta(9)-tetrahydrocannabinol (THC), the primary psychoactive constituent of Cannabis sativa. Importantly, FAAH blockade does not elicit any apparent psychomimetic effects associated with THC or produce reinforcing effects that are predictive of human drug abuse. The beneficial effects caused by FAAH blockade in these models are predominantly mediated through the activation of CB(1) and/or CB(2) receptors, though noncannabinoid mechanisms of actions can also play contributory or even primary roles. Collectively, the current body of scientific literature suggests that activating the endogenous cannabinoid system by targeting FAAH is a promising strategy to treat pain and inflammation but lacks untoward side effects typically associated with Cannabis sativa.

Topics: Amidohydrolases; Analgesics; Animals; Anti-Inflammatory Agents, Non-Steroidal; Arachidonic Acids; Disease Models, Animal; Dronabinol; Drug Delivery Systems; Drug Evaluation, Preclinical; Endocannabinoids; Glycerides; Humans; Inflammation; Mice; Mice, Knockout; Pain; Peroxisome Proliferator-Activated Receptors; Polyunsaturated Alkamides; Rats; Receptors, Cannabinoid; Receptors, Opioid; TRPV Cation Channels

The therapeutic potential of cannabinoids has been the topic of extensive investigation following the discovery of cannabinoid receptors and their endogenous ligands. Cannabinoid receptors and their endogenous ligands are present at supraspinal, spinal and peripheral levels. Cannabinoids suppress behavioral responses to noxious stimulation and suppress nociceptive processing through activation of cannabinoid CB(1) and CB(2) receptor subtypes. Endocannabinoids, the brain's own cannabis-like substances, share the same molecular target as Delta(9)-tetrahydrocannabinol, the main psychoactive component in cannabis. Endocannabinoids serve as synaptic circuit breakers and regulate multiple physiological and pathological conditions, e.g. regulation of food intake, immunomodulation, inflammation, analgesia, cancer, addictive behavior, epilepsy and others. This review will focus on uncovering the roles of anandamide and 2-arachidonoylglycerol, the two best characterized endocannabinoids identified to date, in controlling nociceptive responding. The roles of anandamide and 2-arachidonoylglycerol, released under physiological conditions, in modulating nociceptive responding at different levels of the neuraxis will be emphasized in this review. Effects of modulation of endocannabinoid levels through inhibition of endocannabinoid hydrolysis and uptake is also compared with effects of exogenous administration of synthetic endocannabinoids in acute, inflammatory and neuropathic pain models. Finally, the therapeutic potential of the endocannabinoid signaling system is discussed in the context of identifying novel pharmacotherapies for the treatment of pain.

Topics: Animals; Arachidonic Acids; Brain; Cannabinoid Receptor Modulators; Endocannabinoids; Glycerides; Humans; Pain; Polyunsaturated Alkamides; Receptors, Cannabinoid

Hemp (Cannabis sativa L.) has been used since remotes ages as a herbal remedy. Only recently the medical community highlighted the pharmacological scientific bases of its effects. The most important active principle, Delta-9-tetrahydrocannabinol, was identified in the second half of the last century, and subsequently two receptors were identified and cloned: CB1 that is primarily present in the central nervous system, and CB2 that is present on the cells of the immune system. Endogenous ligands, called endocannabinoids, were characterized. The anandamide was the first one to be discovered. The effectiveness of the cannabinoids in the treatment of nausea and vomit due to anti-neoplastic chemotherapy and in the wasting-syndrome during AIDS is recognized. Moreover, the cannabinoids are analgesic, and their activity is comparable to the weak opioids. Furthermore, parallels exist between opioid and cannabinoid receptors, and evidence is accumulating that the two systems sometimes may operate synergistically. The interest of the pharmaceutical companies led to the production of various drugs, whether synthetic or natural derived. The good ratio between the polyunsatured fatty acids omega-3 and omega-6 of the oil of Cannabis seeds led to reduction of the phlogosis and an improvement of the pain symptoms in patients with chronic musculo-skeletal inflammation.

Topics: Amino Acid Sequence; Analgesics, Non-Narcotic; Anti-Inflammatory Agents, Non-Steroidal; Antiemetics; Arachidonic Acids; Cachexia; Cannabinoids; Cannabis; Dronabinol; Endocannabinoids; Humans; Molecular Sequence Data; Nausea; Pain; Phytotherapy; Polyunsaturated Alkamides; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2

The TRPV1 channel is mainly expressed in sensory nerves. Activation of the channel induces neuropeptide release from central and peripheral sensory nerve terminals, resulting in the sensation of pain, neurogenic inflammation, smooth muscle contraction and cough. The TRPV1 channel can be activated by vanilloids such as capsaicin, as well as endogenous stimulators including H(+), heat, lipoxygenase products and anandamide. TRPV1 channel function is upregulated by several endogenous mediators present in inflammatory conditions, which decreases the threshold for activation of the channel. Under these conditions, TRPV1 can be activated by physiological body temperature, slight acidification or lower concentration of TRPV1 agonists. There is evidence that TRPV1 plays a role in the development of pathophysiological changes and symptoms in several diseases. In this review, we discuss TRPV1 channel activation and regulation in normal and diseased conditions, the role of TRPV1 in pain, cough, asthma and urinary incontinence, and the potential use of TRPV1 antagonists as a novel therapy for these diseases.

Topics: Animals; Arachidonic Acids; Cough; Endocannabinoids; Hot Temperature; Humans; Hydrogen-Ion Concentration; Ion Channels; Lipoxygenase; Lung Diseases; Pain; Polyunsaturated Alkamides; TRPV Cation Channels; Urinary Incontinence

Since the discovery of anandamide in 1992, our knowledge of the endocannabinoid system and its physiological effects has increased greatly, not the least as a result of the availability of compounds affecting endocannabinoid function. In the present review, the pharmacology of the endocannabinoid system is discussed. At present, there are no compounds selectively inhibiting the synthesis of anandamide, and the mechanisms by which anandamide release and reuptake are blocked are a matter for current debate. In contrast, selective agonists and inverse agonists at the CB1 and CB2 receptors have been well characterised, as have inhibitors of the metabolism of anandamide by fatty acid amide hydrolase. Accumulating evidence has suggested that such compounds may be useful for the treatment of a number of disorders. With respect to the treatment of pain, topical CB1 agonists and CB2 agonists may prove therapeutically useful, and there is evidence that the non-steroidal inflammatory agent indomethacin produces effects secondary to activation of the endocannabinoid system. Modulation of the endocannabionid system may also produce neuroprotective effects, although present data would suggest that the observed effects are highly dependent upon the nature of the neurotoxic insult.

Topics: Animals; Arachidonic Acids; Cannabinoid Receptor Modulators; Endocannabinoids; Humans; Neuroprotective Agents; Pain; Polyunsaturated Alkamides; Receptors, Cannabinoid; Signal Transduction

Bayer AG has recently announced that it acquired exclusive rights for the marketing of GW Pharmaceuticals' new medicine Sativex in Europe and in other regions. Sativex is a sublingual spray on Cannabis extract basis, and is equipped with an electronic tool to facilitate accurate dosing and to prevent misuses. It is standardized for the THC and CBD. The new analgesic is proposed for the treatment of muscle spasticity and pains accompanying multiple sclerosis and as an efficient analgetic for neurogenic pain not responding well to opioids and to other therapies available. The entirely new mechanism of action through the recently discovered cannabinoid receptor system may offer a real therapeutic potential to the drug. Although the Government of Netherlands has authorized the sale of pharmaceutical grade Cannabis herb by pharmacies in the Netherlands, the availability on the pharmaceutical market of the registered preparation may render requests for the authorization of the smoking of Cannabis herb (marihuana) by individuals suffering of multiple sclerosis, neurogenic pain, AIDS wasting syndrome unnecessary. Nevertheless, the "old chameleon" plant Cannabis appears to gradually regain its previous status in mainstream therapy and pharmacy. As long as the plant Cannabis and its products continue to be classified as narcotic drugs, medical use of the new preparation will need close supervision.

Topics: Analgesics; Arachidonic Acids; Cannabidiol; Cannabinoids; Cannabis; Dronabinol; Drug Combinations; Drugs, Investigational; Endocannabinoids; Europe; Humans; Multiple Sclerosis; Pain; Plant Extracts; Polyunsaturated Alkamides

Recent work has addressed the role of vanilloid receptor type 1 (VR1) in pain perception. VR1 activity is regulated both directly and indirectly by endogenous factors. For example, protein kinase C sensitizes human VR1 to mild decreases in pH, which are commonly encountered during inflammation, and renders the endocannabinoid anandamide a more potent 'endovanilloid'. Bradykinin and nerve growth factor release VR1 from the inhibitory control of phosphatidylinositol (4,5)-bisphosphate and anti-VR1 serum ameliorates thermal allodynia and hyperalgesia in diabetic mice. There is strong evidence that not only the sensitivity but also the density of expression of VR1 is enhanced during inflammatory conditions. These observations provide an empirical foundation which could explain the reduced inflammatory hyperalgesia in VR1 knockout mice, and they imply an important role for endovanilloid signaling via VR1 in the development of ongoing pain in humans that occurs in most inflammatory conditions. Conversely, downregulation of VR1 expression and/or activity is a promising therapeutic strategy for novel analgesic drugs.

Topics: Animals; Arachidonic Acids; Bradykinin; Calcium Channel Blockers; Cannabinoid Receptor Modulators; Cannabinoids; Cyclic AMP-Dependent Protein Kinases; Down-Regulation; Endocannabinoids; Humans; Hyperalgesia; Inflammation; Nerve Growth Factor; Pain; Polyunsaturated Alkamides; Protein Kinase C; Receptors, Drug; Signal Transduction

Recent advances have dramatically increased our understanding of cannabinoid pharmacology: the psychoactive constituents of Cannabis sativa have been isolated, synthetic cannabinoids described and an endocannabinoid system identified, together with its component receptors, ligands and their biochemistry. Strong laboratory evidence now underwrites anecdotal claims of cannabinoid analgesia in inflammatory and neuropathic pain. Sites of analgesic action have been identified in brain, spinal cord and the periphery, with the latter two presenting attractive targets for divorcing the analgesic and psychotrophic effects of cannabinoids. Clinical trials are now required, but are hindered by a paucity of cannabinoids of suitable bioavailability and therapeutic ratio.

Topics: Amides; Amidohydrolases; Analgesics; Animals; Arachidonic Acids; Benzoxazines; Brain; Camphanes; Cannabinoid Receptor Modulators; Cannabinoids; Cell Membrane; Clinical Trials as Topic; Disease Models, Animal; Drug Design; Drug Interactions; Endocannabinoids; Enzyme Inhibitors; Ethanolamines; Glycerides; Humans; Injections, Spinal; Molecular Structure; Morpholines; Naphthalenes; Pain; Palmitates; Palmitic Acids; Piperidines; Plant Extracts; Polyunsaturated Alkamides; Pyrazoles; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; Spinal Cord

Topics: Amides; Analgesics; Animals; Arachidonic Acids; Brain; Cannabinoids; Drug Interactions; Endocannabinoids; Ethanolamines; Ganglia, Spinal; Glycerides; Humans; Nociceptors; Pain; Palmitic Acids; Polyunsaturated Alkamides; Receptors, Cannabinoid; Receptors, Drug; Signal Transduction

The pain, an unpleasant feeling, induces several central nervous system mechanisms, like sensory-discriminative, motivational-affective activities, behavioral changes and it activates various responses, including antinociceptive actions. Accordingly, signals from the nociceptive neurons in the spinal cord and the sensory trigeminal nucleus ascend in various neuronal pathways and target several brain areas. Here, five ascending pain-conducting neuronal pathways and two spinal reflex routes are briefly summarized. The spinal and supraspinal antinociceptive mechanisms are described in more detail. During the past two decades, endogenous opioids, cannabinoids and their receptors have been discovered, localized and cloned. Five groups of endogenous opiates are known: beta-endorphin, enkephalins, dynorphins, endomorphins, and nociceptin. Two endogenous cannabinoids have already been described in the brain: the anandamide and the 2-arachidonyl-glycerol. The site of their antinociceptive (analgesic) actions in the brain are briefly summarized.

Topics: Afferent Pathways; Animals; Arachidonic Acids; Brain; Cannabinoids; Dynorphins; Efferent Pathways; Endocannabinoids; Endorphins; Glycerides; Humans; Neural Conduction; Neural Pathways; Neurotransmitter Agents; Nociceptin; Opioid Peptides; Pain; Polyunsaturated Alkamides; Receptors, Neurotransmitter; Receptors, Opioid

Multimodal therapies comprising spa applications are widely used as non-pharmaceutical treatment options for musculoskeletal diseases. The purpose of this randomized, controlled, open pilot study was to elucidate the involvement of the endocannabinoid system in a multimodal therapy approach. Twenty-five elderly patients with knee osteoarthritis (OA) received a 2-week spa therapy with or without combination of low-dose radon therapy in the Bad Gastein radon gallery. A 10-point numerical rating scale (pain in motion and at rest), WOMAC questionnaire, and the EuroQol-5D (EQ-5D) questionnaire were recorded at baseline, and during treatment period at weeks one and two, and at 3-month and 6-month follow-ups. Plasma levels of the endocannabinoid anandamide (AEA) were determined at baseline and at 2 weeks, and serum levels of several cartilage metabolism markers at all five time-points. A significant and sustained reduction of self-reported knee pain was observed in the study population, but no further significant effect of the additional radon therapy up and above base therapy. This pain reduction was accompanied by a significant reduction of AEA plasma levels during treatment in both groups. No significant differences were seen in serum marker concentrations between the groups treated with or without radon, but a small reduction of serum cartilage degradation markers was observed during treatment in both groups. This is the first study investigating AEA levels in the context of a non-pharmacological OA treatment. Since the endocannabinoid system represents a potential target for the development of new therapeutics, further studies will have to elucidate its involvement in OA pain.

Topics: Aged; Arachidonic Acids; Combined Modality Therapy; Endocannabinoids; Humans; Osteoarthritis, Knee; Pain; Pilot Projects; Polyunsaturated Alkamides; Radon; Self Report; Treatment Outcome

Nasal transient receptor potential vanilloid 1 (TRPV1) stimulation with capsaicin produces serous and mucinous secretion in the human nasal airway. The primary aim of this study was to examine topical effects of various TRP ion channel agonists on symptoms and secretion of specific mucins: mucin 5 subtype AC (MUC5AC) and B (MUC5B).. Healthy individuals were subjected to nasal challenges with TRPV1 agonists (capsaicin, olvanil and anandamide), TRP ankyrin 1 (TRPA1) agonists (cinnamaldehyde and mustard oil) and a TRP melastatin 8 (TRPM8) agonist (menthol). Symptoms were monitored, and nasal lavages were analysed for MUC5AC and MUC5B, i.e. specific mucins associated with airway diseases. In separate groups of healthy subjects, nasal biopsies and brush samples were analysed for TRPV1 and MUC5B, using immunohistochemistry and RT-qPCR. Finally, calcium responses and ciliary beat frequency were measured on isolated ciliated epithelial cells.. All TRP agonists induced nasal pain or smart. Capsaicin, olvanil and mustard oil also produced rhinorrhea. Lavage fluids obtained after challenge with capsaicin and mustard oil indicated increased levels of MUC5B, whereas MUC5AC was unaffected. MUC5B and TRPV1 immunoreactivities were primarily localized to submucosal glands and peptidergic nerve fibres, respectively. Although trpv1 transcripts were detected in nasal brush samples, functional responses to capsaicin could not be induced in isolated ciliated epithelial cells.. Agonists of TRPV1 and TRPA1 induced MUC5B release in the human nasal airways in vivo. These findings may be of relevance with regard to the regulation of mucin production under physiological and pathophysiological conditions.

Topics: Acrolein; Administration, Intranasal; Adult; Aged; Arachidonic Acids; Biopsy; Calcium; Calcium Channels; Capsaicin; Cilia; Cross-Over Studies; Double-Blind Method; Endocannabinoids; Humans; Immunohistochemistry; Methanol; Middle Aged; Movement; Mucin 5AC; Mucin-5B; Mustard Plant; Nasal Lavage; Nasal Mucosa; Nerve Tissue Proteins; Pain; Pain Measurement; Plant Oils; Polyunsaturated Alkamides; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Sensory System Agents; Sweden; Transient Receptor Potential Channels; TRPA1 Cation Channel; TRPM Cation Channels; TRPV Cation Channels; Young Adult

The endocannabinoid system (ECS) is involved in the pathophysiology of multiple sclerosis (MS), and relief from pain and spasticity has been reported in MS patients self-medicating with marijuana. A cannabis-based medication containing Delta(9)-tetrahydrocannabinol and cannabidiol (Sativex) has been approved in some countries for the treatment of MS-associated pain. The effects of this pharmaceutical preparation on other clinically relevant aspects of MS pathophysiology, however, are still unclear. In 20 MS patients, we measured the effects of Sativex on clinically measured spasticity and on neurophysiological and laboratory parameters that correlate with spasticity severity or with the modulation of the ECS. Sativex failed to affect spasticity and stretch reflex excitability. This compound also failed to affect the synthesis and the degradation of the endocannabinoid anandamide, as well as the expression of both CB1 and CB2 cannabinoid receptors in various subpopulations of peripheral lymphocytes.

Topics: Adult; Arachidonic Acids; Cannabidiol; Cannabinoids; Dronabinol; Drug Combinations; Endocannabinoids; Female; Humans; Lymphocytes; Male; Middle Aged; Multiple Sclerosis; Muscle Spasticity; Pain; Plant Extracts; Polyunsaturated Alkamides; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Reflex, Stretch; Treatment Outcome; Young Adult

Curcumin is one of the compounds present in plants of the genus Curcuma sp., being very used not only as condiment but also with medicinal purposes. As an analgesic, papers highlight the efficacy of curcumin in the treatment of various types of pain.. In this study we evaluated the peripheral antinociceptive effect of curcumin and by which mechanisms this effect is induced.. The mice paw pressure test was used on animals which had increased pain sensitivity by intraplantar injection of carrageenan. All the drugs were administered in the right hind paw.. Curcumin was administered to the right hind paw animals induced antinociceptive effect. Non -selective antagonist of opioid receptors naloxone reverted the antinociceptive effect induced by curcumin. Selective antagonists for μ, δ and κ opioid receptors clocinnamox, naltrindole and nor- binaltorphimine, respectively, reverted the antinociceptive effect induced by curcumin. Bestatin, enkephalinases inhibitor that degrade peptides opioids, did not change the nociceptive response. Selective antagonists for CB. These results suggest that curcumin possibly peripheral antinociception induced by opioid and cannabinoid systems activation and possibly for endocannabinoids and opioids release.

Topics: Analgesics; Animals; Anti-Inflammatory Agents, Non-Steroidal; Arachidonic Acids; Cannabinoid Receptor Agonists; Carrageenan; Cinnamates; Curcumin; Dose-Response Relationship, Drug; Endocannabinoids; Hyperalgesia; Male; Mice; Morphine Derivatives; Narcotic Antagonists; Pain; Polyunsaturated Alkamides; Receptors, Opioid

Fatty acid amide hydrolase inhibition may be used to control bladder function and pain by modulating endocannabinoid levels in cystitis. We studied the effect of the peripherally restricted fatty acid amide hydrolase inhibitor URB937 in bladder reflex activity and bladder pain using the lipopolysaccharide model of cystitis. We also correlated the URB937's effects with tissue levels of the endocannabinoids anandamide and palmitoylethanolamine. URB937 did not change the reflex activity of normal bladders. In inflamed bladders, URB937 had a U-shaped dose-response curve; following an initial cannabinoid receptor type 1-mediated reduction in pain responses and normalisation of bladder reflex activity, URB937 gradually increased both pain responses and bladder reflex activity through the transient receptor potential ion channel subfamily V member 1. Chronic cystitis increased the tissue levels of anandamide and decreased those of palmitoylethanolamine. At the dose that normalised bladder reflex activity and decreased pain responses, URB937 normalised the levels of anandamide and palmitoylethanolamine in the bladder. At high doses that induced excitatory effects, URB937 apparently did not change anandamide and palmitoylethanolamine levels, which therefore were in the range of the inflamed bladder. Fatty acid amide hydrolase inhibition results in complex changes in bladder endocannabinoid levels. The therapeutic effect of fatty acid amide hydrolase inhibitors is not related to increase in anandamide levels but rather a normalisation of the anandamide and palmitoylethanolamine level ratio.

Topics: Amides; Amidohydrolases; Animals; Arachidonic Acids; Cannabinoids; Cystitis; Endocannabinoids; Ethanolamines; Female; Pain; Palmitic Acids; Polyunsaturated Alkamides; Rats, Wistar; Urinary Bladder

The influence of parental support on child pain experiences is well recognised. Accordingly, animal studies have revealed both short- and long-term effects of early life stress on nociceptive responding and neural substrates such as endocannabinoids. The endocannabinoid system plays an important role in mediating and modulating stress, social interaction, and nociception. This study examined the effects of maternal support or acute isolation on nociceptive responding of female rats to a range of stimuli during the juvenile pre-adolescent period and accompanying changes in the endocannabinoid system. The data revealed that juvenile female Sprague Dawley rats (PND21-24) isolated from the dam for 1 h prior to nociceptive testing exhibited increased latency to withdraw in the hot plate test and increased mechanical withdrawal threshold in the Von Frey test, compared to rats tested in the presence of the dam. Furthermore, isolated rats exhibited reduced latency to respond in the acetone drop test and enhanced nociceptive responding in the formalin test when compared to dam-paired counterparts. Anandamide, but not 2-AG, levels were reduced in the prefrontal cortex of dam-paired, but not isolated, juvenile rats following nociceptive testing. There was no change in the expression of CB

Topics: Amidohydrolases; Animals; Arachidonic Acids; Endocannabinoids; Fear; Female; Male; Maternal Deprivation; Monoacylglycerol Lipases; Nociception; Nociceptors; Pain; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1

Parkinson's disease (PD) causes chronic pain in two-thirds of patients, in part originating from sensory neuropathies. The aim of the present study was to describe the phenotype of PD-associated sensory neuropathy and to evaluate its associations with lipid allostasis, the latter motivated by recent genetic studies associating mutations of glucocerebrosidase with PD onset and severity. Glucocerebrosidase catalyzes the metabolism of glucosylceramides.. We used quantitative sensory tests, pain ratings, and questionnaires and analyzed plasma levels of multiple bioactive lipid species using targeted lipidomic analyses. The study comprised 2 sets of patients and healthy controls: the first 128 Israeli PD patients and 224 young German healthy controls for exploration, the second 50/50 German PD patients and matched healthy controls for deeper analyses.. The data showed a 70% prevalence of PD pain and sensory neuropathies with a predominant phenotype of thermal sensory loss plus mechanical hypersensitivity. Multivariate analyses of lipids revealed major differences between PD patients and healthy controls, mainly originating from glucosylceramides and endocannabinoids. Glucosylceramides were increased, whereas anandamide and lysophosphatidic acid 20:4 were reduced, stronger in patients with ongoing pain and with a linear relationship with pain intensity and sensory losses, particularly for glucosylceramide 18:1 and glucosylceramide 24:1.. Our data suggest that PD-associated sensory neuropathies and PD pain are in part caused by accumulations of glucosylceramides, raising the intriguing possibility of reducing PD pain and sensory loss by glucocerebrosidase substituting or refolding approaches. © 2020 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Topics: Arachidonic Acids; Endocannabinoids; Glucosylceramides; Humans; Pain; Parkinson Disease; Polyunsaturated Alkamides

Osteoarthritis (OA) is a degenerative joint disease manifested by movement limitations and chronic pain. Endocannabinoid system (ECS) may modulate nociception via cannabinoid and TRPV1 receptors. The purpose of our study was to examine alterations in the spinal and joint endocannabinoid system during pain development in an animal model of OA. Wistar rats received intra-articular injection of 3mg of sodium monoiodoacetate (MIA) into the knee joint. Animals were sacrificed on day 2, 7, 14, 21, 28 after injection and lumbar spinal cord, cartilage and synovium were collected. Changes in the transcription levels of the ECS elements were measured. At the spinal level, gene expression levels of the cannabinoid and TRPV1 receptors as well as enzymes involved in anandamide synthesis and degradation were elevated in the advanced OA phase. In the joint, an important role of the synovium was demonstrated, since cartilage degeneration resulted in attenuation of the changes in the gene expression. Enzymes responsible for anandamide synthesis and degradation were upregulated particularly in the early stages of OA, presumably in response to early local joint inflammation. The presented study provides missing information about the MIA-induced OA model and encourages the development of a therapy focused on the molecular role of ECS.

Topics: Animals; Arachidonic Acids; Disease Models, Animal; Disease Progression; Endocannabinoids; Gene Expression Regulation; Injections, Intra-Articular; Iodoacetic Acid; Knee Joint; Osteoarthritis; Pain; Polyunsaturated Alkamides; Rats; Rats, Wistar; TRPV Cation Channels

Topics: Amidohydrolases; Animals; Arachidonic Acids; Depression; Endocannabinoids; Glycerides; Hyperalgesia; Interferon-alpha; Male; Mice; Monoacylglycerol Lipases; Nociceptors; Pain; Polyunsaturated Alkamides

The endogenous lipids N-arachidonylglycine and oleoyl-l-carnitine are potential therapeutic leads in the treatment of chronic pain through their inhibition of the glycine transporter GlyT2. However, their mechanism of action is unknown. It has been hypothesized that these "bioactive" lipids either inhibit GlyT2 indirectly, by significantly perturbing the biophysical properties of the membrane, or directly, by binding directly to the transporter (either from a membrane-exposed or solvent-exposed binding site). Here, we used molecular dynamics simulations to study the effects of the lipids anandamide, N-arachidonylglycine, and oleoyl-l-carnitine on (a) the biophysical properties of the bilayer and (b) direct binding interactions with GlyT2. During the simulations, the biophysical properties of the bilayer itself, for example, the area per lipid, bilayer thickness, and order parameters, were not significantly altered by the presence or type of bioactive lipid, regardless of the presence of GlyT2. Our work, together with previous computational and experimental data, suggests that these acyl-inhibitors of GlyT2 inhibit the transporter by directly binding to it. However, these bioactive lipids bound to various parts of GlyT2 and did not prefer a single binding site during 4.5 μs of simulation. We postulate that the binding site is located at the solvent-exposed regions of GlyT2. Understanding the mechanism of action of these and related bioactive lipids is essential in effectively developing high-affinity GlyT2 inhibitors for the treatment of pain.

Topics: Animals; Arachidonic Acids; Biophysical Phenomena; Endocannabinoids; Glycine; Glycine Plasma Membrane Transport Proteins; Lipids; Pain; Polyunsaturated Alkamides; Xenopus laevis

In this study, a taste bud tissue biosensor was prepared by a starch-sodium alginate cross-linking fixation method. Capsaicin was used as a TRPV1 noxious ion channel activator to investigate the antagonism kinetics of six different substances on capsaicin. The results showed that capsazepine, AMG517, loureirin B, and tetrahydropalmatine were all competitive allosteric regulatory ligands for capsaicin, while aconitine and anandamide were mixed allosteric regulatory ligand that combines non-competition and competition effect. Through analyzing the kinetic parameters of capsaicin and its competitive allosteric regulatory ligands, and comparing the structures between spicy substances and endocannabinoids, the importance of amide groups and similar groups in the allosteric regulation of cannabinoids (CB) receptors and analgesic mechanism was elucidated. This indicates that vanilloid activators turn on the TRPV1 ion channel to transmit only pain and other nociceptive signals, while capsaicin and its competitive ligands are capable of activating intracellular G protein/PI3K/PIP2 signaling pathways by binding to endogenous cannabinoid receptors, and then increase intracellular PIP2 levels (the increasing PIP2 can competitively replace capsaicin and other vanilloid activators), thereby closing the TRPV1 channel and exerting the analgesic effect. The elucidation of this mechanism of pain and analgesia will lay the theoretical foundation and new ideas for investigating nociceptive signal and screening potential analgesic drugs.

Topics: Alginates; Allosteric Regulation; Analgesics; Animals; Arachidonic Acids; Biosensing Techniques; Body Temperature; Capsaicin; Cross-Linking Reagents; Electrochemical Techniques; Endocannabinoids; Fever; Pain; Polyunsaturated Alkamides; Rats, Sprague-Dawley; Starch; Taste Buds; TRPV Cation Channels

Ketamine has been widely used as an analgesic and produces dissociative anesthetic effects. The antinociceptive effects of ketamine have been studied, but the involvement of endocannabinoids in these effects has not yet been investigated. In this study, we evaluated the involvement of the endocannabinoid system in the peripheral antinociceptive effects induced by ketamine. All drugs were administered via the intraplantar route. To induce hyperalgesia, rat paws were injected with prostaglandin E. This study suggests that ketamine antinociception depends at least in part on AEA release and CB

Topics: Analgesics; Animals; Arachidonic Acids; Cannabinoid Receptor Agonists; Cannabinoid Receptor Antagonists; Endocannabinoids; Ketamine; Male; Pain; Polyunsaturated Alkamides; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1

Significant age- and experience-dependent remodelling of spinal and supraspinal neural networks occur, resulting in altered pain responses in early life. In adults, endogenous opioid peptide and endocannabinoid (ECs) pain control systems exist which modify pain responses, but the role they play in acute responses to pain and postnatal neurodevelopment is unknown. Here, we have studied the changing role of the ECs in the brainstem nuclei essential for the control of nociception from birth to adulthood in both rats and humans. Using in vivo electrophysiology, we show that substantial functional changes occur in the effect of microinjection of ECs receptor agonists and antagonists in the periaqueductal grey (PAG) and rostroventral medulla (RVM), both of which play central roles in the supraspinal control of pain and the maintenance of chronic pain states in adulthood. We show that in immature PAG and RVM, the orphan receptor, GPR55, is able to mediate profound analgesia which is absent in adults. We show that tissue levels of endocannabinoid neurotransmitters, anandamide and 2-arachidonoylglycerol, within the PAG and RVM are developmentally regulated (using mass spectrometry). The expression patterns and levels of ECs enzymes and receptors were assessed using quantitative PCR and immunohistochemistry. In human brainstem, we show age-related alterations in the expression of key enzymes and receptors involved in ECs function using PCR and in situ hybridisation. These data reveal that significant changes on ECs that to this point have been unknown and which shed new light into the complex neurochemical changes that permit normal, mature responses to pain.

Topics: Age Factors; Aging; Animals; Animals, Newborn; Arachidonic Acids; Disease Models, Animal; Endocannabinoids; Gene Expression Regulation, Developmental; Medulla Oblongata; Microinjections; Neuronal Plasticity; Opioid Peptides; Pain; Pain Measurement; Periaqueductal Gray; Phospholipase D; Polyunsaturated Alkamides; Rats; Receptors, Cannabinoid; Receptors, G-Protein-Coupled; RNA, Messenger

Distraction is used clinically to relieve and manage pain. It is hypothesized that pain demands attention and that exposure to another attention-demanding stimulus causes withdrawal of attention away from painful stimuli, thereby reducing perceived pain. We have recently developed a rat model that provides an opportunity to investigate the neurobiological mechanisms mediating distraction-induced analgesia, as these mechanisms are, at present, poorly understood. Given the well-described role of the endogenous cannabinoid (endocannabinoid; EC) system in the modulation of pain and attentional processing, the present study investigated its role in distraction-induced antinociception in rats.. Animals received the CB1 receptor antagonist/inverse agonist, rimonabant or vehicle intraperitoneally, 30 min prior to behavioural evaluation. Formalin-evoked nociceptive behaviour was measured in the presence or absence of a novel-object distractor. Liquid chromatography-tandem mass spectrometry was used to determine the levels of the endogenous cannabinoids anandamide and 2-arachidonoylglycerol (2-AG) in the ventral hippocampus (vHip).. Exposure to a novel object distractor significantly reduced formalin-evoked nociceptive behaviour. The novel object-induced reduction in nociceptive behaviour was attenuated by rimonabant. Novel object exposure was also associated with increased tissue levels of anandamide and 2-AG in the vHip.. These data suggest that the reduction in formalin-evoked nociceptive behaviour that occurs as a result of exposure to a novel object may be mediated by engagement of the EC system, in particular in the vHip. The results provide evidence that the EC system may be an important neural substrate subserving attentional modulation of pain.

Topics: Animals; Arachidonic Acids; Attention; Behavior, Animal; Cannabinoid Receptor Antagonists; Endocannabinoids; Exploratory Behavior; Fear; Glycerides; Hippocampus; Male; Nociception; Pain; Pain Measurement; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Receptor, Cannabinoid, CB1; Rimonabant

The pharmacological inhibition of anandamide (AEA) hydrolysis by fatty acid amide hydrolase (FAAH) attenuates pain in animal models of osteoarthritis (OA) but has failed in clinical trials. This may have occurred because AEA also activates transient receptor potential vanilloid type 1 (TRPV1), which contributes to pain development. Therefore, we investigated the effectiveness of the dual FAAH-TRPV1 blocker OMDM-198 in an MIA-model of osteoarthritic pain. We first investigated the MIA-induced model of OA by (1) characterizing the pain phenotype and degenerative changes within the joint using X-ray microtomography and (2) evaluating nerve injury and inflammation marker (ATF-3 and IL-6) expression in the lumbar dorsal root ganglia of osteoarthritic rats and differences in gene and protein expression of the cannabinoid CB1 receptors FAAH and TRPV1. Furthermore, we compared OMDM-198 with compounds acting exclusively on FAAH or TRPV1. Osteoarthritis was accompanied by the fragmentation of bone microstructure and destroyed cartilage. An increase of the mRNA levels of ATF3 and IL-6 and an upregulation of AEA receptors and FAAH in the dorsal root ganglia were observed. OMDM-198 showed antihyperalgesic effects in the OA model, which were comparable with those of a selective TRPV1 antagonist, SB-366,791, and a selective FAAH inhibitor, URB-597. The effect of OMDM-198 was attenuated by the CB1 receptor antagonist, AM-251, and by the nonpungent TRPV1 agonist, olvanil, suggesting its action as an "indirect" CB1 agonist and TRPV1 antagonist. These results suggest an innovative strategy for the treatment of OA, which may yield more satisfactory results than those obtained so far with selective FAAH inhibitors in human OA.

Topics: Activating Transcription Factor 3; Amidohydrolases; Anilides; Animals; Arachidonic Acids; Benzamides; Capsaicin; Carbamates; Cinnamates; Disease Models, Animal; Endocannabinoids; Ganglia, Spinal; Gene Expression; Hyperalgesia; Inflammation; Interleukin-6; Lumbar Vertebrae; Male; Osteoarthritis; Pain; Pain Management; Pain Measurement; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; TRPV Cation Channels

Over the past decade, pharmacological manipulation of cannabinoid 1 receptor (CB1R) has become an interesting approach for the management of food ingestion disorders, among other physiological functions. Searching for new substances with similar desirable effects, but fewer side-effects we have synthesized a SR141716A (a cannabinoid receptor inverse agonist also called Rimonabant) analog, 1-(2,4-Difluorophenyl)-4-methyl-N-(1-piperidinyl)-5-[4-(trifluoromethyl)phenyl]-1H-pyrazole-3-carboxamide, ENP11, that so far, as we have previously shown, has induced changes in glucose availability, i.e. hypoglycemia, in rats. In this study we tested the effects, if any, of ENP11 (0.5, 1.0, and 3.0mg/kg) in food ingestion, core temperature, pain perception and motor control in adult Wistar rats. Results showed that ENP11 reduced food ingestion during the first hour immediately after administration. Likewise, ENP11 (1.0mg/kg) blocked anandamide (AEA)-induced hyperphagia during the first 4h of the dark phase of the light-dark cycle, and it also blocked AEA-induced hypothermia. However, none of the ENP11 doses used affected pain perception or motor control. We believe that ENP11 is a potential useful CB1R antagonist that reduces food ingestion and regulates core temperature.

Topics: Animals; Appetite Depressants; Arachidonic Acids; Body Temperature; Body Weight; Cannabinoid Receptor Antagonists; Eating; Endocannabinoids; Feeding Behavior; Hyperphagia; Male; Pain; Pain Perception; Piperidines; Polyunsaturated Alkamides; Postural Balance; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1

A series of piperazine ureas were designed, synthesized, and evaluated for their potential as novel orally efficacious fatty acid amide hydrolase (FAAH) inhibitors for the treatment of neuropathic and inflammatory pain. We carried out an optimization study of compound 5 to improve its in vitro FAAH inhibitory activity, and identified the 2-pyrimidinylpiperazine derivative 21d with potent inhibitory activity, favorable DMPK profile and brain permeability. Compound 21d showed robust and dose-dependent analgesic efficacy in animal models of both neuropathic and inflammatory pain.

Topics: Administration, Oral; Amidohydrolases; Analgesics; Animals; Brain; Disease Models, Animal; Drug Design; Enzyme Inhibitors; Half-Life; Male; Mice; Mice, Inbred ICR; Pain; Piperazine; Piperazines; Pyridazines; Pyrimidines; Rats; Rats, Sprague-Dawley; Urea

In our ongoing program aimed at deeply investigating the endocannabinoid system (ES), a set of new alkyl-resorcinol derivatives was prepared focusing on the nature and the importance of the carboxamide functionality. Binding studies on CB1 and CB2 receptors, monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH) showed that some of the newly developed compounds behaved as very potent cannabinoid receptor ligands (Ki in the nanomolar range) while, however, none of them was able to inhibit MAGL and/or FAAH. Derivative 11 was a potent CB1 and CB2 ligand, with Ki values similar to WIN 55,212, exhibiting a CB1 and CB2 agonist profile in vitro. In the formalin test of peripheral acute and inflammatory pain in mice, this compound showed a weak and delayed antinociceptive effect against the second phase of the nocifensive response, exhibiting, interestingly, a quite potent transient receptor potential ankyrin type-1 (TRPA1) channel agonist activity. Moreover, derivative 14, characterized by lower affinity but higher CB2 selectivity than 11, proved to behave as a weak CB2 competitive inverse agonist.

Topics: Analgesics; Animals; Calcium Channels; Cell Survival; Cells, Cultured; CHO Cells; Cricetulus; Dose-Response Relationship, Drug; Humans; Ligands; Male; Mice; Molecular Structure; Nerve Tissue Proteins; NIH 3T3 Cells; Nociception; Pain; Pain Measurement; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Resorcinols; Structure-Activity Relationship; Transient Receptor Potential Channels; TRPA1 Cation Channel

The endogenous cannabinoid (endocannabinoid) system plays a key role in the modulation of aversive and nociceptive behaviour. The components of the endocannabinoid system are expressed throughout the hippocampus, a brain region implicated in both conditioned fear and pain. In light of evidence that pain can impact on the expression of fear-related behaviour, and vice versa, we hypothesised that exogenous administration of the endocannabinoid 2-arachidonoyl glycerol (2-AG) into the ventral hippocampus (vHip) would differentially regulate fear responding in the absence vs. the presence of formalin-evoked nociceptive tone. Fear-conditioned rats showed significantly increased freezing and a reduction in formalin-evoked nociceptive behaviour upon re-exposure to a context previously paired with footshock. Bilateral microinjection of 2-AG into the vHip significantly reduced contextually induced freezing in non-formalin-treated rats, and reduced formalin-evoked nociceptive behaviour in non-fear-conditioned rats. In contrast, 2-AG microinjection had no effect on fear responding in formalin-treated rats, and no effect on nociceptive behaviour in fear-conditioned rats. The inhibitory effect of 2-AG on fear-related behaviour, but not pain-related behaviour, was blocked by co-administration of the cannabinoid receptor 1 (CB1) antagonist/inverse agonist rimonabant. Tissue levels of the endocannabinoids N-arachidonoylethanolamide (anandamide, AEA) and 2-AG were similar in the vHip of fear-conditioned rats receiving formalin injection and the vHip of fear-conditioned rats receiving saline injection. However, the levels of AEA and 2-AG were significantly lower in the contralateral ventrolateral periaqueductal grey of formalin-treated fear-conditioned rats than in that of their saline-treated counterparts. These data suggest that 2-AG-CB1 receptor signalling in the vHip has an anti-aversive effect, and that this effect is abolished in the presence of a persistent pain state.

Topics: Animals; Arachidonic Acids; Cannabinoid Receptor Agonists; Cannabinoid Receptor Antagonists; Conditioning, Classical; Endocannabinoids; Fear; Freezing Reaction, Cataleptic; Glycerides; Hippocampus; Injections, Intraventricular; Mice; Nociception; Pain; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Receptor, Cannabinoid, CB1; Rimonabant

Cannabinoid receptor (CBR) agonists produce antinociception in conventional preclinical assays of pain-stimulated behavior but are not effective in preclinical assays of pain-depressed behavior. Fatty acid amide hydrolase (FAAH) inhibitors increase physiological levels of the endocannabinoid anandamide, which may confer improved efficacy and safety relative to direct CBR agonists. To further evaluate FAAH inhibitors as candidate analgesics, this study assessed the effects of the FAAH inhibitor URB597 in assays of acute pain-stimulated and pain-depressed behavior in male Sprague-Dawley rats. Intraperitoneal injection of dilute lactic acid served as a noxious stimulus to stimulate a stretching response or depress positively reinforced operant behavior (intracranial self-stimulation), and URB597 was tested 1 and 4 h after administration. Consistent with FAAH inhibitor effects in other assays of pain-stimulated behavior, URB597 (1-10 mg/kg intraperitoneally) produced dose-related and CB1R-mediated decreases in acid-stimulated stretching. Conversely, in the assay of acid-depressed intracranial self-stimulation, URB597 produced a delayed, partial and non-CBR-mediated antinociceptive effect. The antinociceptive dose of URB597 (10 mg/kg) increased plasma and brain anandamide levels. These results suggest that URB597 produces antinociception in these models of 'pain stimulated' and 'pain depressed' behavior, but with different rates of onset and differential involvement of CBRs.

Topics: Amidohydrolases; Analgesics; Animals; Arachidonic Acids; Behavior, Animal; Benzamides; Brain; Carbamates; Conditioning, Operant; Depression; Dose-Response Relationship, Drug; Electric Stimulation; Endocannabinoids; Enzyme Inhibitors; Implantable Neurostimulators; Injections, Intraperitoneal; Lactic Acid; Male; Pain; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley; Receptors, Cannabinoid; Self Administration

Endogenous cannabinoids (endocannabinoids) in the periaqueductal grey (PAG) play a vital role in mediating stress-induced analgesia. This analgesic effect of endocannabinoids is enhanced by pharmacological inhibition of their degradative enzymes. However, the specific effects of endocannabinoids and the inhibitors of their degradation are largely unknown within this pain-modulating region.. In vitro electrophysiological recordings were conducted from PAG neurons in rat midbrain slices. The effects of the major endocannabinoids and their degradation inhibitors on inhibitory GABAergic synaptic transmission were examined.. Exogenous application of the endocannabinoid, anandamide (AEA), but not 2-arachidonoylglycerol (2-AG), produced a reduction in inhibitory GABAergic transmission in PAG neurons. This AEA-induced suppression of inhibition was enhanced by the fatty acid amide hydrolase (FAAH) inhibitor, URB597, whereas a 2-AG-induced suppression of inhibition was unmasked by the monoacylglycerol lipase (MGL) inhibitor, JZL184. In addition, application of the CB1 receptor antagonist, AM251, facilitated the basal GABAergic transmission in the presence of URB597 and JZL184, which was further enhanced by the dual FAAH/MGL inhibitor, JZL195.. Our results indicate that AEA and 2-AG act via disinhibition within the PAG, a cellular action consistent with analgesia. These actions of AEA and 2-AG are tightly regulated by their respective degradative enzymes, FAAH and MGL. Furthermore, individual or combined inhibition of FAAH and/or MGL enhanced tonic disinhibition within the PAG. Therefore, the current findings support the therapeutic potential of FAAH and MGL inhibitors as a novel pharmacotherapy for pain.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Benzamides; Benzodioxoles; Carbamates; Endocannabinoids; Female; Glycerides; In Vitro Techniques; Inhibitory Postsynaptic Potentials; Male; Monoacylglycerol Lipases; Neurons; Pain; Periaqueductal Gray; Piperidines; Polyunsaturated Alkamides; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Synaptic Transmission

In regenerative medicine, platelet by-products containing factors physiologically involved in wound healing, have been successfully used in the form of platelet-rich plasma (PRP) for the topical therapy of various clinical conditions since it produces an improvement in tissue repair as well as analgesic effects. Measurement of endocannabinoids and related compounds in PRP revealed the presence of a significant amount of anandamide, 2-arachidonoylglycerol, palmitoylethanolamide, and oleoylethanolamide. Investigation of the activity of PRP on the keratinocyte cell line NCTC2544 in physiological and inflammatory conditions showed that, under inflammatory conditions, PRP induced in a statistically significant manner the production of these compounds by the cells suggesting that PRP might induce the production of these analgesic mediators particularly in the physiologically inflamed wounded tissue. Studies in a mouse model of acute inflammatory pain induced by formalin injection demonstrated a potent antinociceptive effect against both early and late nocifensive responses. This effect was observed following intrapaw injection of (1) total PRP; (2) lipids extracted from PRP; and (3) an endocannabinoid-enriched lipid fraction of PRP. In all conditions, antagonists of endocannabinoid CB1 and CB2 receptors, injected in the paw, abrogated the antinociceptive effects strongly suggesting for this preparation a peripheral mechanism of action. In conclusion, we showed that PRP and PRP lipid extract exert a potent antinociceptive activity linked, at least in part, to their endocannabinoids and related compound content, and to their capability of elevating the levels of these lipid mediators in cells.

Topics: Amides; Analgesics; Animals; Arachidonic Acids; Blotting, Western; Cell Line, Tumor; Endocannabinoids; Ethanolamines; Glycerides; Humans; Inflammation; Keratinocytes; Mice; Oleic Acids; Pain; Palmitic Acids; Platelet-Rich Plasma; Polyunsaturated Alkamides

Opioids do not effectively manage pain in many patients with advanced cancer. Because anandamide (AEA) activation of cannabinoid type-1 receptors (CB1R) on nociceptors reduces nociception, manipulation of AEA metabolism in the periphery may be an effective alternative or adjuvant therapy in the management of cancer pain. AEA is hydrolyzed by the intracellular enzyme fatty acid amide hydrolase (FAAH), and this enzyme activity contributes to uptake of AEA into neurons and to reduction of AEA available to activate CB1R. We used an in vitro preparation of adult murine dorsal root ganglion (DRG) neurons co-cultured with fibrosarcoma cells to investigate how tumors alter the uptake of AEA into neurons. Evidence that the uptake of [(3)H]AEA into dissociated DRG cells in the co-culture model mimicked the increase in uptake that occurred in DRG cells from tumor-bearing mice supported the utility of the in vitro model to study AEA uptake. Results with the fluorescent AEA analog CAY10455 confirmed that an increase in uptake in the co-culture model occurred in neurons. One factor that contributed to the increase in [(3)H]AEA uptake was an increase in total cellular cholesterol in the cancer condition. Treatment with the FAAH inhibitor URB597 reduced CAY10455 uptake in the co-culture model to the level observed in DRG neurons maintained in the control condition (i.e., in the absence of fibrosarcoma cells), and this effect was paralleled by OMDM-1, an inhibitor of AEA uptake, at a concentration that had no effect on FAAH activity. Maximally effective concentrations of the two drugs together produced a greater reduction than was observed with each drug alone. Treatment with BMS309403, which competes for AEA binding to fatty acid binding protein-5, mimicked the effect of OMDM-1 in vitro. Local injection of OMDM-1 reduced hyperalgesia in vivo in mice with unilateral tumors in and around the calcaneous bone. Intraplantar injection of OMDM-1 (5μg) into the tumor-bearing paw reduced mechanical hyperalgesia through a CB1R-dependent mechanism and also reduced a spontaneous nocifensive behavior. The same dose reduced withdrawal responses evoked by suprathreshold mechanical stimuli in naive mice. These data support the conclusion that OMDM-1 inhibits AEA uptake by a mechanism that is independent of inhibition of FAAH and provide a rationale for the development of peripherally restricted drugs that decrease AEA uptake for the management of cancer pain.

Topics: Animals; Arachidonic Acids; Benzamides; Brain Neoplasms; Cannabinoid Receptor Antagonists; Carbamates; Cells, Cultured; Coculture Techniques; Disease Models, Animal; Endocannabinoids; Enzyme Inhibitors; Fibrosarcoma; Fluorescent Dyes; Ganglia, Spinal; Hyperalgesia; Indoles; Lactones; Male; Mice; Mice, Inbred C3H; Pain; Pain Threshold; Polyunsaturated Alkamides; Sensory Receptor Cells; Statistics, Nonparametric; Tritium

We showed previously that inhibiting fatty acid amide hydrolase (FAAH), an endocannabinoid degrading enzyme, and transient receptor potential vanilloid type-1 (TRPV1) channels with the same molecule, the naturally occurring N-arachidonoyl-serotonin (AA-5-HT), produces more efficacious anti-nociceptive and anti-hyperalgesic actions than the targeting of FAAH or TRPV1 alone. We also reported the synthesis of some piperazinyl carbamates as "dual" FAAH inhibitors and either antagonists at TRPV1 or agonists/desensitizers of the transient receptor potential ankyrin type-1 (TRPA1) cannel, another target for analgesic drugs. We investigated here if two such compounds, the FAAH/TRPV1 blocker OMDM198 and the FAAH inhibitor/TRPA1 agonist, OMDM202, exert anti-nociceptive actions in the formalin test of pain in mice, and through what mechanism. Both compounds inhibited the second phase of the response to formalin, the effect being maximal at 3 mg/kg, i.p. Antagonism of CB1 or CB2 receptors with AM251 or AM630 (1 mg/kg, i.p.), respectively, reversed this effect. A TRPV1 agonist, palvanil (0.1 mg/kg, i.p.), also reversed the analgesic effect of OMDM198. OMDM202 action was also antagonized by a per se inactive dose of the selective TRPA1 blocker, AP-18 (0.05 mg/kg, i.p.), but not by a TRPV1 antagonist. AP-18 at higher doses (0.1-0.2 mg/kg) inhibited both the first and second phase of the formalin response. The effects of OMDM198 and OMDM202 were accompanied by elevation of anandamide levels in the spinal cord. OMDM198 (0.1-5.0 mg/kg, i.p.) also reversed carrageenan-induced oedema and thermal hyperalgesia in mice with efficacy similar to that of AA-5-HT. These data suggest that "dual" fatty acid amide hydrolase and transient receptor potential channel modulators should be clinically evaluated as novel analgesics.

Topics: Amidohydrolases; Analgesics; Animals; Arachidonic Acids; Carbamates; Edema; Endocannabinoids; Male; Mice; Mice, Inbred C57BL; Pain; Pain Measurement; Polyunsaturated Alkamides; Transient Receptor Potential Channels; TRPA1 Cation Channel; TRPV Cation Channels

Activation and sensitization of trigeminovascular nociceptive pathways is believed to contribute to the neural substrate of the severe and throbbing nature of pain in migraine. Endocannabinoids, as well as being physiologically analgesic, are known to inhibit dural trigeminovascular nociceptive responses. They are also involved in the descending modulation of cutaneous-evoked C-fiber spinal nociceptive responses from the brainstem. The purpose of this study was to determine whether endocannabinoids are involved in the descending modulation of dural and/or cutaneous facial trigeminovascular nociceptive responses, from the brainstem ventrolateral periaqueductal gray (vlPAG). CB1 receptor activation in the vlPAG attenuated dural-evoked Aδ-fiber neurons (maximally by 19%) and basal spontaneous activity (maximally by 33%) in the rat trigeminocervical complex, but there was no effect on cutaneous facial receptive field responses. This inhibitory vlPAG-mediated modulation was inhibited by specific CB1 receptor antagonism, given via the vlPAG, and with a 5-HT1B/1D receptor antagonist, given either locally in the vlPAG or systemically. These findings demonstrate for the first time that brainstem endocannabinoids provide descending modulation of both basal trigeminovascular neuronal tone and Aδ-fiber dural-nociceptive responses, which differs from the way the brainstem modulates spinal nociceptive transmission. Furthermore, our data demonstrate a novel interaction between serotonergic and endocannabinoid systems in the processing of somatosensory nociceptive information, suggesting that some of the therapeutic action of triptans may be via endocannabinoid containing neurons in the vlPAG.

Topics: Animals; Arachidonic Acids; Bicuculline; Cannabinoid Receptor Agonists; Disease Models, Animal; Endocannabinoids; GABA-A Receptor Antagonists; Male; Microinjections; Nerve Fibers, Myelinated; Pain; Periaqueductal Gray; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley; Reaction Time; Receptor, Cannabinoid, CB1; Receptors, Serotonin; Serotonin Agents; Serotonin Antagonists; Skin; Trigeminal Nuclei

The endocannabinoid system regulates nociception and aversion and mediates fear-conditioned analgesia (FCA). We investigated the effects of the fatty acid amide hydrolase (FAAH) inhibitor URB597, which inhibits the catabolism of the endocannabinoid anandamide and related N-acylethanolamines, on expression of FCA and fear and pain related behaviour per se in rats. We also examined associated alterations in the expression of the signal transduction molecule phospho-Akt in the periaqueductal grey (PAG) by immunoblotting. FCA was modelled by assessing formalin-evoked nociceptive behaviour in an arena previously paired with footshock. URB597 (0.3 mg/kg, i.p.) enhanced FCA and increased fear-related behaviour in formalin-treated rats. Conditioned fear per se in non-formalin-treated rats was associated with increased expression of phospho-Akt in the PAG. URB597 reduced the expression of fear-related behaviour in the early part of the trial, an effect that was accompanied by attenuation of the fear-induced increase in phospho-Akt expression in the PAG. Intra-plantar injection of formalin also reduced the fear-induced increase in phospho-Akt expression. These data provide evidence for a role of FAAH in FCA, fear responding in the presence or absence of nociceptive tone, and fear-evoked increases in PAG phospho-Akt expression. In addition, the results suggest that fear-evoked activation of Akt signalling in the PAG is abolished in the presence of nociceptive tone.

Topics: Amidohydrolases; Analgesia; Animals; Arachidonic Acids; Behavior, Animal; Benzamides; Cannabinoid Receptor Modulators; Carbamates; Conditioning, Psychological; Endocannabinoids; Ethanolamines; Exploratory Behavior; Fear; Formaldehyde; Hippocampus; Male; Motor Activity; Nociception; Pain; Periaqueductal Gray; Polyunsaturated Alkamides; Proto-Oncogene Proteins c-akt; Rats; Signal Transduction

It was suggested that endocannabinoids are metabolized by cyclooxygenase (COX)-2 in the spinal cord of rats with kaolin/λ-carrageenan-induced knee inflammation, and that this mechanism contributes to the analgesic effects of COX-2 inhibitors in this experimental model. We report the development of a specific method for the identification of endocannabinoid COX-2 metabolites, its application to measure the levels of these compounds in tissues, and the finding of prostamide F(2α) (PMF(2α)) in mice with knee inflammation. Whereas the levels of spinal endocannabinoids were not significantly altered by kaolin/λ-carrageenan-induced knee inflammation, those of the COX-2 metabolite of AEA, PMF(2α), were strongly elevated. The formation of PMF(2α) was reduced by indomethacin (a non-selective COX inhibitor), NS-398 (a selective COX-2 inhibitor) and SC-560 (a selective COX-1 inhibitor). In healthy mice, spinal application of PMF(2α) increased the firing of nociceptive (NS) neurons, and correspondingly reduced the threshold of paw withdrawal latency (PWL). These effects were attenuated by the PMF(2α) receptor antagonist AGN211336, but not by the FP receptor antagonist AL8810. Also prostaglandin F(2α) increased NS neuron firing and reduced the threshold of PWL in healthy mice, and these effects were antagonized by AL8810, and not by AGN211336. In mice with kaolin/λ-carrageenan-induced knee inflammation, AGN211336, but not AL8810, reduced the inflammation-induced NS neuron firing and reduction of PWL. These findings suggest that inflammation-induced, and prostanoid-mediated, enhancement of dorsal horn NS neuron firing stimulates the production of spinal PMF(2α), which in turn contributes to further NS neuron firing and pain transmission by activating specific receptors.

Topics: Action Potentials; Animals; Arachidonic Acids; Cannabinoid Receptor Modulators; Chromatography, Liquid; Cyclooxygenase 1; Cyclooxygenase 2; Dinoprost; Dinoprostone; Endocannabinoids; Evoked Potentials; Hindlimb; Inflammation; Mass Spectrometry; Membrane Proteins; Mice; Nociceptors; Pain; Polyunsaturated Alkamides; Posterior Horn Cells; Rats

Inhibitors of fatty acid amide hydrolase (FAAH) and anandamide (AEA) uptake, which limit the degradation of endogenous cannabinoids, have received interest as potential therapeutics for pain. There is also evidence that endogenous cannabinoids mediate the antinociceptive effects of opioids. Assays of pain-elicited and pain-suppressed behavior have been used to differentiate the effects of drugs that specifically alter nociception from drugs that alter nociception caused by nonspecific effects such as catalepsy or a general suppression of activity. Using such procedures, this study examines the effects of the direct cannabinoid type 1 (CB1) agonist (-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol (CP55940), the FAAH inhibitor cyclohexylcarbamic acid 3'-carbamoylbiphenyl-3-yl ester (URB597), and the AEA uptake inhibitor N-(4-hydroxyphenyl) arachidonylamide (AM404). Additional experiments examined these compounds in combination with morphine. CP55940 produced antinociception in assays of pain-elicited, but not pain-suppressed, behavior and disrupted responding in an assay of schedule-controlled behavior. URB597 and AM404 produced antinociception in assays of pain-elicited and pain-suppressed behavior in which acetic acid was the noxious stimulus, but had no effect on the hotplate and schedule-controlled responding. CP55940 in combination with morphine resulted in effects greater than those of morphine alone in assays of pain-elicited and scheduled-controlled behavior but not pain-suppressed behavior. URB597 in combination with morphine resulted in enhanced morphine effects in assays of pain-elicited and pain-suppressed behavior in which diluted acetic acid was the noxious stimulus, but did not alter morphine's effects on the hotplate or schedule-controlled responding. These studies suggest that, compared with direct CB1 agonists, manipulations of endogenous cannabinoid signaling have enhanced clinical potential; however, their effects depend on the type of noxious stimulus.

Topics: Amidohydrolases; Analgesics; Animals; Arachidonic Acids; Benzamides; Cannabinoid Receptor Modulators; Carbamates; Cyclohexanols; Endocannabinoids; Male; Mice; Mice, Inbred C57BL; Morphine; Nociception; Pain; Polyunsaturated Alkamides; Receptor, Cannabinoid, CB1

Elevating levels of endocannabinoids with inhibitors of fatty acid amide hydrolase (FAAH) is a major focus of pain research, purported to be a safer approach devoid of cannabinoid receptor-mediated side effects. Here, we have determined the effects of sustained pharmacological inhibition of FAAH on inflammatory pain behaviour and if pharmacological inhibition of FAAH was as effective as genetic deletion of FAAH on pain behaviour.. Effects of pre-treatment with a single dose, versus 4 day repeated dosing with the selective FAAH inhibitor, URB597 (i.p. 0.3 mg·kg⁻¹), on carrageenan-induced inflammatory pain behaviour and spinal pro-inflammatory gene induction were determined in rats. Effects of pain induction and of the drug treatments on levels of arachidonoyl ethanolamide (AEA), palmitoyl ethanolamide (PEA) and oleolyl ethanolamide (OEA) in the spinal cord were determined.. Single, but not repeated, URB597 treatment significantly attenuated the development of inflammatory hyperalgesia (P < 0.001, vs. vehicle-treated animals). Neither mode of URB597 treatment altered levels of AEA, PEA and OEA in the hind paw, or carrageenan-induced paw oedema. Single URB597 treatment produced larger increases in AEA, PEA and OEA in the spinal cord, compared with those after repeated administration. Single and repeated URB597 treatment decreased levels of immunoreactive N-acylphosphatidylethanolamine phospholipase D (NAPE-PLD) in the spinal cord and attenuated carrageenan-induced spinal pro-inflammatory gene induction.. Changes in the endocannabinoid system may contribute to the loss of analgesic effects following repeated administration of low dose URB597 in this model of inflammatory pain.

Topics: Amides; Amidohydrolases; Animals; Arachidonic Acids; Behavior, Animal; Benzamides; Carbamates; Disease Models, Animal; Drug Administration Schedule; Endocannabinoids; Ethanolamines; Inflammation; Male; Oleic Acids; Pain; Palmitic Acids; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley; Spinal Cord

URB937 is a peripherally restricted inhibitor of the anandamide-deactivating enzyme fatty-acid amide hydrolase (FAAH). Despite its limited access to the CNS, URB937 produces marked antinociceptive effects in rodents. URB937 is actively extruded from the CNS by the ATP-binding cassette (ABC) membrane transporter, Abcg2. Tissue Abcg2 levels are markedly different between males and females, and this transporter is known to limit the access of xenobiotics to the fetoplacental unit in gestating female rodents. In the present study, we investigated the tissue distribution and antinociceptive properties of URB937 in female mice and rats.. We studied the systemic disposition of URB937 in female mice and the antinociceptive effects of this compound in models of visceral (acetic acid-induced writhing) and inflammatory nociception (carrageenan-induced hyperalgesia) in female mice and rats. Furthermore, we evaluated the interaction of URB937 with the blood-placenta barrier in gestating mice and rats.. Abcg2 restricted the access of URB937 to the CNS of female mice and rats. Nevertheless, URB937 produced a high degree of antinociception in female mice and rats in models of visceral and inflammatory pain. Moreover, the compound displayed a restricted access to placental and fetal tissues in pregnant mice and rats.. Peripheral FAAH blockade with URB937 reduces nociception in female mice and rats, as previously shown for males of the same species. In female mice and rats, Abcg2 limits the access of URB937, not only to the CNS, but also to the fetoplacental unit. LINKED ARTICLES This article is part of a themed section on Cannabinoids. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.167.issue-8.

Topics: Acetic Acid; Amidohydrolases; Analgesics; Animals; Arachidonic Acids; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Cannabinoids; Carrageenan; Endocannabinoids; Enzyme Inhibitors; Female; Gene Expression Regulation; Inflammation; Male; Mice; Mice, Inbred C57BL; Pain; Placenta; Polyunsaturated Alkamides; Pregnancy; Rats; Rats, Sprague-Dawley; Sex Characteristics; Tissue Distribution

The cannabinoid receptor-mediated analgesic effects of 2-arachidonoylglycerol (2-AG) are limited by monoacylglycerol lipase (MAGL). 4-nitrophenyl 4-[bis (1,3-benzodioxol-5-yl) (hydroxy) methyl] piperidine-1-carboxylate (JZL184) is a potent inhibitor of MAGL in the mouse, though potency is reportedly reduced in the rat. Here we have assessed the effects of spinal inhibition of MAGL with JZL184 on nociceptive processing in rats.. In vivo spinal electrophysiological assays in anaesthetized rats were used to determine the effects of spinal administration of JZL184 on spinal nociceptive processing in the presence and absence of hindpaw inflammation. Contributions of CB(1) receptors to these effects was assessed with AM251. Inhibition of 2-oleoylglycerol hydrolytic activity and alterations of 2-AG in the spinal cord after JZL 184 were also assessed.. Spinal JZL184 dose-dependently inhibited mechanically evoked responses of wide dynamic range (WDR) neurones in naïve anaesthetized rats, in part via the CB(1) receptor. A single spinal administration of JZL184 abolished inflammation-induced expansion of the receptive fields of spinal WDR neurones. However, neither spinal nor systemic JZL184 altered levels of 2-AG, or 2-oleoylglycerol hydrolytic activity in the spinal cord, although JZL184 displayed robust inhibition of MAGL when incubated with spinal cord tissue in vitro.. JZL184 exerted robust anti-nociceptive effects at the level of the spinal cord in vivo and inhibited rat spinal cord MAGL activity in vitro. The discordance between in vivo and in vitro assays suggests that localized sites of action of JZL184 produce these profound functional inhibitory effects.. This article is part of a themed section on Cannabinoids. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.167.issue-8.

Topics: Amidohydrolases; Analgesics; Animals; Anti-Inflammatory Agents; Arachidonic Acids; Benzodioxoles; Carrageenan; Central Nervous System Sensitization; Drug Administration Routes; Endocannabinoids; Ethanolamines; Glycerides; Inflammation; Lipoprotein Lipase; Male; Mice; Mice, Inbred C57BL; Monoacylglycerol Lipases; Pain; Piperidines; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Species Specificity; Spinal Cord

BACKGROUND AND PURPOSE The endocannabinoid 2-arachidonoylglycerol (2-AG) is degraded primarily by monoacylglycerol lipase (MGL). We compared peripheral antinociceptive effects of JZL184, a novel irreversible MGL inhibitor, with the reversible MGL-preferring inhibitor URB602 and exogenous 2-AG in rats. EXPERIMENTAL APPROACH Nociception in the formalin test was assessed in groups receiving dorsal paw injections of vehicle, JZL184 (0.001-300 µg), URB602 (0.001-600 µg), 2-AG (ED(50)), 2-AG + JZL184 (at their ED(50)), 2-AG + URB602 (at their ED(50)), AM251 (80 µg), AM251 + JZL184 (10 µg), AM630 (25 µg) or AM630 + JZL184 (10 µg). Effects of MGL inhibitors on endocannabinoid accumulation and on activities of endocannabinoid-metabolizing enzymes were assessed. KEY RESULTS Intra-paw administration of JZL184, URB602 and 2-AG suppressed early and late phases of formalin pain. JZL184 and URB602 acted through a common mechanism. JZL184 (ED(50) Phase 1: 0.06 ± 0.028; Phase 2: 0.03 ± 0.011 µg) produced greater antinociception than URB602 (ED(50) Phase 1: 120 ± 51.3; Phase 2: 66 ± 23.9 µg) or 2-AG. Both MGL inhibitors produced additive antinociceptive effects when combined with 2-AG. Antinociceptive effects of JZL184, like those of URB602, were blocked by cannabinoid receptor 1 (CB(1)) and cannabinoid receptor 2 (CB(2)) antagonists. JZL184 suppressed MGL but not fatty-acid amide hydrolase or N-arachidonoyl-phosphatidylethanolamine phospholipase D activities ex vivo. URB602 increased hind paw 2-AG without altering anandamide levels. CONCLUSIONS AND IMPLICATIONS MGL inhibitors suppressed formalin-induced pain through peripheral CB(1) and CB(2) receptor mechanisms. MGL inhibition increased paw skin 2-AG accumulation to mediate these effects. MGL represents a target for the treatment of inflammatory pain.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Benzodioxoles; Biphenyl Compounds; Cannabinoid Receptor Modulators; Drug Interactions; Drug Therapy, Combination; Endocannabinoids; Glycerides; Male; Monoacylglycerol Lipases; Pain; Pain Measurement; Phospholipase D; Piperidines; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2

N-acyl-vanillamide (NAVAM) analogues of the natural pungent principle of capsicum, capsaicin, were developed several years ago as potential non-pungent analgesic compounds. N-oleoyl-vanillamide (olvanil) and N-arachidonoy-vanillamide (arvanil), in particular, were described in several publications and patents to behave as potent anti-hyperalgesic compounds in experimental models of chronic and inflammatory pain, and to activate both "capsaicin receptors", i.e. the transient receptor potential of vanilloid type-1 (TRPV1) channel, and, either directly or indirectly, cannabinoid receptors of type-1. Here we report the biochemical and pharmacological characterization of a so far neglected NAVAM, N-palmitoyl-vanillamide (palvanil), and propose its possible use instead of capsaicin, as a possible topical analgesic. Palvanil exhibited a kinetics of activation of human recombinant TRPV1-mediated intracellular calcium elevation significantly slower than that of capsaicin (t(1/2)=21s and 8s, respectively at 1μM). Slow kinetics of TRPV1 agonists were previously found to be associated with stronger potencies as TRPV1 desensitizing agents, which in turn are usually associated with lower pungency and stronger anti-hyperalgesic activity. Accordingly, palvanil desensitized the human recombinant TRPV1 to the effect of capsaicin (10nM) with significantly higher potency than capsaicin (IC(50)=0.8nM and 3.8nM, respectively), this effect reaching its maximum more rapidly (50 and 250min, respectively). Palvanil was also more potent than capsaicin at desensitizing the stimulatory effect of TRPV1 by low pH together with anandamide, which mimics conditions occurring during inflammation. In the eye-wiping assay carried out in mice, palvanil was not pungent and instead caused a strong and long-lasting inhibition of capsaicin-induced eye-wiping. Finally, intraplantar palvanil inhibited the second phase of the nociceptive response to formalin in mice. In conclusion, palvanil appears to be a non-pungent analogue of capsaicin with stronger desensitizing effects on TRPV1 and hence potentially higher anti-hyperalgesic activity.

Topics: Administration, Topical; Analgesics; Animals; Arachidonic Acids; Calcium; Capsaicin; Cell Line; Endocannabinoids; Eye; Humans; Male; Mice; Pain; Polyunsaturated Alkamides; TRPV Cation Channels

We hypothesize that innate immune signals from infectious organisms and/or injured tissues may activate peripheral neuronal pain signals. In this study, we demonstrated that TLRs 3, 7, and 9 are expressed by human dorsal root ganglion neurons (DRGNs) and in cultures of primary mouse DRGNs. Stimulation of murine DRGNs with TLR ligands induced expression and production of proinflammatory chemokines and cytokines CCL5 (RANTES), CXCL10 (IP-10), IL-1α, IL-1β, and PGE(2), which have previously been shown to augment pain. Further, TLR ligands upregulated the expression of a nociceptive receptor, transient receptor potential vanilloid type 1 (TRPV1), and enhanced calcium flux by TRPV1-expressing DRGNs. Using a tumor-induced temperature sensitivity model, we showed that in vivo administration of a TLR9 antagonist, known as a suppressive oligodeoxynucleotide, blocked tumor-induced temperature sensitivity. Taken together, these data indicate that stimulation of peripheral neurons by TLR ligands can induce nerve pain.

Topics: Aminoquinolines; Anilides; Animals; Arachidonic Acids; Blotting, Western; Calcium; Capsaicin; Cells, Cultured; Cinnamates; Cytokines; Dinoprostone; Dose-Response Relationship, Drug; Endocannabinoids; Ganglia, Spinal; Humans; Imidazoles; Mice; Microscopy, Confocal; Neurons; Pain; Poly I-C; Polyunsaturated Alkamides; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Toll-Like Receptor 3; Toll-Like Receptor 7; Toll-Like Receptor 9; Toll-Like Receptors; TRPV Cation Channels

The present studies examined the effect of chronic neuropathic pain on cannabinoid receptor density and receptor-mediated G-protein activity within supraspinal brain areas involved in pain processing and modulation in mice. Chronic constriction injury (CCI) produced a significant decrease in WIN 55,212-2-stimulated [(35)S]GTPgammaS binding in membranes prepared from the rostral anterior cingulate cortex (rACC) of CCI mice when compared to sham-operated controls. Saturation binding with [(3)H]SR 141716A in membranes of the rACC showed no significant differences in binding between CCI and sham mice. Analysis of levels of the endocannabinoids anandamide (AEA) or 2-arachidonoylglycerol (2-AG) in the rACC following CCI showed no significant differences between CCI and sham mice. These data suggest that CCI produced desensitization of the cannabinoid 1 receptor in the rACC in the absence of an overall decrease in cannabinoid 1 receptor density or change in levels of AEA or 2-AG. These data are the first to show alterations in cannabinoid receptor function in the rostral anterior cingulate cortex in response to a model of neuropathic pain.

Topics: Analgesics; Animals; Arachidonic Acids; Benzoxazines; Cannabinoid Receptor Modulators; Cell Membrane; Constriction; Disease Models, Animal; Endocannabinoids; Glycerides; Guanosine 5'-O-(3-Thiotriphosphate); Male; Mice; Mice, Inbred Strains; Models, Neurological; Morpholines; Naphthalenes; Pain; Piperidines; Polyunsaturated Alkamides; Prefrontal Cortex; Pyrazoles; Receptor, Cannabinoid, CB1; Rimonabant; Sulfur Radioisotopes; Tritium

To investigate the impact of an experimental model of osteoarthritis (OA) on spinal nociceptive processing and the role of the inhibitory endocannabinoid system in regulating sensory processing at the spinal level.. Experimental OA was induced in rats by intraarticular injection of sodium mono-iodoacetate (MIA), and the development of pain behavior was assessed. Extracellular single-unit recordings of wide dynamic range (WDR) neurons in the dorsal horn were obtained in MIA-treated rats and saline-treated rats. The levels of endocannabinoids and the protein and messenger RNA levels of the main synthetic enzymes for the endocannabinoids (N-acyl phosphatidylethanolamine phospholipase D [NAPE-PLD] and diacylglycerol lipase α [DAGLα]) in the spinal cord were measured.. Low-weight (10 gm) mechanically evoked responses of WDR neurons were significantly (P < 0.05) facilitated 28 days after MIA injection compared with the responses in saline-treated rats, and spinal cord levels of anandamide and 2-arachidonoyl glycerol (2-AG) were increased in MIA-treated rats. Protein levels of NAPE-PLD and DAGLα, which synthesize anandamide and 2-AG, respectively, were elevated in the spinal cords of MIA-treated rats. The functional role of endocannabinoids in the spinal cords of MIA-treated rats was increased via activation of cannabinoid 1 (CB(1) ) and CB(2) receptors, and blockade of the catabolism of anandamide had significantly greater inhibitory effects in MIA-treated rats compared with control rats.. Our findings provide new evidence for altered spinal nociceptive processing indicative of central sensitization and for adaptive changes in the spinal cord endocannabinoid system in an experimental model of OA. The novel control of spinal cord neuronal responses by spinal cord CB(2) receptors suggests that this receptor system may be an important target for the modulation of pain in OA.

Topics: Animals; Arachidonic Acids; Cannabinoid Receptor Modulators; Disease Models, Animal; Endocannabinoids; Glycerides; Iodoacetates; Lipoprotein Lipase; Male; Neurons; Osteoarthritis; Pain; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Spinal Cord

Peripheral cannabinoid receptors exert a powerful inhibitory control over pain initiation, but the endocannabinoid signal that normally engages this intrinsic analgesic mechanism is unknown. To address this question, we developed a peripherally restricted inhibitor (URB937) of fatty acid amide hydrolase (FAAH), the enzyme responsible for the degradation of the endocannabinoid anandamide. URB937 suppressed FAAH activity and increased anandamide levels outside the rodent CNS. Despite its inability to access brain and spinal cord, URB937 attenuated behavioral responses indicative of persistent pain in rodent models of peripheral nerve injury and inflammation and prevented noxious stimulus-evoked neuronal activation in spinal cord regions implicated in nociceptive processing. CB₁ cannabinoid receptor blockade prevented these effects. These results suggest that anandamide-mediated signaling at peripheral CB₁ receptors controls the access of pain-related inputs to the CNS. Brain-impenetrant FAAH inhibitors, which strengthen this gating mechanism, might offer a new approach to pain therapy.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Cannabinoid Receptor Modulators; Cannabinoids; Carrageenan; Chromatography, Liquid; Disease Models, Animal; Drug Administration Routes; Drug Administration Schedule; Endocannabinoids; Enzyme Inhibitors; Escape Reaction; Ethylene Glycols; Feeding Behavior; Formaldehyde; Gene Expression Regulation; Hyperalgesia; Indoles; Male; Mass Spectrometry; Mice; Mice, Inbred C57BL; Mice, Knockout; Monoacylglycerol Lipases; Motor Activity; Oncogene Proteins v-fos; Pain; Pain Measurement; Pain Threshold; Peripheral Nervous System Diseases; Piperidines; Polyunsaturated Alkamides; PPAR alpha; Pyrazoles; Rats; Rats, Sprague-Dawley; Rimonabant; Sciatica; Spinal Cord; Statistics, Nonparametric; Time Factors; Tissue Distribution; Tritium

Both anandamide and adenosine have significant roles in pain mechanisms, but no data are available concerning their interaction at the spinal level. The goal of this study was to determine how adenosine and the adenosine receptor antagonist caffeine affect the antinociceptive effect of anandamide. The pain sensitivity was assessed by the acute tail-flick test and by paw withdrawal test after carrageenan-induced inflammation. The substances were administered intrathecally to male Wistar rats. Anandamide alone (1, 30 and 100 microg) dose-dependently decreased the hyperalgesia, however it had low potency in the tail-flick test. Neither adenosine (100 microg) nor caffeine (400 microg) alone changed the pain sensitivity markedly. Their combination caused a short-lasting antihyperalgesia, but it did not influence the tail-flick latency. Both adenosine and caffeine decreased the antihyperalgesic potential of 100 microg anandamide, while adenosine-caffeine pretreatment temporarily enhanced its effect. As regards acute heat pain sensitivity, no combination with anandamide influenced the effect of anandamide. These findings provide new data concerning the interaction between two endogenous ligands and caffeine. Since these substances may exert effects on several receptors and/or systems, their interaction in vivo must be very complex and the net outcome after their coadministration could not been predicted from the in vitro results.

Topics: Adenosine; Analgesics; Animals; Arachidonic Acids; Caffeine; Dose-Response Relationship, Drug; Drug Interactions; Endocannabinoids; Hot Temperature; Inflammation; Injections, Spinal; Male; Pain; Pain Measurement; Polyunsaturated Alkamides; Purinergic P1 Receptor Antagonists; Rats; Rats, Wistar; Reaction Time

1. Although it is well known that the combined administration of synthetic or plant-originated opioids with cannabinoids (CB) results in synergistic antinociception, the effects of combined administration of endogenous ligands acting at micro-opioid and CB receptors are not known. The aim of the present study was to determine the interaction between anandamide (AEA; a CB(1) receptor agonist) and endomorphin-1 (EM-1; a micro-opioid receptor agonist) after intrathecal administration. 2. Nociception was assessed by the paw-withdrawal test after carrageenan-induced inflammation in male Wistar rats. 3. Endomorphin-1 (16.4 pmol to 16.4 nmol) and AEA (4.3-288 nmol) alone dose-dependently decreased carrageenan-induced thermal hyperalgesia, although the highest dose of AEA also exhibited pain-inducing potential. The potency of AEA was approximately 59-fold lower than that of EM-1 (35% effective dose (ED(35)) 194.4 vs 3.3 nmol, respectively). Coadministration of these ligands revealed that combinations of 16.4 pmol EM-1 plus 28.8 or 86.5 nmol AEA were more effective than either drug alone, but other combinations were no more effective than the administration of EM-1 itself. Therefore, coadministration of AEA did not significantly shift the dose-response curve to EM-1. 4. The results of the present study indicate that the coadministration of AEA and EM-1 results in potentiated antihyperalgesia only for a combination of specific doses. Because AEA activates other receptor types (e.g. TRPV1) in addition to CB(1) receptors, the results of the present suggest that, after the coadministration of EM-1 and AEA, complex interactions ensue that may lead to different outcomes compared with those seen following the injection of exogenous ligands.

Topics: Analgesics; Animals; Arachidonic Acids; Carrageenan; Drug Combinations; Drug Evaluation, Preclinical; Drug Interactions; Endocannabinoids; Hyperalgesia; Inflammation; Injections, Spinal; Male; Oligopeptides; Pain; Polyunsaturated Alkamides; Rats; Rats, Wistar; Spine; Time Factors

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

Anandamide has been characterized as both an endocannabinoid and endovanilloid. Consistent with its actions as an endovanilloid, previous studies have demonstrated that anandamide can excite primary sensory neurons in vitro via transient receptor potential vanilloid type one (TRPV1) receptors. In the present study, we sought to determine if anandamide excited cutaneous C nociceptors in vivo and if this excitation correlated with nocifensive behaviors. Using teased-fiber electrophysiological methods in the rat, C nociceptors isolated from the tibial nerve with receptive fields (RFs) on the plantar surface of the hindpaw were studied. Injection of anandamide into the RF dose-dependently excited nociceptors at doses of 10 and 100 microg. The TRPV1 receptor antagonists, capsazepine or SB 366791, were applied to the RF to determine if excitation by anandamide was mediated through TRPV1 receptors. Intraplantar injection of either capsazepine (10 microg) or SB 366791 (3 microg) attenuated the excitation produced by 100 microg anandamide. We also determined whether excitation of C nociceptors by anandamide was associated with nocifensive behaviors. Intraplantar injection of 100 microg anandamide produced nocifensive behaviors that were attenuated by pre-treatment with either capsazepine or SB 366791. Furthermore, we determined if intraplantar injection of anandamide altered withdrawal responses to radiant heat. Neither intraplantar injection of anandamide nor vehicle produced antinociception or hyperalgesia to radiant heat. Our results indicate that anandamide excited cutaneous C nociceptors and produced nocifensive behaviors via activation of TRPV1 receptors.

Topics: Action Potentials; Anilides; Animals; Arachidonic Acids; Calcium Channel Blockers; Capsaicin; Cinnamates; Dose-Response Relationship, Drug; Endocannabinoids; Hindlimb; Hot Temperature; Male; Nociceptors; Pain; Pain Measurement; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley; Skin; Tibial Nerve; TRPV Cation Channels

Endocannabinoids are lipid signaling molecules that regulate a wide range of mammalian behaviors, including pain, inflammation, and cognitive/emotional state. The endocannabinoid anandamide is principally degraded by the integral membrane enzyme fatty acid amide hydrolase (FAAH), and there is currently much interest in developing FAAH inhibitors to augment endocannabinoid signaling in vivo. Here, we report the discovery and detailed characterization of a highly efficacious and selective FAAH inhibitor, PF-3845. Mechanistic and structural studies confirm that PF-3845 is a covalent inhibitor that carbamylates FAAH's serine nucleophile. PF-3845 selectively inhibits FAAH in vivo, as determined by activity-based protein profiling; raises brain anandamide levels for up to 24 hr; and produces significant cannabinoid receptor-dependent reductions in inflammatory pain. These data thus designate PF-3845 as a valuable pharmacological tool for in vivo characterization of the endocannabinoid system.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Brain; Crystallography, X-Ray; Endocannabinoids; Enzyme Inhibitors; Humans; Male; Pain; Piperazine; Piperazines; Piperidines; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley; Receptors, Cannabinoid; Structure-Activity Relationship; Urea

Acupuncture is widely used clinically to treat acute and chronic pain conditions, but the mechanisms underlying its effect are not fully understood. Although endocannabinoids are involved in modulation of nociception in animal models and in humans, their role in acupuncture analgesia has not been assessed. In this report, we determined the effect of electroacupuncture (EA) on the level of anandamide in the skin tissue and the role of cannabinoid CB1 and CB2 receptors in the analgesic effect of EA in an animal model of inflammatory pain. Inflammatory pain was induced by local injection of complete Freund's adjuvant (CFA) into the hind paw of rats. Thermal hyperalgesia was tested with a radiant heat stimulus, and mechanical allodynia was quantified with von Frey filaments. The anandamide concentration in the skin tissue was measured by using high-performance liquid chromatography. EA, applied to GB30 and GB34, at 2 and 100Hz significantly reduced thermal hyperalgesia and mechanical allodynia induced by CFA injection. Compared with the sham group, EA significantly increased the anandamide level in the inflamed skin tissue. Local pretreatment with a specific CB2 receptor antagonist, AM630, significantly attenuated the antinociceptive effect of EA. However, the effect of EA was not significantly altered by AM251, a selective CB1 receptor antagonist. These findings suggest that EA potentiates the local release of endogenous anandamide from inflamed tissues. Activation of peripheral CB2 receptors contributes to the analgesic effect of EA on inflammatory pain.. This study shows that electroacupuncture increases the anandamide level in inflammatory skin tissues, and CB2 receptors contribute to the analgesic effect of electroacupuncture in a rat model of inflammatory pain. This information improves our understanding of the mechanisms involved in the analgesic effect of acupuncture.

Topics: Animals; Arachidonic Acids; Chromatography, High Pressure Liquid; Electroacupuncture; Endocannabinoids; Freund's Adjuvant; Hindlimb; Hot Temperature; Hyperalgesia; Indoles; Male; Pain; Pain Management; Physical Stimulation; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Skin

Direct-acting cannabinoid receptor agonists are well known to reduce hyperalgesic responses and allodynia after nerve injury, although their psychoactive side effects have damped enthusiasm for their therapeutic development. Alternatively, inhibiting fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), the principal enzymes responsible for the degradation of the respective endogenous cannabinoids, anandamide (AEA) and 2-arachydonylglycerol (2-AG), reduce nociception in a variety of nociceptive assays, with no or minimal behavioral effects. In the present study we tested whether inhibition of these enzymes attenuates mechanical allodynia, and acetone-induced cold allodynia in mice subjected to chronic constriction injury of the sciatic nerve. Acute administration of the irreversible FAAH inhibitor, cyclohexylcarbamic acid 3'-carbamoylbiphenyl-3-yl ester (URB597), or the reversible FAAH inhibitor, 1-oxo-1-[5-(2-pyridyl)-2-yl]-7-phenylheptane (OL-135), decreased allodynia in both tests. This attenuation was completely blocked by pretreatment with either CB(1) or CB(2) receptor antagonists, but not by the TRPV1 receptor antagonist, capsazepine, or the opioid receptor antagonist, naltrexone. The novel MAGL inhibitor, 4-nitrophenyl 4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate (JZL184) also attenuated mechanical and cold allodynia via a CB(1), but not a CB(2), receptor mechanism of action. Whereas URB597 did not elicit antiallodynic effects in FAAH(-/-) mice, the effects of JZL184 were FAAH-independent. Finally, URB597 increased brain and spinal cord AEA levels, whereas JZL184 increased 2-AG levels in these tissues, but no differences in either endo-cannabinoid were found between nerve-injured and control mice. These data indicate that inhibition of FAAH and MAGL reduces neuropathic pain through distinct receptor mechanisms of action and present viable targets for the development of analgesic therapeutics.

Topics: Amidohydrolases; Analgesics, Non-Narcotic; Animals; Arachidonic Acids; Benzamides; Benzodioxoles; Cannabinoid Receptor Modulators; Carbamates; Cold Temperature; Endocannabinoids; Enzyme Inhibitors; Glycerides; Hyperalgesia; Male; Mice; Mice, Inbred C57BL; Monoacylglycerol Lipases; Narcotic Antagonists; Pain; Pain Measurement; Peripheral Nervous System Diseases; Piperidines; Polyunsaturated Alkamides; Pyridines; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; TRPV Cation Channels

Cannabinoid ligands have been shown to be anti-nociceptive in animal models of acute and chronic pain by acting at the two known cannabinoid receptors, cannabinoid-1 receptor (CB-1) and cannabinoid-2 receptor (CB-2). A major concern with the use of cannabinoids for pain relief is that they activate receptors at sites other than those involved in the transmission of nociceptive stimuli. An alternative approach is to target the naturally occurring endocannabinoids, such as anandamide (AEA), 2-arachidonylglycerol (2-AG) and N-arachidonylglycine (N-AG). However in vivo results obtained with these compounds appear to be weak, most probably due to their rapid degradation and subsequent short half-life. The predominant enzyme responsible for the hydrolysis of anandamide (and some other endocannabinoids) in the brain is fatty acid amide hydrolase (FAAH). Recently, the alpha-ketoheterocycle OL135 has been synthesized and shown to be a highly potent and selective inhibitor of FAAH with efficacy in pain models in vivo. In the present study, we have adapted the mild thermal injury (MTI) model of acute pain for the mouse and pharmacologically characterized this model by showing significant reversal of the tactile allodynia by morphine (3, 5 and 10 mg kg(-1) s.c.), gabapentin (100 and 300 mg kg(-1) i.p.), ibuprofen (100 mg kg(-1) i.p.) and OL135 (10, 30 and 100 mg kg(-1) i.p.). Furthermore we have demonstrated, using this model, that a subtherapeutic dose of OL135 can enable the endocannabinoids AEA and 2-AG, but not N-AG to be active at doses where they are otherwise nonanalgesic (20 mg kg(-1) i.p.). The implications of this model in the study of pain in mice, and the therapeutic potential of FAAH inhibition to provide analgesia without the undesirable side effects of direct agonism of cannabinoid receptors are discussed.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Burns; Cannabinoid Receptor Modulators; Disease Models, Animal; Endocannabinoids; Enzyme Inhibitors; Glycerides; Glycine; Male; Mice; Mice, Congenic; Mice, Inbred C57BL; Pain; Polyunsaturated Alkamides

Tetrazoles were recently developed as inhibitors of the cellular uptake of the endocannabinoid anandamide or of its hydrolysis by fatty acid amide hydrolase (FAAH), but were proposed to act also on non-endocannabinoid-related serine hydrolases.. We tested, in a model of inflammatory pain induced in mice by formalin, five chemically similar inhibitors: (i) OMDM119 and OMDM122, two potent carbamoyl tetrazole FAAH inhibitors with no effect on anandamide uptake; (ii) LY2183240, a carbamoyl tetrazole with activity as both FAAH and uptake inhibitor; (iii) OMDM132, a non-carbamoyl tetrazole with activity only as uptake inhibitor and iv) OMDM133, a non-carbamoyl tetrazole with no activity at either FAAH or uptake.. All compounds (2.5-10 mg kg(-1), i.p.) inhibited the second phase of the nocifensive response induced by intraplantar injection of formalin. The effects of OMDM119, OMDM122 and OMDM133 were not antagonized by pretreatment with cannabinoid CB(1) receptor antagonists, such as rimonabant or AM251 (1-3 mg kg(-1), i.p.). The effects of LY2183240 and OMDM132 were fully or partially antagonized by rimonabant, respectively, and the latter compound was also partly antagonized by the CB(2) receptor antagonist, AM630.. (i) non-FAAH hydrolases might be entirely responsible for the antinociceptive activity of some, but not all, tetrazole FAAH inhibitors, (ii) the presence of a carbamoylating group is neither necessary nor sufficient for such compounds to act through targets other than FAAH and (iii) inhibition of anandamide uptake is responsible for part of this antinociceptive activity, independently of effects on FAAH.

Topics: Amidohydrolases; Analgesics; Animals; Arachidonic Acids; Cannabinoid Receptor Antagonists; Cannabinoid Receptor Modulators; Dose-Response Relationship, Drug; Endocannabinoids; Male; Mice; Mice, Inbred Strains; Pain; Pain Measurement; Polyunsaturated Alkamides; Structure-Activity Relationship; Tetrazoles

Lack of involvement of the opioid system with the endocannabinoid, arachidonylethanolamide (anandamide) was possibly due to hydrolysis by fatty acid amide hydrolase (FAAH). Cyclohexylcarbamic acid 3'-carbamoyl-biphenyl-3-yl ester (URB597) is an inhibitor of FAAH, increases brain anandamide levels and enhances anandamide-induced antinociception in male ICR mice (25-30 g). The combination of URB597 (10 mg/kg, i.p.) and anandamide (40 mg/kg, i.p.) produced maximal antinociception in the mouse tail-flick test [68.7+/-16.8 percent maximum possible effect (%MPE)], versus either substance alone (27.3+/-7.9%MPE and 4.6+/-2.3%MPE, respectively) and is significantly blocked (p<0.05) by the cannabinoid CB(1) receptor antagonist, SR141716A (rimonabant), the kappa opioid receptor-selective antagonist, nor-Binaltorphimine (10 microg i.t.; 12.7+/-4.0%MPE) and the mu opioid receptor antagonist, naloxone (1 mg/kg, s.c.; 6.0+/-3.8%MPE), but not by the delta opioid receptor-selective antagonist, naltrindole (2 mg/kg, s.c.; 29.7+/-8.2%MPE) or the cannabinoid CB(2) receptor antagonist, SR144528. In addition, nor-BNI (10 microg i.t) administration to FAAH(-/-) knockout mice produced a nociceptive response. The URB597/anandamide combination was not active in the CB(1)(-/-) knockout mice, but retained activity in the MOR(-/-) knockout mice. The sub-active combination of (URB597 10 mg/kg, i.p/anandamide 10 mg/kg, i.p.; 15.5+/-4.3%MPE) shifted the dose response curve of morphine to the left (morphine alone ED(50)=4.6 mg/kg [3.7-5.6] versus morphine/URB597/anandamide (ED(50)=2.5 mg/kg [1.9-3.4]). These data are the first demonstration that anandamide, if protected from degradation, acts via the CB(1) receptor to interact with kappa opioid receptor systems in opioid analgesia.

Topics: Amidohydrolases; Analgesics, Opioid; Animals; Arachidonic Acids; Benzamides; Cannabinoid Receptor Modulators; Carbamates; Dose-Response Relationship, Drug; Drug Therapy, Combination; Endocannabinoids; Enzyme Inhibitors; Male; Mice; Mice, Inbred ICR; Mice, Knockout; Morphine; Pain; Polyunsaturated Alkamides; Receptor, Cannabinoid, CB1; Receptors, Opioid, kappa

Tumors in bone are associated with pain in humans. Data generated in a murine model of bone cancer pain suggest that a disturbance of local endocannabinoid signaling contributes to the pain. When tumors formed after injection of osteolytic fibrosarcoma cells into the calcaneus bone of mice, cutaneous mechanical hyperalgesia was associated with a decrease in the level of anandamide (AEA) in plantar paw skin ipsilateral to tumors. The decrease in AEA occurred in conjunction with increased degradation of AEA by fatty acid amide hydrolase (FAAH). Intraplantar injection of AEA reduced the hyperalgesia, and intraplantar injection of URB597, an inhibitor of FAAH, increased the local level of AEA and also reduced hyperalgesia. An increase in FAAH mRNA and enzyme activity in dorsal root ganglia (DRG) L3-L5 ipsilateral to the affected paw suggests DRG neurons contribute to the increased FAAH activity in skin in tumor-bearing mice. Importantly, the anti-hyperalgesic effects of AEA and URB597 were blocked by a CB1 receptor antagonist. Increased expression of CB1 receptors by DRG neurons ipsilateral to tumor-bearing limbs may contribute to the anti-hyperalgesic effect of elevated AEA levels. Furthermore, CB1 receptor protein-immunoreactivity as well as inhibitory effects of AEA and URB597 on the depolarization-evoked Ca(2+) transient were increased in small DRG neurons cocultured with fibrosarcoma cells indicating that fibrosarcoma cells are sufficient to evoke phenotypic changes in AEA signaling in DRG neurons. Together, the data provide evidence that manipulation of peripheral endocannabinoid signaling is a promising strategy for the management of bone cancer pain.

Topics: Animals; Arachidonic Acids; Bone Neoplasms; Cannabinoids; Cells, Cultured; Disease Models, Animal; Endocannabinoids; Hyperalgesia; Male; Mice; Mice, Inbred C3H; Pain; Physical Stimulation; Polyunsaturated Alkamides; Receptor, Cannabinoid, CB1; Signal Transduction; Skin; Touch; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

The opioid and endocannabinoid systems mediate analgesia expressed upon re-exposure to a contextually aversive stimulus (fear-conditioned analgesia; FCA), and modulate the mitogen-activated protein kinase (MAPK) pathway. However, an interaction between the opioid and endocannabinoid systems during FCA has not been investigated at the behavioural or molecular level. FCA was modeled in male Lister-hooded rats by assessing formalin-evoked nociceptive behaviour in an arena previously paired with footshock. Administration of the fatty acid amide hydrolase and endocannabinoid catabolism inhibitor, URB597 (0.3 mg/kg, i.p.), enhanced expression of FCA. The opioid receptor antagonist, naloxone, attenuated FCA and attenuated the URB597-induced enhancement of FCA. SR141716A (CB(1) antagonist) and SR144528 (CB(2) antagonist) also attenuated the URB597-mediated enhancement of FCA. Expression of FCA was associated with increased relative phospho-ERK2 expression in the amygdala, an effect blocked by naloxone, SR141716A, and SR144528. Furthermore, URB597-mediated enhancement of FCA was associated with reduced phospho-ERK1 and phospho-ERK2 in the amygdala. Phospho-ERK1/2 expression in the hippocampus, prefrontal cortex, and thalamus was unchanged following FCA and drug treatment. None of the drugs affected formalin-evoked nociceptive behaviour or phospho-ERK1/2 expression in non-fear-conditioned rats. These data suggest that endocannabinoid-mediated enhancement of FCA is abolished by pharmacological blockade of opioid receptors as well as CB(1) or CB(2) receptors. Both pharmacological enhancement (with URB597) and attenuation (with naloxone) of this form of endogenous analgesia were associated with reduced expression of phospho-ERK1/2 in the amygdaloid complex arguing against a causal role for ERK1/2 signaling in the amygdala during expression of FCA or its modulation by opioids or cannabinoids.

Topics: Amidohydrolases; Amygdala; Analgesia; Animals; Arachidonic Acids; Avoidance Learning; Benzamides; Cannabinoid Receptor Modulators; Carbamates; Conditioning, Psychological; Endocannabinoids; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Fear; Male; Narcotic Antagonists; Neural Inhibition; Pain; Pain Measurement; Polyunsaturated Alkamides; Rats; Receptor, Cannabinoid, CB1; Receptors, Opioid; Signal Transduction

We describe herein the discovery of LASSBio-881 (3c) as a novel in vivo antinociceptive, anti-inflammatory, and in vitro antiproliferative and antioxidant compound, with a cannabinoid ligand profile. We observed that LASSBio-881 (3c) was able to bind to CB1 receptors (71% at 100microM) and also to inhibit T-cell proliferation (66% at 10microM) probably by binding to CB2 receptors, in a non-proapoptotic manner, different from anandamide (1). It was also demonstrated that LASSBio-881 (3c) had an important antioxidant profile toward free radicals (DPPH and hydroxyl), probably due to its particular redox behavior, which reflects the presence of both nitro and 3,5-di-tert-butyl-4-hydroxyphenyl sub-units, as demonstrated by cyclic voltammetry studies. In addition, we showed that these structural sub-units are essential for the observed pharmacological activity.

Topics: Analgesics; Animals; Anti-Inflammatory Agents; Antioxidants; Arachidonic Acid; Arachidonic Acids; Biphenyl Compounds; Brain; Cannabinoid Receptor Modulators; Carrageenan; Cell Proliferation; Edema; Endocannabinoids; Female; Formaldehyde; Free Radical Scavengers; Hydrazines; Hydrazones; Ligands; Male; Mice; Models, Molecular; Pain; Picrates; Polyunsaturated Alkamides; Pyridines; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Structure-Activity Relationship; Superoxides; T-Lymphocytes

The analgesic properties of cannabinoid receptor agonists are well characterized. However, numerous side effects limit the therapeutic potential of these agents. Here we report a synergistic antinociceptive interaction between the endogenous cannabinoid receptor agonist anandamide and the synthetic peroxisome proliferator-activated receptor-alpha (PPAR-alpha) agonist 2-(4-(2-(1-Cyclohexanebutyl)-3-cyclohexylureido)ethyl)phenylthio)-2-methylpropionic acid (GW7647) in a model of acute chemical-induced pain. Moreover, we show that anandamide synergistically interacts with the large-conductance potassium channel (KCa1.1, BK) activator isopimaric acid. These findings reveal a synergistic interaction between the endocannabinoid and PPAR-alpha systems that might be exploited clinically and identify a new pharmacological effect of the BK channel activator isopimaric acid.

Topics: Analgesics; Animals; Arachidonic Acids; Behavior, Animal; Butyrates; Cannabinoid Receptor Agonists; Drug Synergism; Endocannabinoids; Formaldehyde; Male; Mice; Pain; Phenylurea Compounds; Polyunsaturated Alkamides; PPAR alpha

Cannabinoids, such as anandamide, are involved in pain transmission. We evaluated the effects of AM404 (N-(4-hydroxyphenyl)-5Z,8Z,11Z,14Z-eicosatetraenamide), an anandamide reuptake inhibitor, monitoring the expression of c-fos, a marker of activated neurons and the pain-related behaviours using formalin test. The study was carried out in an experimental model of inflammatory pain made by a single injection of formalin in rat hind paws. Formalin test showed that the antinociceptive effect of AM404 was evident in phase I. We found that Fos-positive neurons in dorsal superficial and deep laminae of the lumbar spinal cord increased in formalin-injected animals and that AM404 significantly reduced Fos induction. Co-administration of cannabinoid CB(1) receptor antagonist (AM251), cannabinoid CB(2) receptor antagonist (AM630) and transient receptor potential vanilloid type 1 (TRPV-1) antagonist (capsazepine), attenuate the inhibitory effect of AM404 and this effect was higher using cannabinoid CB(2) and vanilloid TRPV-1 receptor antagonists. These results suggest that AM404 could be a useful drug to reduce inflammatory pain in our experimental model and that cannabinoid CB(2) receptor and vanilloid TRPV-1 receptor, and to a lesser extent, the cannabinoid CB(1) receptor are involved.

Topics: Animals; Arachidonic Acids; Capsaicin; Endocannabinoids; Immunohistochemistry; Indoles; Inflammation; Male; Pain; Pain Measurement; Piperidines; Polyunsaturated Alkamides; Proto-Oncogene Proteins c-fos; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Spinal Cord; TRPV Cation Channels

Anandamide (AEA) activates both cannabinoid CB(1) and TRPV1 receptors, which are expressed on cultured dorsal root ganglion neurones. Increased levels of nerve growth factor (NGF) are associated with chronic pain states.. The aim of this study was to compare of the effects of AEA on CB(1) receptor signalling and TRPV1-CB(1) crosstalk in low and high concentrations of NGF, using voltage-clamp electrophysiology and Fura-2 calcium imaging.. Chronic exposure to high NGF (200 ng ml(-1)) as compared to low NGF (20 ng ml(-1)) increases the proportion of neurones that exhibit an inward current in response to AEA (1 microM), from 7 to 29%. In contrast, inhibition of voltage-gated calcium currents by AEA is not significantly different in low NGF (33+/-9%, compared to high NGF 28+/-6%). Crosstalk between CB and TRPV1 receptors is modulated by exposure to high NGF. In low NGF, exposure to the CB(1) receptor antagonist, SR141716A, (100 nM) increases the percentage of neurones in which AEA elicits an increase in [Ca(2+)](i), from 10 to 23%. In high NGF, the antagonist does not alter the percentage of responders (33 to 30%). In low NGF, exposure to the CB receptor agonist, WIN55 (1 microM) reduces capsaicin-mediated increases in [Ca(2+)](i) to 28+/-8% of control as compared to an enhancement to 172+/-26% of control observed in high NGF.. We conclude that cannabinoid-mediated modulation of TRPV1 receptor activation is altered after exposure to high NGF.

Topics: Animals; Animals, Newborn; Arachidonic Acids; Calcium; Calcium Channels; Cannabinoid Receptor Modulators; Cells, Cultured; Chronic Disease; Dose-Response Relationship, Drug; Electrophysiology; Endocannabinoids; Fura-2; Ganglia, Spinal; Nerve Growth Factor; Pain; Patch-Clamp Techniques; Polyunsaturated Alkamides; Rats; Receptor, Cannabinoid, CB1; Signal Transduction; TRPV Cation Channels

1. Although cannabinoid receptor agonists have analgesic activity in chronic pain states, they produce a spectrum of central cannabinoid CB(1) receptor-mediated motor and psychotropic side-effects. The actions of endocannabinoids, such as anandamide, are terminated by uptake and subsequent intracellular enzymatic degradation. In the present study, we examined the effect of acute administration of the anandamide transport inhibitor AM404 in rat models of chronic neuropathic and inflammatory pain. 2. Systemic administration of AM404 (10 mg/kg) reduced mechanical allodynia in the partial sciatic nerve ligation (PNL) model of neuropathic pain, but not in the complete Freund's adjuvant (CFA) model of inflammatory pain. 3. The effect of AM404 in the PNL model was abolished by coapplication with the selective cannabinoid CB(1) receptor antagonist AM251 (1 mg/kg). AM404 did not produce a reduction in motor performance in either the PNL or CFA models. 4. These findings suggest that acute administration of AM404 reduces allodynia in a neuropathic pain model via cannabinoid CB(1) receptor activation, without causing the undesirable motor disruption associated with cannabinoid receptor agonists.

Topics: Analgesics; Animals; Arachidonic Acids; Cannabinoid Receptor Modulators; Carrier Proteins; Disease Models, Animal; Endocannabinoids; Freund's Adjuvant; Inflammation; Ligation; Male; Motor Activity; Pain; Pain Measurement; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Sciatic Nerve; Sciatic Neuropathy; Time Factors

N-arachidonoylserotonin (AA-5-HT, 1a) is an inhibitor of fatty acid amide hydrolase (FAAH) that acts also as an antagonist of transient receptor potential vanilloid-type 1 (TRPV1) channels and is analgesic in rodents. We modified the chemical structure of 1a with the aim of developing "hybrid" FAAH/TRPV1 blockers more potent than the parent compound or obtaining analogues with single activity at either of the two targets to study the mechanism of the analgesic action of 1a. Thirty-eight AA-5-HT analogues, containing a serotonin "head" bound to a variety of lipophilic moieties via amide, urea, or carbamate functionalities, were synthesized. Unlike 1a, most of the new compounds possessed activity at only one of the two considered targets. The amides 1b and 1c of alpha- and gamma-linolenic acid, however, showed "hybrid" activity similar to 1a. The carbamate 3f (OMDM106), although unable to antagonize TRPV1 receptors, was the most potent FAAH inhibitor in this study (IC50=0.5 microM). Compounds 3f and 1m (OMDM129), which exhibited activity at only FAAH or TRPV1, respectively, were 10-fold less potent than 1a at preventing formalin-induced hyperalgesia in mice.

Topics: Amidohydrolases; Analgesics; Animals; Arachidonic Acids; Biphenyl Compounds; Brain; Calcium; Carbamates; Cell Line; Endocannabinoids; Humans; Hydrolysis; Hyperalgesia; Indoles; Mice; Pain; Pain Measurement; Polyunsaturated Alkamides; Rats; Serotonin; Structure-Activity Relationship; TRPV Cation Channels

In this issue, Alvin King, Daniele Piomelli, and colleagues publish another interesting paper on inhibition of monoacylglycerol lipase (MGL). MGL is a hot target for antinociceptive agents, being the chief degrading enzyme of the endocannabinoid 2-arachidonoylglycerol.

Topics: Amidohydrolases; Analgesics; Animals; Arachidonic Acids; Biphenyl Compounds; Brain; Cannabinoid Receptor Agonists; Cannabinoid Receptor Modulators; Endocannabinoids; Enzyme Inhibitors; Glycerides; Humans; Monoacylglycerol Lipases; Organophosphonates; Pain; Polyunsaturated Alkamides; Rats; Receptors, Cannabinoid

Topics: Animals; Anti-Obesity Agents; Anxiety; Arachidonic Acids; Blood Pressure; Brain; Cannabinoid Receptor Antagonists; Cannabinoid Receptor Modulators; Controlled Clinical Trials as Topic; Drug Evaluation, Preclinical; Endocannabinoids; Humans; Inflammation; Neurons; Obesity; Osteoporosis; Pain; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptors, Cannabinoid; Rimonabant; Smoking Cessation

Anandamide, an endocannabinoid, is degraded by the enzyme fatty acid amide hydrolase which can be inhibited by nonsteroidal anti-inflammatory drugs (NSAIDs). The present work was designed to study the peripheral interactions between anandamide and ibuprofen (a non-specific cyclooxygenase inhibitor) in the rat formalin test. We first determined the ED50 for anandamide (0.018 microg +/- 0.009), ibuprofen (0.18 microg +/- 0.09), and their combination (0.006 microg +/- 0.002). Drugs were given 15 min before a 2.5% formalin injection into the dorsal surface of the right hind paw. Results were analyzed using isobolographic analysis. The antinociceptive interaction between anandamide and ibuprofen was synergistic. To further investigate the mechanisms by which the combination of anandamide with ibuprofen produced their antinociceptive effects, we used specific antagonists for the cannabinoid CB1 (AM251; 80 microg) and CB2 (AM630; 25 microg) receptors. We demonstrated that the antinociceptive effects of ibuprofen were not antagonized by either AM251 or AM630 and that those of anandamide were antagonized by AM251 but not by AM630. The synergistic antinociceptive effects of the combination of anandamide with ibuprofen were completely antagonized by AM251 but only partially inhibited by AM630. In conclusion, locally (hind paw) injected anandamide, ibuprofen or combination thereof decreased pain behavior in the formalin test. The combination of anandamide with ibuprofen produced synergistic antinociceptive effects involving both cannabinoid CB1 and CB2 receptors. Comprehension of the mechanisms involved needs further investigation.

Topics: Acute Disease; Analysis of Variance; Animals; Anti-Inflammatory Agents, Non-Steroidal; Arachidonic Acids; Area Under Curve; Cannabinoid Receptor Modulators; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Combinations; Drug Interactions; Endocannabinoids; Ibuprofen; Indoles; Inflammation; Male; Pain; Pain Measurement; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Random Allocation; Rats

1 The reversible fatty acid amide hydrolase (FAAH) inhibitor OL135 reverses mechanical allodynia in the spinal nerve ligation (SNL) and mild thermal injury (MTI) models in the rat. The purpose of this study was to investigate the role of the cannabinoid and opioid systems in mediating this analgesic effect. 2 Elevated brain concentrations of anandamide (350 pmol g(-1) of tissue vs 60 pmol g(-1) in vehicle-treated controls) were found in brains of rats given OL135 (20 mg kg(-1)) i.p. 15 min prior to 20 mg kg(-1) i.p. anandamide. 3 Predosing rats with OL135 (2-60 mg kg(-1) i.p.) 30 min before administration of an irreversible FAAH inhibitor (URB597: 0.3 mg kg(-1) intracardiac) was found to protect brain FAAH from irreversible inactivation. The level of enzyme protection was correlated with the OL135 concentrations in the same brains. 4 OL135 (100 mg kg(-1) i.p.) reduced by 50% of the maximum possible efficacy (MPE) mechanical allodynia induced by MTI in FAAH(+/+)mice (von Frey filament measurement) 30 min after dosing, but was without effect in FAAH(-/-) mice. 5 OL135 given i.p. resulted in a dose-responsive reversal of mechanical allodynia in both MTI and SNL models in the rat with an ED(50) between 6 and 9 mg kg(-1). The plasma concentration at the ED(50) in both models was 0.7 microM (240 ng ml(-1)). 6 In the rat SNL model, coadministration of the selective CB(2) receptor antagonist SR144528 (5 mg kg(-1) i.p.), with 20 mg kg(-1) OL135 blocked the OL135-induced reversal of mechanical allodynia, but the selective CB(1) antagonist SR141716A (5 mg kg(-1) i.p.) was without effect. 7 In the rat MTI model neither SR141716A or SR144528 (both at 5 mg kg(-1) i.p.), or a combination of both antagonists coadministered with OL135 (20 mg kg(-1)) blocked reversal of mechanical allodynia assessed 30 min after dosing. 8 In both the MTI model and SNL models in rats, naloxone (1 mg kg(-1), i.p. 30 min after OL135) reversed the analgesia (to 15% of control levels in the MTI model, to zero in the SNL) produced by OL135.

Topics: Amidohydrolases; Analgesics; Animals; Arachidonic Acids; Brain; Dose-Response Relationship, Drug; Endocannabinoids; Enzyme Inhibitors; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Models, Animal; Naloxone; Narcotic Antagonists; Pain; Pain Measurement; Polyunsaturated Alkamides; Pyridines; Rats; Rats, Sprague-Dawley; Spinal Cord

An attractive alternative to the use of direct agonists at the cannabinoid receptor type 1 (CB1) in the control of neuropathic pain may be to potentiate the actions of endogenous cannabinoids. Thus, the effects of AM404, an inhibitor of anandamide uptake, were assessed in an experimental model of neuropathic pain in rats. Daily treatment with AM404 prevented, time- and dose-dependently, the development of thermal hyperalgesia and mechanical allodynia in neuropathic rats. Antagonists at cannabinoid CB1 or CB2 receptors, or at the transient receptor potential vanilloid type 1 receptor, each partially reversed effects induced by AM404. A complete reversal was obtained when the three antagonists were given together, suggesting that all three receptors are involved. AM404 treatment affected two pathways involved in the generation and maintenance of neuropathic pain, one mediated by nitric oxide (NO) and the other by cytokines. AM404 completely prevented the overproduction of NO and the overexpression of nNOS, inhibited the increase in tumour necrosis factor alpha (TNFalpha) and enhanced the production of interleukin-10. Both NO and TNFalpha are known to contribute to the apoptotic process, which plays an important role in the establishment of chronic pain states. AM404 treatment prevented the increase in the ratio between pro- and anti-apoptotic gene bax/bcl-2 expression observed in the spinal cord of neuropathic rats. Taken together, these findings suggest that inhibition of endocannabinoid uptake, by blocking the putative anandamide carrier, results in the relief of neuropathic pain and may represent a novel strategy for treating chronic pain.

Topics: Analgesics; Animals; Apoptosis; Arachidonic Acids; bcl-2-Associated X Protein; Behavior, Animal; Blotting, Western; Calcium Channel Blockers; Cannabinoid Receptor Antagonists; Cytokines; Endocannabinoids; Genes, bcl-2; Hot Temperature; Hyperalgesia; Male; Motor Activity; NF-kappa B; Nitrates; Nitrites; Pain; Peripheral Nervous System Diseases; Physical Stimulation; Polyunsaturated Alkamides; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction

We studied the effect of cannabinoid receptor agonists anandamide and WIN 55,212-2 on the central pain syndrome induced by intraspinal injection of penicillin sodium salt in rats. Cannabinoids suppressed allodynia and spontaneous attacks in rats with the central pain syndrome. The analgesic effect was most pronounced after intrathecal injection of cannabinoid receptor agonist in a dose of 100 microg in 10 microl. After systemic treatment the analgesic effect was produced by only WIN 55,212-2 in a dose of 1 mg/kg. WIN 55,212-2 was superior to anandamide by the duration and intensity of the effect on allodynia and spontaneous attacks.

Topics: Analysis of Variance; Animals; Arachidonic Acids; Benzoxazines; Cannabinoid Receptor Agonists; Dose-Response Relationship, Drug; Endocannabinoids; Injections, Spinal; Male; Morpholines; Naphthalenes; Pain; Pain Measurement; Penicillin V; Polyunsaturated Alkamides; Rats; Rats, Wistar

Transient receptor potential (TRP) channels detect diverse sensory stimuli, including alterations in osmolarity. However, a molecular detector of noxious hypertonic stimuli has not yet been identified. We show here that acute pain-related behavior evoked by elevated ionic strength is abolished in TRP vanilloid subtype 1 (TRPV1)-null mice and inhibited by iodoresiniferatoxin, a potent TRPV1 antagonist. Electrophysiological recordings demonstrate a novel form of ion channel modulation by which extracellular Na+, Mg2+, and Ca2+ ions sensitize and activate the capsaicin receptor, TRPV1. At room temperature, increasing extracellular Mg2+ (from 1 to 5 mM) or Na+ (+50 mM) increased ligand-activated currents up to fourfold, and 10 mM Mg2+ reduced the EC50 for activation by capsaicin from 890 to 450 nM. Moreover, concentrations of divalent cations >10 mM directly gate the receptor. These effects occur via electrostatic interactions with two glutamates (E600 and E648) formerly identified as proton-binding residues. Furthermore, phospholipase C-mediated signaling enhances the effects of cations, and physiological concentrations of cations contribute to the bradykinin-evoked activation of TRPV1 and the sensitization of the receptor to heat. Thus, the modulation of TRPV1 by cationic strength may contribute to inflammatory pain signaling.

Topics: Adenosine Triphosphate; Aminobutyrates; Animals; Arachidonic Acids; Behavior, Animal; Bradykinin; Calcium; Calcium Channel Blockers; Capsaicin; Cations; Cells, Cultured; Dose-Response Relationship, Drug; Dose-Response Relationship, Radiation; Drug Interactions; Electric Stimulation; Endocannabinoids; Extracellular Space; Humans; Hydrogen-Ion Concentration; Magnesium Sulfate; Membrane Potentials; Mice; Mice, Inbred C57BL; Mice, Knockout; Microinjections; Models, Biological; Mutagenesis; Neurons; Nodose Ganglion; Oocytes; Pain; Patch-Clamp Techniques; Phorbol 12,13-Dibutyrate; Polyunsaturated Alkamides; Protein Kinase C; Signal Transduction; Temperature; Time Factors; Transfection; TRPV Cation Channels

Arachidonylethanolamide (anandamide, AEA) is believed to be the endogenous ligand of the cannabinoid CB(1) and CB(2) receptors. CB(1) receptors have been found localized on fibers in the spinal trigeminal tract and spinal trigeminal nucleus caudalis. Known behavioral effects of anandamide are antinociception, catalepsy, hypothermia, and depression of motor activity, similar to Delta(9)-tetrahydocannanbinol, the psychoactive constituent of cannabis. It may be a possible therapeutic target for migraine. In this study, we looked at the possible role of the CB(1) receptor in the trigeminovascular system, using intravital microscopy to study the effects of anandamide against various vasodilator agents. Anandamide was able to inhibit dural blood vessel dilation brought about by electrical stimulation by 50%, calcitonin gene-related peptide (CGRP) by 30%, capsaicin by 45%, and nitric oxide by 40%. CGRP(8-37) was also able to attenuate nitric oxide (NO)-induced dilation by 50%. The anandamide inhibition was reversed by the CB(1) receptor antagonist AM251. Anandamide also reduced the blood pressure changes caused by CGRP injection, this effect was not reversed by AM251. It would seem that anandamide acts both presynaptically, to prevent CGRP release from trigeminal sensory fibers, and postsynaptically to inhibit the CGRP-induced NO release in the smooth muscle of dural arteries. CB(1) receptors seem to be involved in the NO/CGRP relationship that exists in causing headache and dural blood vessel dilation. It also seems that some of the blood pressure changes caused by anandamide are mediated by a noncannabinoid receptor, as AM251 was unable to reverse these effects. It can be suggested that anandamide is tonically released to play some form of modulatory role in the trigeminovascular system.

Topics: Animals; Arachidonic Acids; Blood Pressure; Calcitonin Gene-Related Peptide; Capsaicin; Electric Stimulation; Endocannabinoids; Hypotension; Male; Neurons; Nitroprusside; Pain; Peptide Fragments; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Trigeminal Caudal Nucleus; Vasodilation

Although the N-arachidonoyl ethanolamine (anandamide) binds to cannabinoid receptors and has been implicated in the suppression of pain, its rapid catabolism in vivo by fatty acid amide hydrolase (FAAH) has presented a challenge in investigating the physiological functions of this endogenous cannabinoid. In order to test whether anandamide and other non-cannabinoid fatty amides modulate nociception, we compared FAAH (+/+) and (-/-) mice in the tail immersion, hot plate, and formalin tests, as well as for thermal hyperalgesia in the carrageenan and the chronic constriction injury (CCI) models. FAAH (-/-) mice exhibited a CB1 receptor-mediated phenotypic hypoalgesia in thermal nociceptive tests. These mice also exhibited CB1 receptor-mediated hypoalgesia in both phases of the formalin test accompanied with a phenotypic anti-edema effect, which was not blocked by either CB1 or CB2 antagonists. Additionally, FAAH (-/-) mice displayed thermal anti-hyperalgesic and anti-inflammatory effects in the carrageenan model that were mediated, in part, by CB2, but not CB1 receptors. In contrast, no genotype differences in pain behavior were evident following CCI, which was instead found to obliterate the phenotypic hypoalgesia displayed by FAAH (-/-) mice in the tail immersion and hot plate tests, suggesting that nerve injury may promote adaptive changes in these animals. Collectively, these findings demonstrate a cannabinoid receptor-mediated analgesic phenotype in FAAH (-/-) mice. In more general terms, these findings suggest that selective inhibitors of FAAH might represent a viable pharmacological approach for the clinical treatment of pain disorders.

Topics: Adaptation, Physiological; Amidohydrolases; Animals; Arachidonic Acids; Cannabinoid Receptor Antagonists; Disease Models, Animal; Endocannabinoids; Enzyme Inhibitors; Female; Genotype; Inflammation; Male; Mice; Mice, Knockout; Pain; Pain Measurement; Pain Threshold; Peripheral Nerve Injuries; Peripheral Nerves; Phenotype; Polyunsaturated Alkamides; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Receptors, Cannabinoid; Somatosensory Disorders

The role of anandamide in the development of inflammatory hyperalgesia and visceral hyperreflexia was studied in the rat urinary bladder. Animals were given intraperitoneal cyclophosphamide injection, which evokes painful hemorrhagic cystitis accompanied by increased bladder reflex activity. The vanilloid receptor 1 [transient receptor potential vanilloid 1 (TRPV1)] antagonist capsazepine, applied onto the serosal surface of bladders, significantly reduced the hyperreflexia. Mass spectrometric analysis revealed that cyclophosphamide injection significantly and persistently increased the anandamide content of bladder tissues. The increase in the anandamide content paralleled the development of reflex hyperactivity. Anandamide (1-100 microm), applied onto the serosal surface of naive bladders, increased the reflex activity in a concentration-dependent manner. Repeated anandamide applications did not produce desensitization of the response. The anandamide-evoked effect was blocked by capsazepine or by instillation of resiniferatoxin, the ultrapotent TRPV1 agonist, into the bladders 24 hr before the anandamide challenge. The cannabinoid 1 receptor antagonist SR141716A [N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methylpyrazole-3-carboxamide] significantly increased the potency of anandamide in enhancing bladder reflex activity in naive but not in cyclophosphamide-injected animals. Application of the fatty acid amide hydrolyze inhibitor palmitoylisopropylamine onto the serosal surface of bladders also increased the reflex activity both in naive and cyclophosphamide-injected rats. This latter effect in naive animals was blocked by capsazepine and by resiniferatoxin pretreatment. Finally, intravesical instillation of anandamide (50 microm) increased c-fos expression in the spinal cord, which was reduced by capsazepine or by resiniferatoxin pretreatment. These results suggest that anandamide, through activating TRPV1, contributes to the development of hyperreflexia and hyperalgesia during cystitis.

Topics: Acrolein; Animals; Arachidonic Acids; Capsaicin; Cyclophosphamide; Cystitis; Endocannabinoids; Female; Hydrolysis; Ion Channels; Pain; Polyunsaturated Alkamides; Posterior Horn Cells; Proto-Oncogene Proteins c-fos; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Reflex, Abnormal; TRPV Cation Channels; Urinary Bladder

Cannabinoids have been shown to increase the release of arachadonic acid, whereas nonsteroidal anti-inflammatory drugs (NSAIDs) have been shown to decrease the analgesic effects of cannabinoids. We evaluated the antinociceptive effects of chronic administration of Delta(9)-tetrahydrocannabinol (Delta(9)-THC), anandamide (an endogenous cannabinoid), arachadonic acid, ethanolamine, and methanandamide on several NSAIDs via p.o. and/or i.p. routes of administration using the mouse p-phenylquinone (PPQ) test, a test for visceral nociception. Our studies with a cannabinoid receptor (CB1) antagonist [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride (SR141716A)], a CB2 antagonist [N-((1S)-endo-1,3,3-trimethyl-bicyclo-heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide) (SR144528)], and an another CB2 agonist [1,1-dimethylbutyl-1-deoxy-Delta(9)-THC (JWH-133)] were performed to better characterize PPQ interactions with cannabinoid receptors. The acute affects of Delta(9)-THC were blocked by SR141716A (i.p.) and partially blocked by SR144528 (i.p.). When NSAIDs (p.o.) were administered, the ED(50) values were as follows: 23 mg/kg aspirin, 3 mg/kg indomethacin, 5 mg/kg celecoxib, 3 mg/kg ketorolac, 57 mg/kg acetaminophen (32.3-99.8), and 0.8 mg/kg diclofenac (0.1-4.9). In animals given chronic Delta(9)-THC, only diclofenac and acetaminophen were active. Conversely, chronic methanandamide (i.p.) did not alter the antinociceptive effects of the NSAIDs. Neither the CB1 or CB2 antagonist blocked the effects of the NSAIDs. The effects of chronic arachadonic acid, ethanolamine, and anandamide could not be evaluated. In summary, our data indicate that chronic Delta(9)-THC alters the cyclooxygenase system. Alternatively, the data suggest that this alteration is not due to chronic endogenous cannabinoid release. Based upon these data, we hypothesize that human subjects who are chronic users of Delta(9)-THC may not respond to analgesic treatment with the above NSAIDs.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Arachidonic Acid; Arachidonic Acids; Aspirin; Camphanes; Diclofenac; Dose-Response Relationship, Drug; Dronabinol; Endocannabinoids; Ethanolamines; Indomethacin; Kinetics; Male; Mice; Mice, Inbred ICR; Pain; Polyunsaturated Alkamides; Pyrazoles; Receptors, Cannabinoid; Receptors, Drug

Mounting in vitro and in vivo data suggest that the endocannabinoids anandamide and 2-arachidonoyl glycerol, as well as some plant and synthetic cannabinoids, have neuroprotective effects following brain injury. Cannabinoid receptor agonists inhibit glutamatergic synaptic transmission and reduce the production of tumour necrosis factor-alpha and reactive oxygen intermediates, which are factors in causing neuronal damage. The formation of the endocannabinoids anandamide and 2-arachidonoyl glycerol is strongly enhanced after brain injury, and there is evidence that these compounds reduce the secondary damage incurred. Some plant and synthetic cannabinoids, which do not bind to the cannabinoid receptors, have also been shown to be neuroprotective, possibly through their direct effect on the excitatory glutamate system and/or as antioxidants.

Topics: Animals; Arachidonic Acids; Brain; Brain Injuries; Cannabinoid Receptor Modulators; Cannabinoids; Clinical Trials, Phase III as Topic; Dronabinol; Endocannabinoids; Glycerides; Humans; Neuroprotective Agents; Pain; Polyunsaturated Alkamides; Rats

Actions of anandamide (10 microM) were examined on monosynaptic glutamatergic transmission from the periphery to substantia gelatinosa (SG) neurones in adult rat spinal cord slices using the whole-cell patch-clamp technique. In 64% of neurones examined, Adelta-fibre-evoked excitatory postsynaptic currents (EPSCs) were depressed (in amplitude; by 32 +/- 4%, n=21) by anandamide. On the contrary, an inhibitory action on C-fibre-evoked EPSCs was observed in only 31% of neurones tested; this magnitude (17 +/- 3%, n=4) was less than that of Adelta-fibre EPSCs (P<0.05). A cannabinoid-receptor agonist, WIN 55,212-2 (5 microM), exhibited similar actions on the EPSCs. In a neurone with minimal effects of anandamide on C-fibre EPSCs, capsaicin (1 microM) largely depressed the EPSCs (n=3); Adelta-fibre EPSCs were little affected by capsaicin. It is concluded that unlike capsaicin, anandamide inhibits more effectively Adelta-fibre than C-fibre-mediated excitatory transmission in the SG, possibly through the activation of the cannabinoid receptor.

Topics: Animals; Arachidonic Acids; Calcium Channel Blockers; Capsaicin; Electric Stimulation; Endocannabinoids; Excitatory Postsynaptic Potentials; Glutamic Acid; Nerve Fibers; Nerve Fibers, Myelinated; Neural Conduction; Nociceptors; Pain; Polyunsaturated Alkamides; Rats; Substantia Gelatinosa; Synaptic Transmission

Anandamide (N-arachidonoylethanolamine, AEA) and 2-arachidonoylglycerol (2-AG) are the most active endocannabinoids at brain (CB1) cannabinoid receptors. CD1 mice lacking the CB1 receptors ("knockout" [KO] mutants) were compared with wildtype (WT) littermates for their ability to degrade AEA through an AEA membrane transporter (AMT) and an AEA hydrolase (fatty acid amide hydrolase, FAAH). The age dependence of AMT and FAAH activity were investigated in 1- or 4-month-old WT and KO animals, and found to increase with age in KO, but not WT, mice and to be higher in the hippocampus than in the cortex of all animals. AEA and 2-AG were detected in nmol/mg protein (microm) concentrations in both regions, though the hippocampus showed approximately twice the amount found in the cortex. In the same regions, 2-AG failed to change across groups, while AEA was significantly decreased (approximately 30%) in hippocampus, but not in cortex, of old KO mice, when compared with young KO or age-matched WT animals. In the open-field test under bright light and in the lit-dark exploration model of anxiety, young KO mice, compared with old KO, exhibited a mild anxiety-related behaviour. In contrast, neither the increase in memory performance assessed by the object recognition test, nor the reduction of morphine withdrawal symptoms, showed age dependence in CB1 KO mice. These results suggest that invalidation of the CB1 receptor gene is associated with age-dependent adaptive changes of endocannabinoid metabolism which appear to correlate with the waning of the anxiety-like behaviour exhibited by young CB1 KO mice.

Topics: Adaptation, Physiological; Aging; Amidohydrolases; Animals; Arachidonic Acids; Behavior, Animal; Brain; Brain Chemistry; Cannabinoid Receptor Modulators; Carrier Proteins; Cerebral Cortex; Down-Regulation; Endocannabinoids; Hippocampus; Mice; Mice, Knockout; Neurons; Pain; Pain Threshold; Polyunsaturated Alkamides; Receptors, Cannabinoid; Receptors, Drug; Recognition, Psychology; Substance Withdrawal Syndrome; Synaptosomes

The endogenous fatty acid ethanolamide, palmitylethanolamide, alleviated, in a dose-dependent manner, pain behaviors elicited in mice by injections of formalin (5%, intraplantar), acetic acid (0.6%, 0.5 ml per animal, intraperitoneal, i.p.), kaolin (2.5 mg per animal, i.p.), and magnesium sulfate (120 mg per kg, i.p.). The antinociceptive effects of palmitylethanolamide were prevented by the cannabinoid CB2 receptor antagonist SR144528 [N-([1s]-endo-1.3.3-trimethylbicyclo[2.3.1]heptan-2-yl)-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide], not by the cannabinoid CB1 receptor antagonist SR141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide x HCl]. By contrast, palmitylethanolamide had no effect on capsaicin-evoked pain behavior or thermal nociception. The endogenous cannabinoid, anandamide (arachidonylethanolamide), alleviated nociception in all tests (formalin, acetic acid, kaolin, magnesium sulfate, capsaicin and hot plate). These effects were prevented by the cannabinoid CB1 receptor antagonist SR141716A, not the cannabinoid CB2 receptor antagonist SR141716A. Additional fatty acid ethanolamides (oleylethanolamide, myristylethanolamide, palmitoleylethanolamide, palmitelaidylethanolamide) had little or no effect on formalin-evoked pain behavior, and were not investigated in other pain models. These results support the hypothesis that endogenous palmitylethanolamide participates in the intrinsic control of pain initiation. They also suggest that the putative receptor site activated by palmitylethanolamide may provide a novel target for peripherally acting analgesic drugs.

Topics: Amides; Analgesics; Analysis of Variance; Animals; Arachidonic Acids; Calcium Channel Blockers; Camphanes; Dose-Response Relationship, Drug; Drug Synergism; Endocannabinoids; Ethanolamines; Formaldehyde; Male; Mice; Pain; Palmitic Acids; Polyunsaturated Alkamides; Pyrazoles

Topics: Adjuvants, Immunologic; Animals; Arachidonic Acids; Cannabinoids; Endocannabinoids; Mice; Pain; Plant Extracts; Polyunsaturated Alkamides

In mammals, specific lipids and amino acids serve as crucial signaling molecules. In bacteria, conjugates of lipids and amino acids (referred to as lipoamino acids) have been identified and found to possess biological activity. Here, we report that mammals also produce lipoamino acids, specifically the arachidonyl amino acids. We show that the conjugate of arachidonic acid and glycine (N-arachidonylglycine (NAGly)) is present in bovine and rat brain as well as other tissues and that it suppresses tonic inflammatory pain. The biosynthesis of NAGly and its degradation by the enzyme fatty acid amide hydrolase can be observed in rat brain tissue. In addition to NAGly, bovine brain produces at least two other arachidonyl amino acids: N-arachidonyl gamma-aminobutyric acid (NAGABA) and N-arachidonylalanine. Like NAGly, NAGABA inhibits pain. These findings open the door to the identification of other members of this new class of biomolecules, which may be integral to pain regulation and a variety of functions in mammals.

Topics: Alanine; Amidohydrolases; Amino Acids; Animals; Arachidonic Acid; Arachidonic Acids; Brain; Calcium; Cattle; Dose-Response Relationship, Drug; Endocannabinoids; Formaldehyde; gamma-Aminobutyric Acid; Gas Chromatography-Mass Spectrometry; Glycine; Kinetics; Models, Chemical; Pain; Polyunsaturated Alkamides; Protein Binding; Rats; Signal Transduction; Time Factors; Tissue Distribution

Topics: Amidohydrolases; Animals; Arachidonic Acids; Brain; Dronabinol; Endocannabinoids; Humans; Mice; Pain; Polyunsaturated Alkamides

The endogenous opioid dynorphin B was evaluated for its role in cannabinoid-induced antinociception. Previous work in our laboratory has shown that the synthetic, bicyclic cannabinoid, CP55,940, induces the release of dynorphin B whilst the naturally occurring cannabinoid, Delta(9)-tetrahydrocannabinol (Delta(9)-THC), releases dynorphin A. The dynorphins contribute in part to the antinociceptive effects of both cannabinoids at the level of the spinal cord. The present study compares dynorphin B released from perfused rat spinal cord in response to acute administration of anandamide (AEA), Delta(9)-THC and CP55,940 at two time points, 10 min and 30 min post administration, and attempts to correlate such release with antinociceptive effects of the drugs. Dynorphin B was collected from spinal perfusates of rats pretreated with Delta(9)-THC, CP55,940 or AEA. The supernatant was lyophilized and the concentrations of dynorphin B were measured via radioimmunoassay. At a peak time of antinociception (10 min), CP55,940 and Delta(9)-THC induced significant two-fold increases in the release of dynorphin B. AEA did not significantly release dynorphin B. Upon a 30-min pretreatment with the drugs, no significant dynorphin B release was observed, although antinociceptive effects persisted for CP55,940 and Delta(9)-THC. Previous work indicates that Delta(9)-THC releases dynorphin A while AEA releases no dynorphin A. This study confirms that although all three test drugs produced significant antinociception at 10 min, the endocannabinoid, AEA, does not induce antinociception via dynorphin release. Thus, our data indicate a distinct mechanism which underlies AEA-induced antinociception.

Topics: Analgesia; Analgesics; Analgesics, Non-Narcotic; Animals; Arachidonic Acids; Calcium Channel Blockers; Cannabinoid Receptor Modulators; Cannabinoids; Cyclohexanols; Dronabinol; Dynorphins; Endocannabinoids; Endorphins; Male; Pain; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley; Spinal Cord

The vanilloid receptor-1 (VR1) is a ligand-gated, non-selective cation channel expressed predominantly by sensory neurons. VR1 responds to noxious stimuli including capsaicin, the pungent component of chilli peppers, heat and extracellular acidification, and it is able to integrate simultaneous exposure to these stimuli. These findings and research linking capsaicin with nociceptive behaviours (that is, responses to painful stimuli in animals have led to VR1 being considered as important for pain sensation. Here we have disrupted the mouse VR1 gene using standard gene targeting techniques. Small diameter dorsal root ganglion neurons isolated from VR1-null mice lacked many of the capsaicin-, acid- and heat-gated responses that have been previously well characterized in small diameter dorsal root ganglion neurons from various species. Furthermore, although the VR1-null mice appeared normal in a wide range of behavioural tests, including responses to acute noxious thermal stimuli, their ability to develop carrageenan-induced thermal hyperalgesia was completely absent. We conclude that VR1 is required for inflammatory sensitization to noxious thermal stimuli but also that alternative mechanisms are sufficient for normal sensation of noxious heat.

Topics: Adenosine Triphosphate; Animals; Arachidonic Acids; Behavior, Animal; Capsaicin; Carrageenan; Cells, Cultured; Electrophysiology; Endocannabinoids; Female; gamma-Aminobutyric Acid; Ganglia, Spinal; Gene Targeting; Hot Temperature; Hydrogen-Ion Concentration; Hyperalgesia; Inflammation; Male; Mice; Mice, Inbred C57BL; Neurons, Afferent; Pain; Polyunsaturated Alkamides; Receptors, Drug; Stem Cells; TRPV Cation Channels

Intracerebroventricular (i.c.v.) administration to mice of delta9-tetrahydrocannabinol (delta9-THC), WIN 55,212-2 or the endogenous cannabinoid anandamide induced dose-related antinociception in the 55 degrees C warm-water tail-flick test. Pretreatment (24 h, i.c.v.) with pertussis toxin dose-dependently reduced the antinociceptive effect of delta9-THC (955 nmol), WIN 55,212-2 (30 nmol) and anandamide (135 nmol) (IC50 = 0.13, 5.5, and 0.32 nmol, respectively). In contrast, pretreatment (24 h, i.c.v.) with cholera toxin (0.1-3.0 mg) reduced the antinociception of WIN 55,212-2, had minimal effect on delta9-THC, and dose-dependently increased the antinociception of anandamide (ED50 = 0.50 nmol). These data suggest differences in the receptor-effector coupling of delta9-THC, WIN 55,212-2 and anandamide in supraspinal-induced antinociception in mice.

Topics: Analgesics; Animals; Arachidonic Acids; Benzoxazines; Cannabinoids; Cerebral Ventricles; Cholera Toxin; Endocannabinoids; Injections, Intraventricular; Male; Mice; Mice, Inbred ICR; Morpholines; Naphthalenes; Pain; Polyunsaturated Alkamides; Spinal Cord

There is an abundance of cannabinoid (CB) receptors for derivatives of cannabis plants in the brain and throughout the body, and several naturally occurring arachidonic acid derivatives can activate these receptors. The specific objective of this study was to activate these CB receptors in castrated male calves through administration of several CB agonists and to measure immediate changes in concentrations of several serum hormones, respiration rate, and sensitivity to pain. The rationale for the study was that exogenous activation of CB receptors might reveal whether the endogenous CB system (consisting of receptors and endogenous ligands) plays a role in the stress response of animals and specifically whether the activated CB system might be part of a coping mechanism to combat stress. Intravenous administration of three CB agonists (anandamide, methanandamide and WIN 55212-2) to nine castrated male calves under non-stress conditions provoked immediate increases of serum cortisol and respiration rate as well as rapidly caused hypoalgesia to cutaneous pain and thermal stimuli. Although anandamide and methanandamide did not affect serum prolactin, administration of another CB agonist (WIN 55212-2) did increase serum prolactin abruptly. None of the CB agonists affected serum growth hormone. In summary, many of the changes following administration of CB agonists were similar to a stress response in this species, but there were some agonist-specific differences, notably regarding prolactin secretion, as well as differences between calves and observations made in other species. Although CB receptors in calves may be activated by endogenous ligands during exposure to some stressors, the present results are also consistent with this CB system being part of a coping mechanism that helps animals deal with imposed stressors.

Topics: Adjuvants, Immunologic; Animals; Arachidonic Acids; Behavior, Animal; Benzoxazines; Calcium Channel Blockers; Cannabinoids; Cattle; Endocannabinoids; Growth Hormone; Hydrocortisone; Injections, Intravenous; Male; Morpholines; Naphthalenes; Pain; Polyunsaturated Alkamides; Prolactin; Receptors, Cannabinoid; Receptors, Drug; Stress, Psychological

Synthetic cannabinoids produce behavioral analgesia and suppress pain neurotransmission, raising the possibility that endogenous cannabinoids serve naturally to modulate pain. Here, the development of a sensitive method for measuring cannabinoids by atmospheric pressure-chemical ionization mass spectrometry permitted measurement of the release of the endogenous cannabinoid anandamide in the periaqueductal gray (PAG) by in vivo microdialysis in the rat. Electrical stimulation of the dorsal and lateral PAG produced CB1 cannabinoid receptor-mediated analgesia accompanied by a marked increase in the release of anandamide in the PAG, suggesting that endogenous anandamide mediates the behavioral analgesia. Furthermore, pain triggered by subcutaneous injections of the chemical irritant formalin substantially increased the release of anandamide in the PAG. These findings indicate that the endogenous cannabinoid anandamide plays an important role in a cannabinergic pain-suppression system existing within the dorsal and lateral PAG. The existence of a cannabinergic pain-modulatory system may have relevance for the treatment of pain, particularly in instances where opiates are ineffective.

Topics: Analgesia; Animals; Arachidonic Acids; Calcium Channel Blockers; Cannabinoids; Electrophysiology; Endocannabinoids; Formaldehyde; Gas Chromatography-Mass Spectrometry; Humans; Male; Microdialysis; Models, Biological; Pain; Periaqueductal Gray; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Sprague-Dawley; Rimonabant; Time Factors

The potent analgesic effects of cannabis-like drugs and the presence of CB1-type cannabinoid receptors in pain-processing areas of the brain and spinal cord indicate that endogenous cannabinoids such as anandamide may contribute to the control of pain transmission within the central nervous system (CNS). Here we show that anandamide attenuates the pain behaviour produced by chemical damage to cutaneous tissue by interacting with CB1-like cannabinoid receptors located outside the CNS. Palmitylethanolamide (PEA), which is released together with anandamide from a common phospholipid precursor, exerts a similar effect by activating peripheral CB2-like receptors. When administered together, the two compounds act synergistically, reducing pain responses 100-fold more potently than does each compound alone. Gas-chromatography/mass-spectrometry measurements indicate that the levels of anandamide and PEA in the skin are enough to cause a tonic activation of local cannabinoid receptors. In agreement with this possibility, the CB1 antagonist SR141716A and the CB2 antagonist SR144528 prolong and enhance the pain behaviour produced by tissue damage. These results indicate that peripheral CB1-like and CB2-like receptors participate in the intrinsic control of pain initiation and that locally generated anandamide and PEA may mediate this effect.

Topics: Amides; Analgesics; Animals; Arachidonic Acids; Cannabinoids; Drug Synergism; Endocannabinoids; Ethanolamines; Formaldehyde; Gas Chromatography-Mass Spectrometry; Male; Mice; Pain; Palmitic Acids; Polyunsaturated Alkamides; Rats; Rats, Wistar; Receptor, Cannabinoid, CB2; Receptors, Cannabinoid; Receptors, Drug

This study assessed the effects of two N-acylethanolamides in established rat models of visceral and somatic inflammatory pain. (1) The therapeutic effects of the cannabinoid anandamide and the putative CB2 agonist palmitoylethanolamide were tested in a model of persistent visceral pain (turpentine inflammation of the urinary bladder). Both anandamide (at a dose of 25 mg/kg) and palmitoylethanolamide (at doses of 10-30 mg/kg) were able to attenuate the viscero-visceral hyper-reflexia (VVH) induced by inflammation of the urinary bladder. (2) The effects of the same compounds on the behavioural response to subcutaneous formalin injection were assessed. The characteristic biphasic response was observed in control animals. Anandamide (dose range 5-25 mg/kg) and palmitoylethanolamide (dose range 5-10 mg/kg) both reduced the second phase of the response. The results confirm the analgesic potential of endogenous ligands at cannabinoid receptor sites. The anti-nociceptive effect of the putative CB2 receptor agonist, palmitoylethanolamide, is particularly interesting since it is believed to be a peripherally mediated effect. This observation might be exploited to separate central psychotropic effects from peripheral analgesic actions of the cannabinoids, under inflammatory conditions.

Topics: Amides; Analgesics; Animals; Arachidonic Acids; Cannabinoids; Capillary Permeability; Cystitis; Electrophysiology; Endocannabinoids; Ethanolamines; Female; Formaldehyde; Hyperalgesia; Inflammation; Nociceptors; Pain; Pain Measurement; Palmitic Acids; Polyunsaturated Alkamides; Rats; Rats, Wistar; Receptor, Cannabinoid, CB2; Receptors, Cannabinoid; Receptors, Drug; Urinary Bladder