pf-3845 and glyceryl-2-arachidonate

Increasing the available repertoire of effective treatments for mood and anxiety disorders represents a critical unmet need. Pharmacological augmentation of endogenous cannabinoid (eCB) signaling has been suggested to represent a novel approach to the treatment of anxiety disorders; however, the functional interactions between two canonical eCB pathways mediated via anandamide (N-arachidonylethanolamine [AEA]) and 2-arachidonoylglycerol (2-AG) in the regulation of anxiety are not well understood.. We utilized pharmacological augmentation and depletion combined with behavioral and electrophysiological approaches to probe the role of 2-AG signaling in the modulation of stress-induced anxiety and the functional redundancy between AEA and 2-AG signaling in the modulation of anxiety-like behaviors in mice.. Selective 2-AG augmentation reduced anxiety in the light/dark box assay and prevented stress-induced increases in anxiety associated with limbic AEA deficiency. In contrast, acute 2-AG depletion increased anxiety-like behaviors, which was normalized by selective pharmacological augmentation of AEA signaling and via direct cannabinoid receptor 1 stimulation with Δ. Although AEA and 2-AG likely subserve distinct physiological roles, a pharmacological and functional redundancy between these canonical eCB signaling pathways exists in the modulation of anxiety-like behaviors. These data support development of eCB-based treatment approaches for mood and anxiety disorders and suggest a potentially wider therapeutic overlap between AEA and 2-AG augmentation approaches than was previously appreciated.

Topics: Adaptation, Ocular; Animals; Anti-Anxiety Agents; Anxiety; Arachidonic Acids; Benzodioxoles; Brain; Cannabinoid Receptor Agonists; Cyclohexanols; Disease Models, Animal; Dronabinol; Endocannabinoids; Excitatory Postsynaptic Potentials; Glycerides; Heterocyclic Compounds, 1-Ring; Locomotion; Male; Mice; Mice, Inbred ICR; Piperidines; Polyunsaturated Alkamides; Pyridines; Signal Transduction

N-Palmitoylethanolamine or palmitoylethanolamide (PEA) is an anti-inflammatory compound that was recently shown to exert peroxisome proliferator-activated receptor-α-dependent beneficial effects on colon inflammation. The actions of PEA are terminated following hydrolysis by 2 enzymes: fatty acid amide hydrolase (FAAH), and the less-studied N-acylethanolamine-hydrolyzing acid amidase (NAAA). This study aims to investigate the effects of inhibiting the enzymes responsible for PEA hydrolysis in colon inflammation in order to propose a potential therapeutic target for inflammatory bowel diseases (IBDs). Two murine models of IBD were used to assess the effects of NAAA inhibition, FAAH inhibition, and PEA on macroscopic signs of colon inflammation, macrophage/neutrophil infiltration, and the expression of proinflammatory mediators in the colon, as well as on the colitis-related systemic inflammation. NAAA inhibition increases PEA levels in the colon and reduces colon inflammation and systemic inflammation, similarly to PEA. FAAH inhibition, however, does not increase PEA levels in the colon and does not affect the macroscopic signs of colon inflammation or immune cell infiltration. This is the first report of an anti-inflammatory effect of a systemically administered NAAA inhibitor. Because NAAA is the enzyme responsible for the control of PEA levels in the colon, we put forth this enzyme as a potential therapeutic target in chronic inflammation in general and IBD in particular.

Topics: Amides; Amidohydrolases; Animals; Anti-Inflammatory Agents; Arachidonic Acids; Chromatography, High Pressure Liquid; Colitis; Colon; Cytokines; Disease Models, Animal; Endocannabinoids; Enzyme-Linked Immunosorbent Assay; Ethanolamines; Gene Expression Regulation; Glycerides; Inflammation; Inflammatory Bowel Diseases; Male; Mice; Mice, Inbred C57BL; Neutrophils; Palmitic Acids; Peroxidase; Piperidines; Pyridines; Taurine

Persistent CB1 cannabinoid receptor activity limits neurotransmitter release at various synapses throughout the brain. However, it is not fully understood how constitutively active CB1 receptors, tonic endocannabinoid signaling, and its regulation by multiple serine hydrolases contribute to the synapse-specific calibration of neurotransmitter release probability. To address this question at perisomatic and dendritic GABAergic synapses in the mouse hippocampus, we used a combination of paired whole-cell patch-clamp recording, liquid chromatography/tandem mass spectrometry, stochastic optical reconstruction microscopy super-resolution imaging, and immunogold electron microscopy. Unexpectedly, application of the CB1 antagonist and inverse agonist AM251 [N-1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide], but not the neutral antagonist NESS0327 [8-chloro-1-(2,4-dichlorophenyl)-N-piperidin-1-yl-5,6-dihydro-4H-benzo[2,3]cyclohepta[2,4-b]pyrazole-3-carboxamine], significantly increased synaptic transmission between CB1-positive perisomatic interneurons and CA1 pyramidal neurons. JZL184 (4-nitrophenyl 4-[bis(1,3-benzodioxol-5-yl)(hydroxy)methyl]piperidine-1-carboxylate), a selective inhibitor of monoacylglycerol lipase (MGL), the presynaptic degrading enzyme of the endocannabinoid 2-arachidonoylglycerol (2-AG), elicited a robust increase in 2-AG levels and concomitantly decreased GABAergic transmission. In contrast, inhibition of fatty acid amide hydrolase (FAAH) by PF3845 (N-pyridin-3-yl-4-[[3-[5-(trifluoromethyl)pyridin-2-yl]oxyphenyl]methyl]piperidine-1-carboxamide) elevated endocannabinoid/endovanilloid anandamide levels but did not change GABAergic synaptic activity. However, FAAH inhibitors attenuated tonic 2-AG increase and also decreased its synaptic effects. This antagonistic interaction required the activation of the transient receptor potential vanilloid receptor TRPV1, which was concentrated on postsynaptic intracellular membrane cisternae at perisomatic GABAergic symmetrical synapses. Interestingly, neither AM251, JZL184, nor PF3845 affected CB1-positive dendritic interneuron synapses. Together, these findings are consistent with the possibility that constitutively active CB1 receptors substantially influence perisomatic GABA release probability and indicate that the synaptic effects of tonic 2-AG release are tightly controlled by presynaptic MGL activity and also by postsynaptic endovanilloid signaling and. Tonic cannabinoid signaling plays a critical role in the regulation of synaptic transmission. However, the mechanistic details of how persistent CB1 cannabinoid receptor activity inhibits neurotransmitter release have remained elusive. Therefore, electrophysiological recordings, lipid measurements, and super-resolution imaging were combined to elucidate those signaling molecules and mechanisms that underlie tonic cannabinoid signaling. The findings indicate that constitutive CB1 activity has pivotal function in the tonic control of hippocampal GABA release. Moreover, the endocannabinoid 2-arachidonoylglycerol (2-AG) is continuously generated postsynaptically, but its synaptic effect is regulated strictly by presynaptic monoacylglycerol lipase activity. Finally, anandamide signaling antagonizes tonic 2-AG signaling via activation of postsynaptic transient receptor potential vanilloid TRPV1 receptors. This unexpected mechanistic diversity may be necessary to fine-tune GABA release probability under various physiological and pathophysiological conditions.

Topics: Animals; Arachidonic Acids; Cannabinoid Receptor Modulators; Endocannabinoids; Enzyme Inhibitors; Female; gamma-Aminobutyric Acid; Glycerides; Hippocampus; Inhibitory Postsynaptic Potentials; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neurons; Piperidines; Pyrazoles; Pyridines; Receptor, Cannabinoid, CB1; Signal Transduction; Synapses; TRPV Cation Channels

Inflammatory pain presents a problem of clinical relevance and often elicits allodynia, a condition in which non-noxious stimuli are perceived as painful. One potential target to treat inflammatory pain is the endogenous cannabinoid (endocannabinoid) system, which is comprised of CB1 and CB2 cannabinoid receptors and several endogenous ligands, including anandamide (AEA). Blockade of the catabolic enzyme fatty acid amide hydrolase (FAAH) elevates AEA levels and elicits antinociceptive effects, without the psychomimetic side effects associated with Δ(9) -tetrahydrocannabinol (THC).. Allodynia was induced by intraplantar injection of LPS. Complementary genetic and pharmacological approaches were used to determine the strategy of blocking FAAH to reverse LPS-induced allodynia. Endocannabinoid levels were quantified using mass spectroscopy analyses.. FAAH (-/-) mice or wild-type mice treated with FAAH inhibitors (URB597, OL-135 and PF-3845) displayed an anti-allodynic phenotype. Furthermore, i.p. PF-3845 increased AEA levels in the brain and spinal cord. Additionally, intraplantar PF-3845 produced a partial reduction in allodynia. However, the anti-allodynic phenotype was absent in mice expressing FAAH exclusively in the nervous system under a neural specific enolase promoter, implicating the involvement of neuronal fatty acid amides (FAAs). The anti-allodynic effects of FAAH-compromised mice required activation of both CB1 and CB2 receptors, but other potential targets of FAA substrates (i.e. µ-opioid, TRPV1 and PPARα receptors) had no apparent role.. AEA is the primary FAAH substrate reducing LPS-induced tactile allodynia. Blockade of neuronal FAAH reverses allodynia through the activation of both cannabinoid receptors and represents a promising target to treat inflammatory pain.. This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue-7.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Brain; Endocannabinoids; Enzyme Inhibitors; Female; Glycerides; Hyperalgesia; Inflammation; Lipopolysaccharides; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Peripheral Nervous System; Piperidines; Polyunsaturated Alkamides; Pyridines; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Spinal Cord