ABSTRACT
Extracellular adenosine 5'-triphosphate (ATP) acts as an autocrine and paracrine agent, the actions of which on affected cells are mediated by P2 receptors (P2R), which include trans cell-membrane cationic channels (P2XRs), and G protein coupled receptors (P2YRs). The mammalian P2X receptors form homotrimeric or heterotrimeric cationic channels, each of which contains three ATP-binding sites. There are seven homotrimeric P2X receptors (P2X1-7) and three heteromeric (P2X2/P2X3, P2X4/P2X6, P2X1/P2X5). In the lungs and airways, ATP activates P2X3 and P2X2/3 receptors (P2X3R, P2X2/3R, respectively) localized on vagal sensory nerve terminals resulting in bronchoconstriction, and cough, and probably also localized release of pro-inflammatory neuropeptides via the axon reflex. Currently, several P2X3R and P2X2/3R antagonists are being developed as drug-candidates for the treatment of chronic cough. This report presents the receptor affinity data of a novel water-soluble small molecule, DT-0111, that acts as a selective P2X3R antagonist.
Subject(s)
Cough , Receptors, Purinergic P2X3 , Animals , Purinergic P2X Receptor Antagonists/pharmacology , Adenosine Triphosphate/metabolism , Lung/metabolism , Receptors, Purinergic P2X2 , Mammals/metabolismABSTRACT
Nicotine, the primary psychoactive component in tobacco, plays a major role in the initiation and maintenance of tobacco dependence and addiction, a leading cause of preventable death worldwide. An essential need thus exists for more effective pharmacotherapies for nicotine-use cessation. Previous reports suggest that pharmacological and genetic blockade of CB1 receptors attenuate nicotine reinforcement and reward; while exogenous agonists enhanced these abuse-related behaviors. In this study, we utilized complementary genetic and pharmacologic approaches to test the hypothesis that increasing the levels of the endocannabinoid 2-arachindonoylglycerol (2-AG), will enhance nicotine reward by stimulating neuronal CB1 receptors. Contrary to our hypothesis, we found that inhibition of monoacylglycerol lipase (MAGL), the primary catabolic enzyme of 2-AG, attenuates nicotine conditioned place preference (CPP) in mice, through a non-CB1 receptor-mediated mechanism. MAGL inhibition did not alter palatable food reward or Lithium Chloride (LiCl) aversion. In support of our findings, repeated MAGL inhibition did not induce a reduction in CB1 brain receptor levels or hinder function. To explore the potential mechanism of action, we investigated if MAGL inhibition affected other fatty acid levels in our CPP paradigm. Indeed, MAGL inhibition caused a concomitant decrease in arachidonic acid (AA) levels in various brain regions of interest, suggesting an AA cascade-dependent mechanism. This idea is supported by dose-dependent attenuation of nicotine preference by the selective COX-2 inhibitors valdecoxib and LM-4131. Collectively, these findings, along with our reported studies on nicotine withdrawal, suggest that inhibition of MAGL represents a promising new target for the development of pharmacotherapies to treat nicotine dependence.
Subject(s)
Conditioning, Classical/drug effects , Monoacylglycerol Lipases/antagonists & inhibitors , Monoacylglycerol Lipases/metabolism , Nicotine/administration & dosage , Reward , Tobacco Use Disorder/metabolism , Animals , Anti-Anxiety Agents/pharmacology , Arachidonic Acids/pharmacology , Benzodioxoles/pharmacology , Cannabinoid Receptor Agonists/pharmacology , Conditioning, Classical/physiology , Endocannabinoids/pharmacology , Enzyme Inhibitors/pharmacology , Glycerides/pharmacology , Male , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Inbred ICR , Mice, Knockout , Piperidines/pharmacology , Receptor, Cannabinoid, CB1/agonists , Receptor, Cannabinoid, CB1/metabolism , Tobacco Use Disorder/drug therapy , Tobacco Use Disorder/psychologyABSTRACT
BACKGROUND AND PURPOSE: Neuropathic pain is commonly treated with GABA analogues, steroids or non-steroidal anti-inflammatory drugs (NSAIDs). NSAIDs inhibit one or more COX isozymes but chronic COX inhibition paradoxically increases gastrointestinal inflammation and risk of unwanted cardiovascular events. The cannabinoids also have analgesic and anti-inflammatory properties and reduce neuropathic pain in animal models. The present study investigated the analgesic effects of inhibiting both monoacylglycerol lipase (MAGL) and COX enzymes, using low doses of both inhibitors. EXPERIMENTAL APPROACH: Mice subjected to chronic constriction injury (CCI) were tested for mechanical and cold allodynia after administration of the MAGL inhibitor, JZL184, or the non-selective COX inhibitor diclofenac. Then, both drugs were co-administered at fixed dose proportions of 1:3, 1:1 and 3:1, based on their ED50 values. PGs, endocannabinoids and related lipids were quantified in lumbar spinal cord. KEY RESULTS: Combining low doses of JZL184 and diclofenac synergistically attenuated mechanical allodynia and additively reduced cold allodynia. The cannabinoid CB1 receptor antagonist, rimonabant, but not the CB2 receptor antagonist, SR144528, blocked the analgesic effects of the JZL184 and diclofenac combination on mechanical allodynia, implying that CB1 receptors were primarily responsible for the anti-allodynia. Diclofenac alone and with JZL184 significantly reduced PGE2 and PGF2α in lumbar spinal cord tissue, whereas JZL184 alone caused significant increases in the endocannabinoid metabolite, N-arachidonoyl glycine. CONCLUSIONS AND IMPLICATIONS: Combining COX and MAGL inhibition is a promising therapeutic approach for reducing neuropathic pain with minimal side effects.
Subject(s)
Analgesics/therapeutic use , Benzodioxoles/therapeutic use , Cyclooxygenase Inhibitors/therapeutic use , Diclofenac/therapeutic use , Monoacylglycerol Lipases/antagonists & inhibitors , Neuralgia/drug therapy , Piperidines/therapeutic use , Animals , Cannabinoid Receptor Antagonists/pharmacology , Drug Synergism , Drug Therapy, Combination , Eicosanoids/metabolism , Endocannabinoids/metabolism , Fatty Acids/metabolism , Hyperalgesia/drug therapy , Hyperalgesia/metabolism , Male , Mice, Inbred C57BL , Neuralgia/metabolism , Piperidines/pharmacology , Prostaglandin-Endoperoxide Synthases , Pyrazoles/pharmacology , Rimonabant , Spinal Cord/metabolismABSTRACT
BACKGROUND AND PURPOSE: The psychoactive cannabinoid Δ(9) -tetrahydrocannabinol (THC) and the non-psychoactive cannabinoid cannabidiol (CBD) can both reduce cancer progression, each through distinct anti-tumour pathways. Our goal was to discover a compound that could efficiently target both cannabinoid anti-tumour pathways. EXPERIMENTAL APPROACH: To measure breast cancer cell proliferation/viability and invasion, MTT and Boyden chamber assays were used. Modulation of reactive oxygen species (ROS) and apoptosis was measured using dichlorodihydrofluorescein and annexin/propidium iodide, respectively, in combination with cell flow cytometry. Changes in protein levels were evaluated using Western analysis. Orthotopic and i.v. mouse models of breast cancer metastasis were used to test the activity of cannabinoids in vivo. KEY RESULTS: CBD reduced breast cancer metastasis in advanced stages of the disease as the direct result of down-regulating the transcriptional regulator Id1. However, this was associated with moderate increases in survival. We therefore screened for analogues that could co-target cannabinoid anti-tumour pathways (CBD- and THC-associated) and discovered the compound O-1663. This analogue inhibited Id1, produced a marked stimulation of ROS, up-regulated autophagy and induced apoptosis. Of all the compounds tested, it was the most potent at inhibiting breast cancer cell proliferation and invasion in culture and metastasis in vivo. CONCLUSIONS AND IMPLICATIONS: O-1663 prolonged survival in advanced stages of breast cancer metastasis. Developing compounds that can simultaneously target multiple cannabinoid anti-tumour pathways efficiently may provide a novel approach for the treatment of patients with metastatic breast cancer.
Subject(s)
Antineoplastic Agents/therapeutic use , Breast Neoplasms/drug therapy , Cannabidiol/therapeutic use , Lung Neoplasms/drug therapy , Resorcinols/therapeutic use , Animals , Antineoplastic Agents/pharmacology , Breast Neoplasms/pathology , Cannabidiol/pharmacology , Cell Line, Tumor , Female , Humans , Inhibitor of Differentiation Protein 1/metabolism , Lung Neoplasms/secondary , Mice, Inbred BALB C , Mice, Nude , Reactive Oxygen Species/metabolism , Receptor, Cannabinoid, CB2/agonists , Receptor, Cannabinoid, CB2/metabolism , Resorcinols/pharmacologyABSTRACT
BACKGROUND AND PURPOSE: The development of potent and selective inhibitors of the biosynthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG) via DAG lipases (DAGL) α and ß is just starting to be considered as a novel and promising source of pharmaceuticals for the treatment of disorders that might benefit from a reduction in endocannabinoid tone, such as hyperphagia in obese subjects. EXPERIMENTAL APPROACH: Three new fluorophosphonate compounds O-7458, O-7459 and O-7460 were synthesized and characterized in various enzymatic assays. The effects of O-7460 on high-fat diet intake were tested in mice. KEY RESULTS: Of the new compounds, O-7460 exhibited the highest potency (IC50 = 690 nM) against the human recombinant DAGLα, and selectivity (IC50 > 10 µM) towards COS-7 cell and human monoacylglycerol lipase (MAGL), and rat brain fatty acid amide hydrolase. Competitive activity-based protein profiling confirmed that O-7460 inhibits mouse brain MAGL only at concentrations ≥ 10 µM, and showed that this compound has only one major 'off-target', that is, the serine hydrolase KIAA1363. O-7460 did not exhibit measurable affinity for human recombinant CB1 or CB2 cannabinoid receptors (Ki > 10 µM). In mouse neuroblastoma N18TG2 cells stimulated with ionomycin, O-7460 (10 µM) reduced 2-AG levels. When administered to mice, O-7460 dose-dependently (0-12 mg·kg⻹, i.p.) inhibited the intake of a high-fat diet over a 14 h observation period, and, subsequently, slightly but significantly reduced body weight. CONCLUSIONS AND IMPLICATIONS: O-7460 might be considered a useful pharmacological tool to investigate further the role played by 2-AG both in vitro and in vivo under physiological as well as pathological conditions.
Subject(s)
Anti-Obesity Agents/therapeutic use , Arachidonic Acids/antagonists & inhibitors , Endocannabinoids/antagonists & inhibitors , Enzyme Inhibitors/therapeutic use , Glycerides/antagonists & inhibitors , Glycerophospholipids/therapeutic use , Lipoprotein Lipase/antagonists & inhibitors , Obesity/drug therapy , Oleic Acids/therapeutic use , Organophosphonates/therapeutic use , Animals , Anti-Obesity Agents/administration & dosage , Anti-Obesity Agents/pharmacology , Arachidonic Acids/metabolism , Behavior, Animal/drug effects , Cell Line , Chlorocebus aethiops , Dose-Response Relationship, Drug , Endocannabinoids/metabolism , Energy Intake/drug effects , Enzyme Inhibitors/pharmacology , Glycerides/metabolism , Glycerophospholipids/administration & dosage , Glycerophospholipids/pharmacology , Humans , Hypothalamus/drug effects , Hypothalamus/enzymology , Hypothalamus/metabolism , Lipoprotein Lipase/genetics , Lipoprotein Lipase/metabolism , Liver/drug effects , Liver/enzymology , Liver/metabolism , Male , Mice , Mice, Inbred C57BL , Nerve Tissue Proteins/antagonists & inhibitors , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Neurons/drug effects , Neurons/enzymology , Neurons/metabolism , Obesity/enzymology , Obesity/metabolism , Oleic Acids/administration & dosage , Oleic Acids/pharmacology , Organophosphonates/administration & dosage , Organophosphonates/pharmacology , Rats , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sterol Esterase/antagonists & inhibitors , Sterol Esterase/metabolismABSTRACT
AIM: The present study tested whether the selective monoacylglycerol lipase (MAGL) inhibitor JZL184 would reduce allodynia and paw edema in the carrageenan test. MAIN METHODS: The anti-edematous and anti-allodynic effects of JZL184 were compared to those of PF-3845, an inhibitor of fatty acid amide hydrolase (FAAH), and diclofenac, a non-selective cyclooxygenase inhibitor. Cannabinoid receptor involvement in the anti-edematous and anti-allodynic effects of JZL184 was evaluated by administration of the respective CB1 and CB2 receptor antagonists rimonabant and SR144528 as well as with CB1(-/-) and CB2(-/-) mice. JZL184 (1.6, 4, 16, or 40mg/kg) was administered for six days to assess tolerance. KEY FINDINGS: JZL184 administered before or after carrageenan significantly attenuated carrageenan-induced paw edema and mechanical allodynia. Complementary genetic and pharmacological approaches revealed that the anti-allodynic effects of JZL184 required both CB1 and CB2 receptors, but only CB2 receptors mediated its anti-edematous actions. Importantly, both the anti-edematous and anti-allodynic effects underwent tolerance following repeated injections of high dose JZL184 (16 or 40mg/kg), but repeated administration of low dose JZL184 (4mg/kg) retained efficacy. SIGNIFICANCE: These results suggest that the MAGL inhibitor JZL184 reduces inflammatory nociception through the activation of both CB1 and CB2 receptors, with no evidence of tolerance following repeated administration of low doses.
Subject(s)
Benzodioxoles/pharmacology , Inflammation/drug therapy , Monoacylglycerol Lipases/antagonists & inhibitors , Pain/drug therapy , Piperidines/pharmacology , Amidohydrolases/antagonists & inhibitors , Animals , Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Carrageenan , Diclofenac/therapeutic use , Edema/chemically induced , Edema/drug therapy , Hyperalgesia/chemically induced , Hyperalgesia/drug therapy , Inflammation/chemically induced , Inflammation/pathology , Mice , Mice, Inbred C57BL , Mice, Knockout , Pain/chemically induced , Pain/pathology , Pain Measurement/drug effects , Physical Stimulation , Pyridines/pharmacology , Receptor, Cannabinoid, CB1/antagonists & inhibitors , Receptor, Cannabinoid, CB1/genetics , Receptor, Cannabinoid, CB2/antagonists & inhibitors , Receptor, Cannabinoid, CB2/geneticsABSTRACT
Obesity contributes to a multitude of serious health problems. Given the demonstrated role of the endogenous cannabinoid system in appetite regulation, the purpose of the present study was to evaluate structural analogs of two cannabinoids, rimonabant (cannabinoid CB(1) receptor antagonist) and O-2050 (sulfonamide analog of Δ(8)-tetrahydrocannabinol), that showed appetite suppressant effects in previous studies. Structure-activity relationships of these two lead compounds were examined in several assays, including cannabinoid CB(1) and CB(2) receptor binding, food intake, and an in vivo test battery (locomotor activity, antinociception, ring immobility, and body temperature) in mice. Rimonabant and O-2050 reliably decreased feeding in mice; however, their analogs decreased feeding only at higher doses, even though some compounds had quite good cannabinoid CB(1) binding affinity. Results of the in vivo test battery were inconsistent, with some of the compounds producing effects characteristic of cannabinoid agonists while other compounds were inactive or were antagonists against an active dose of Δ(9)-tetrahydrocannabinol. These results demonstrate that reduction of food intake is not a characteristic effect of pyrazole and sulfonamide cannabinoid analogs with favorable cannabinoid CB(1) binding affinity, suggesting that development of these classes of cannabinoids for the treatment of obesity will require evaluation of their effects in a broad spectrum of pharmacological assays.
Subject(s)
Dronabinol/analogs & derivatives , Eating/drug effects , Piperidines/chemistry , Piperidines/pharmacology , Pyrans/chemistry , Pyrans/pharmacology , Pyrazoles/chemistry , Pyrazoles/pharmacology , Animals , Dronabinol/chemistry , Dronabinol/metabolism , Dronabinol/pharmacology , Male , Mice , Mice, Inbred ICR , Piperidines/metabolism , Pyrans/metabolism , Pyrazoles/metabolism , Receptor, Cannabinoid, CB1/metabolism , Receptor, Cannabinoid, CB2/metabolism , RimonabantABSTRACT
Previous studies have shown that modulation of the receptor-mediated cannabinoid system during neuroinflammation can produce potent neuroprotective and anti-inflammatory effects. However, in this context, little is known about how selective activation of the cannabinoid type-2 receptor (CB2R) affects the activated state of the brain endothelium and blood-brain barrier (BBB) function. Using human brain tissues and primary human brain microvascular endothelial cells (BMVECs), we demonstrate that the CB2R is highly upregulated during inflammatory insult. We then examined whether the CB2R agonists could attenuate inflammatory responses at the BBB using a mouse model of LPS-induced encephalitis and highly selective CB2R agonists. Visualization by intravital microscopy revealed that administration of JWH133 [(6aR,10aR)-3-(1,1-dimethylbutyl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran] or a novel resorcinol-based compound, O-1966 (1-[4-(1,1-dimethyl-heptyl)-2,6-dimethoxy-phenyl]-3-methyl-cyclohexanol), greatly attenuated leukocyte adhesion in surface pial vessels and in deep ascending cortical postcapillary venules. BBB permeability assessments with small and large fluorescent tracers showed that CB2R agonists were effective at preventing barrier leakiness after LPS administration. To determine whether the effects by CB2R agonists on barrier protection are not only due to the CB2R modulation of immune cell function, we tested the agonists in vitro with barrier-forming primary BMVECs. Remarkably, the addition of CB2R agonist increased transendothelial electrical resistance and increased the amount of tight junction protein present in membrane fractions. Furthermore, CB2R agonists decreased the induction of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 surface expression in BMVECs exposed to various proinflammatory mediators. Together, these results suggest that pharmacological CB2R ligands offer a new strategy for BBB protection during neuroinflammation.
Subject(s)
Blood-Brain Barrier/physiopathology , Encephalitis/pathology , Endothelial Cells/physiology , Leukocytes/physiology , Receptor, Cannabinoid, CB2/metabolism , Animals , Anisoles/pharmacology , Anisoles/therapeutic use , Blood-Brain Barrier/drug effects , Camphanes/pharmacology , Cannabinoids/pharmacology , Capillary Permeability/drug effects , Capillary Permeability/physiology , Cell Adhesion/drug effects , Cell Adhesion/genetics , Cells, Cultured , Cyclohexanols , Dextrans/metabolism , Disease Models, Animal , Electric Impedance , Encephalitis/chemically induced , Endothelium/metabolism , Flow Cytometry , Fluorescein-5-isothiocyanate/analogs & derivatives , Fluorescein-5-isothiocyanate/metabolism , Gene Expression Regulation/drug effects , Gene Expression Regulation/genetics , Humans , Intercellular Adhesion Molecule-1/metabolism , Lipopolysaccharides/adverse effects , Membrane Proteins/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Phosphoproteins/metabolism , Platelet Endothelial Cell Adhesion Molecule-1/metabolism , Pyrazoles/pharmacology , Receptor, Cannabinoid, CB2/agonists , Receptor, Cannabinoid, CB2/antagonists & inhibitors , Receptor, Cannabinoid, CB2/deficiency , Statistics, Nonparametric , Tumor Necrosis Factor-alpha/pharmacology , Vascular Cell Adhesion Molecule-1/metabolism , Zonula Occludens-1 ProteinABSTRACT
BACKGROUND AND PURPOSE: Activation of cannabinoid CB(2) receptors protects against various forms of ischaemia-reperfusion (I/R) injury. Δ(8) -Tetrahydrocannabivarin (Δ(8) -THCV) is a synthetic analogue of the plant cannabinoid Δ(9) -tetrahydrocannabivarin, which exhibits anti-inflammatory effects in rodents involving activation of CB(2) receptors. Here, we assessed effects of Δ(8) -THCV and its metabolite 11-OH-Δ(8) -THCV on CB(2) receptors and against hepatic I/R injury. EXPERIMENTAL APPROACH: Effects in vitro were measured with human CB(2) receptors expressed in CHO cells. Hepatic I/R injury was assessed in mice with 1h ischaemia and 2, 6 or 24h reperfusion in vivo. KEY RESULTS: Displacement of [(3) H]CP55940 by Δ(8) -THCV or 11-OH-Δ(8) -THCV from specific binding sites in CHO cell membranes transfected with human CB(2) receptors (hCB(2) ) yielded K(i) values of 68.4 and 59.95 nM respectively. Δ(8) -THCV or 11-OH-Δ(8) -THCV inhibited forskolin-stimulated cAMP production by hCB(2) CHO cells (EC(50) = 12.95 and 14.3 nM respectively). Δ(8) -THCV, given before induction of I/R, attenuated hepatic injury (measured by serum alanine aminotransferase and aspartate aminotransferase levels), decreased tissue protein carbonyl adducts, 4-hydroxy-2-nonenal, the chemokines CCL3 and CXCL2,TNF-α, intercellular adhesion molecule 1 (CD54) mRNA levels, tissue neutrophil infiltration, caspase 3/7 activity and DNA fragmentation. Protective effects of Δ(8) -THCV against liver damage were still present when the compound was given at the beginning of reperfusion. Pretreatment with a CB(2) receptor antagonist attenuated the protective effects of Δ(8) -THCV, while a CB(1) antagonist tended to enhance it. CONCLUSIONS AND IMPLICATIONS: Δ(8) -THCV activated CB(2) receptors in vitro, and decreased tissue injury and inflammation in vivo, associated with I/R partly via CB(2) receptor activation. LINKED ARTICLES: 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.
Subject(s)
Dronabinol/analogs & derivatives , Dronabinol/therapeutic use , Protective Agents/therapeutic use , Receptor, Cannabinoid, CB2/agonists , Reperfusion Injury/drug therapy , Alanine Transaminase/blood , Aldehydes/metabolism , Animals , Apoptosis/drug effects , Aspartate Aminotransferases/blood , CHO Cells , Cricetinae , Cricetulus , Cytokines/genetics , DNA Fragmentation , Dronabinol/metabolism , Humans , Inflammation/drug therapy , Inflammation/metabolism , Inflammation/pathology , Liver/drug effects , Liver/metabolism , Liver/pathology , Male , Mice , Mice, Inbred C57BL , Oxidative Stress/drug effects , Protective Agents/metabolism , RNA, Messenger/metabolism , Receptor, Cannabinoid, CB2/metabolism , Reperfusion Injury/metabolism , Reperfusion Injury/pathologyABSTRACT
We reported previously that both a cannabinoid receptor 2 (CB2R) agonist and a cannabinoid receptor 1 (CB1R) antagonist were protective in the treatment of transient middle cerebral artery occlusion/reperfusion injury (MCAO/R) and that they acted in a synergistic manner when administered in combination. The goal of the current study was to determine which of the potential cannabinoid receptors participate in the protective effects of this drug combination in a mouse model of MCAO/R. The effects of administration of the CB2R agonist/CB1R antagonist combination on infarct size and cerebral blood flow during a 1-h occlusion were tested in CB1R-deficient animals, CB2R-deficient animals, and animals treated with capsazepine, the antagonist for the vanilloid receptor type I (TRPV1) and WAY100135, the antagonist for the hydroxytryptamine1A receptor (5-HT1A). The protective effect of the CB2R agonist/CB1R antagonist combination on infarct size was not influenced by the absence of the CB1R nor by blocking the TRPV1 receptor, but was attenuated by the absence of CB2R and by blocking the 5-HT1A receptor. Increases in cerebral blood flow and arteriolar diameter were also found to be independent of the CB1R and TRPV1 receptor. In conclusion, administration of the CB2R agonist/CB1R antagonist combination causes a significant reduction in infarct size in the MCAO/R model. The protective effect involves both the CB2R and the 5-HT1A receptor. Neither the CB1R nor the TRPV1 receptors appear to participate in this response.
ABSTRACT
Nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly used analgesics, but can cause gastric and esophageal hemorrhages, erosion, and ulceration. The endogenous cannabinoid (endocannabinoid; eCB) system possesses several potential targets to reduce gastric inflammatory states, including cannabinoid receptor type 1 (CB(1)), cannabinoid receptor type 2 (CB(2)), and enzymes that regulate the eCB ligands 2-arachidonoylglycerol (2-AG) and N-arachidonoyl ethanolamine (anandamide; AEA). In the presented study, we tested whether 4-nitrophenyl 4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate (JZL184), a selective inhibitor of the primary catabolic enzyme of 2-AG, monoacylglycerol lipase (MAGL), would protect against NSAID-induced gastric damage. Food-deprived mice administered the nonselective cyclooxygenase inhibitor diclofenac sodium displayed gastric hemorrhages and increases in proinflammatory cytokines. JZL184, the proton pump inhibitor omeprazole (positive control), or the primary constituent of marijuana, Δ(9)-tetrahydrocannabinol (THC), significantly prevented diclofenac-induced gastric hemorrhages. JZL184 also increased stomach levels of 2-AG, but had no effect on AEA, arachidonic acid, or the prostaglandins E(2) and D(2). MAGL inhibition fully blocked diclofenac-induced increases in gastric levels of proinflammatory cytokines interleukin (IL)-1ß, IL-6, tumor necrosis factor α, and granulocyte colony-stimulating factor, as well as IL-10. Pharmacological inhibition or genetic deletion of CB(1) or CB(2) revealed that the gastroprotective effects of JZL184 and THC were mediated via CB(1). The antihemorrhagic effects of JZL184 persisted with repeated administration, indicating a lack of tolerance. These data indicate that increasing 2-AG protects against gastric damage induced by NSAIDs, and its primary catabolic enzyme MAGL offers a promising target for the development of analgesic therapeutics possessing gastroprotective properties.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal , Enzyme Inhibitors/therapeutic use , Gastrointestinal Hemorrhage/chemically induced , Gastrointestinal Hemorrhage/prevention & control , Monoacylglycerol Lipases/antagonists & inhibitors , Amidohydrolases/antagonists & inhibitors , Animals , Benzodioxoles/pharmacology , Cannabinoid Receptor Modulators/metabolism , Cytokines/metabolism , Diclofenac , Dronabinol/pharmacology , Enzyme-Linked Immunosorbent Assay , Food Deprivation , Gastric Mucosa/metabolism , Gastrointestinal Hemorrhage/pathology , Male , Mice , Mice, Inbred C57BL , Omeprazole/pharmacology , Piperidines/pharmacology , Prostaglandins/metabolism , Pyridines/pharmacology , Receptor, Cannabinoid, CB1/drug effects , Receptor, Cannabinoid, CB2/drug effects , Stomach/drug effects , Stomach/pathologyABSTRACT
Rimonabant, the prototypic antagonist of cannabinoid CB(1) receptors, has been reported to have inverse agonist properties at higher concentrations, which may complicate its use as a tool for mechanistic evaluation of cannabinoid pharmacology. Consequently, recent synthesis efforts have concentrated on discovery of a neutral antagonist using a variety of structural templates. The purpose of this study was to evaluate the pharmacological properties of the putative neutral cannabinoid CB(1) receptor antagonist O-2050, a sulfonamide side chain analog of Δ(8)-tetrahydrocannabinol. O-2050 and related sulfonamide cannabinoids exhibited good affinity for both cannabinoid CB(1) and CB(2) receptors. While the other sulfonamide analogs produced cannabinoid agonist effects in vivo (e.g., activity suppression, antinociception, and hypothermia), O-2050 stimulated activity and was inactive in the other two tests. O-2050 also decreased food intake in mice, an effect that was reminiscent of that produced by rimonabant. Unlike rimonabant, however, O-2050 did not block the effects of cannabinoid agonists in vivo, even when administered i.c.v. In contrast, O-2050 antagonized the in vitro effects of cannabinoid agonists in [(35)S]GTPγS and mouse vas deferens assays without having activity on its own in either assay. Further evaluation revealed that O-2050 fully and dose-dependently substituted for Δ(9)-tetrahydrocannabinol in a mouse drug discrimination procedure (a cannabinoid agonist effect) and that it inhibited forskolin-stimulated cyclic AMP signaling with a maximum efficacy of approximately half that of the full agonist CP55,940 [(-)-cis-3-[2-hydroxy-4(1,1-dimethyl-heptyl)phenyl]-trans-4-(3-hydroxy-propyl)cyclohexanol]. Together, these results suggest that O-2050 is not a viable candidate for classification as a neutral cannabinoid CB(1) receptor antagonist.
Subject(s)
Dronabinol/analogs & derivatives , Pyrans/chemistry , Pyrans/pharmacology , Receptor, Cannabinoid, CB1/antagonists & inhibitors , Animals , Cyclic AMP/metabolism , Dronabinol/chemistry , Dronabinol/metabolism , Dronabinol/pharmacology , Guanosine 5'-O-(3-Thiotriphosphate)/metabolism , Male , Mice , Pyrans/metabolism , Receptor, Cannabinoid, CB1/metabolism , Vas Deferens/drug effects , Vas Deferens/metabolismABSTRACT
Although Δ(9)-tetrahydrocannabinol (THC) and other mixed CB(1)/CB(2) receptor agonists are well established to elicit antinociceptive effects, their psychomimetic actions and potential for abuse have dampened enthusiasm for their therapeutic development. Conversely, CB(2) receptor-selective agonists have been shown to reduce pain and inflammation, without eliciting apparent cannabinoid behavioral effects. In the present study, we developed a novel ethyl sulfonamide THC analog, O-3223, and compared its pharmacological effects to those of the potent, mixed CB(1)/CB(2) receptor agonist, CP55,940, in a battery of preclinical pain models. Competitive cannabinoid receptor binding experiments revealed that O-3223 was approximately 80-fold more selective for CB(2) than CB(1) receptors. Additionally, O-3223 behaved as a full CB(2) receptor agonist in [(35)S]GTPγS binding. O-3223 reduced nociceptive behavior in both phases of the formalin test, reduced thermal hyperalgesia in the chronic constriction injury of the sciatic nerve (CCI) model, and reduced edema and thermal hyperalgesia elicited by intraplantar injection of LPS. These effects were blocked by pretreatment with the CB(2) receptor-selective antagonist SR144528, but not by the CB(1) receptor antagonist, rimonabant. Unlike CP55,940, O-3223 did not elicit acute antinociceptive effects in the hot-plate test, hypothermia, or motor disturbances, as assessed in the rotarod test. These data indicate that the CB(2) receptor-selective agonist, O-3223, reduces inflammatory and neuropathic nociception, without affecting basal nociception or eliciting overt behavioral effects. Moreover, this compound can serve as a template to develop new CB(2) receptor agonists with increased receptor selectivity and increased potency in treating inflammatory and neuropathic pain.
Subject(s)
Cannabinoids/therapeutic use , Dronabinol/analogs & derivatives , Dronabinol/therapeutic use , Pain/drug therapy , Receptor, Cannabinoid, CB2/agonists , Animals , Cannabinoids/pharmacology , Cyclohexanols/pharmacology , Cyclohexanols/therapeutic use , Dose-Response Relationship, Drug , Dronabinol/chemistry , Dronabinol/pharmacology , Inflammation/drug therapy , Inflammation/metabolism , Inflammation/psychology , Male , Mice , Pain/physiopathology , Pain Measurement , Piperidines/pharmacology , Pyrazoles/pharmacology , Receptor, Cannabinoid, CB2/antagonists & inhibitors , Receptor, Cannabinoid, CB2/physiology , RimonabantABSTRACT
Considerable preclinical research has demonstrated the efficacy of Delta(9)-tetrahydrocannabinol (Delta(9)-THC), the primary psychoactive constituent of Cannabis sativa, in a wide variety of animal models of pain, but few studies have examined other phytocannabinoids. Indeed, other plant-derived cannabinoids, including cannabidiol (CBD), cannabinol (CBN), and cannabichromene (CBC) elicit antinociceptive effects in some assays. In contrast, tetrahydrocannabivarin (THCV), another component of cannabis, antagonizes the pharmacological effects of Delta(9)-THC. These results suggest that various constituents of this plant may interact in a complex manner to modulate pain. The primary purpose of the present study was to assess the antinociceptive effects of these other prevalent phytocannabinoids in the acetic acid stretching test, a rodent visceral pain model. Of the cannabinoid compounds tested, Delta(9)-THC and CBN bound to the CB(1) receptor and produced antinociceptive effects. The CB(1) receptor antagonist, rimonabant, but not the CB(2) receptor antagonist, SR144528, blocked the antinociceptive effects of both compounds. Although THCV bound to the CB(1) receptor with similar affinity as Delta(9)-THC, it had no effects when administered alone, but antagonized the antinociceptive effects of Delta(9)-THC when both drugs were given in combination. Importantly, the antinociceptive effects of Delta(9)-THC and CBN occurred at lower doses than those necessary to produce locomotor suppression, suggesting motor dysfunction did not account for the decreases in acetic acid-induced abdominal stretching. These data raise the intriguing possibility that other constituents of cannabis can be used to modify the pharmacological effects of Delta(9)-THC by either eliciting antinociceptive effects (i.e., CBN) or antagonizing (i.e., THCV) the actions of Delta(9)-THC.
Subject(s)
Acetic Acid , Analgesics , Cannabinoids/pharmacology , Pain Measurement/drug effects , Pain/prevention & control , Analgesics/pharmacology , Animals , Anti-Obesity Agents/pharmacology , Camphanes/pharmacology , Cannabinoids/antagonists & inhibitors , Cyclohexanols/pharmacology , Dose-Response Relationship, Drug , Dronabinol/pharmacology , Male , Mice , Mice, Inbred ICR , Motor Activity/drug effects , Pain/chemically induced , Piperidines/pharmacology , Pyrazoles/pharmacology , RNA, Messenger/biosynthesis , Receptor, Cannabinoid, CB1/antagonists & inhibitors , Receptor, Cannabinoid, CB1/drug effects , Receptor, Cannabinoid, CB1/metabolism , Receptor, Cannabinoid, CB2/antagonists & inhibitors , Receptor, Cannabinoid, CB2/drug effects , Receptor, Cannabinoid, CB2/metabolism , RimonabantABSTRACT
Biosynthesis Inhibition: O-5596, a new inhibitor of the biosynthesis of the endocannabinoid, 2-arachidonoylglycerol, was synthesized and found to be potent (IC(50)=100 nM) and selective versus other proteins and enzymes of the endocannabinoid system in vitro and active in vivo at reducing food intake in mice.
Subject(s)
Alkenes/chemical synthesis , Alkenes/pharmacology , Arachidonic Acids/biosynthesis , Enzyme Inhibitors/chemical synthesis , Glycerides/biosynthesis , Organophosphorus Compounds/chemical synthesis , Organophosphorus Compounds/pharmacology , Alkenes/chemistry , Animals , Arachidonic Acids/chemistry , Cell Line , Drug Stability , Eating/drug effects , Endocannabinoids , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Glycerides/chemistry , Humans , Lipoprotein Lipase/antagonists & inhibitors , Lipoprotein Lipase/metabolism , Mice , Organophosphorus Compounds/chemistry , Receptor, Cannabinoid, CB1/antagonists & inhibitors , Receptor, Cannabinoid, CB1/metabolism , Receptor, Cannabinoid, CB2/antagonists & inhibitors , Receptor, Cannabinoid, CB2/metabolismABSTRACT
Inhibition of the metabolism of the endocannabinoids, anandamide (AEA) and 2-arachidonyl glycerol (2-AG), by their primary metabolic enzymes, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively, has the potential to increase understanding of the physiological functions of the endocannabinoid system. To date, selective inhibitors of FAAH, but not MAGL, have been developed. The purpose of this study was to determine the selectivity and efficacy of N-arachidonyl maleimide (NAM), a putative MAGL inhibitor, for modulation of the effects of 2-AG. Our results showed that NAM unmasked 2-AG activity in a tetrad of in vivo tests sensitive to the effects of cannabinoids in mice. The efficacy of 2-AG (and AEA) to produce hypothermia was reduced compared with Delta(9)-tetrahydrocannabinol; however, 2-AG differed from AEA by its lower efficacy for catalepsy. All tetrad effects were partially CB(1) receptor-mediated because they were attenuated (but not eliminated) by SR141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-H-pyrazole-3-carboxamide HCl] and in CB(1)(-/-) mice. In vitro, NAM increased endogenous levels of 2-AG in the brain. Furthermore, NAM raised the potency of 2-AG, but not AEA, in agonist-stimulated guanosine 5'-O-(3-[(35)S]thio)triphosphate binding assay, a measure of G-protein activation. These results suggest that NAM is an MAGL inhibitor with in vivo and in vitro efficacy. NAM and other MAGL inhibitors are valuable tools to elucidate the biological functions of 2-AG and to examine the consequences of dysregulation of this endocannabinoid. In addition, NAM's unmasking of 2-AG effects that are only partially reversed by SR141716A offers support for the existence of non-CB(1), non-CB(2) cannabinoid receptors.
Subject(s)
Arachidonic Acids/pharmacology , Glycerides/pharmacology , Maleimides/pharmacology , Animals , Drug Synergism , Endocannabinoids , Female , Guanosine 5'-O-(3-Thiotriphosphate)/metabolism , Mice , Mice, Inbred ICR , Monoacylglycerol Lipases/antagonists & inhibitors , Motor Activity/drug effects , Piperidines/pharmacology , Pyrazoles/pharmacology , Receptor, Cannabinoid, CB1/drug effects , Receptor, Cannabinoid, CB1/physiology , RimonabantABSTRACT
Endocannabinoids, including anandamide (arachidonoyl ethanolamide) have been implicated in the regulation of a growing number of physiological and pathological processes. Anandamide can be generated from its membrane phospholipid precursor N-arachidonoyl phosphatidylethanolamine (NAPE) through hydrolysis by a phospholipase D (NAPE-PLD). Recent evidence indicates, however, the existence of two additional, parallel pathways. One involves the sequential deacylation of NAPE by alpha,beta-hydrolase 4 (Abhd4) and the subsequent cleavage of glycerophosphate to yield anandamide, and the other one proceeds through phospholipase C-mediated hydrolysis of NAPE to yield phosphoanandamide, which is then dephosphorylated by phosphatases, including the tyrosine phosphatase PTPN22 and the inositol 5' phosphatase SHIP1. Conversion of synthetic NAPE to AEA by brain homogenates from wild-type and NAPE-PLD(-/-) mice can proceed through both the PLC/phosphatase and Abdh4 pathways, with the former being dominant at shorter (<10 min) and the latter at longer (60 min) incubations. In macrophages, the endotoxin-induced synthesis of anandamide proceeds uniquely through the phospholipase C/phosphatase pathway.
Subject(s)
Arachidonic Acids/biosynthesis , Metabolic Networks and Pathways/physiology , Animals , Cell Line, Transformed , Chromatography, High Pressure Liquid , Chromatography, Thin Layer , Drug Interactions , Endocannabinoids , Glycerophosphates/metabolism , Hydrolases/metabolism , Hydrolysis/drug effects , Inositol Polyphosphate 5-Phosphatases , Lipopolysaccharides/pharmacology , Macrophages/drug effects , Macrophages/metabolism , Metabolic Networks and Pathways/drug effects , Mice , Mice, Knockout , Neomycin/pharmacology , Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases , Phospholipase D/deficiency , Phosphoric Monoester Hydrolases/deficiency , Polyunsaturated Alkamides , Protein Synthesis Inhibitors/pharmacology , Protein Tyrosine Phosphatase, Non-Receptor Type 22/deficiency , RNA, Small Interfering/metabolism , Transfection/methods , Type C Phospholipases/metabolismABSTRACT
The structure-activity relationship (SAR) of the end pentyl chain in anandamide (AEA) has been established to be very similar to that of Delta(9)-tetrahydrocannabinol (Delta(9)-THC). In order to broaden our understanding of the structural similarities between AEA and THC, hybrid structures 1-3 were designed. In these hybrids the aromatic ring of THC-DMH was linked to the AEA moiety through an ether linkage with the oxygen of the phenol of THC. Hybrid 1 (O-2220) was found to have very high binding affinity to CB1 receptors (K(i)=8.5 nM), and it is interesting to note that the orientation of the side chain with respect to the oxygen in the phenol is the same as in THCs. To further explore the SAR in this series the terminal carbon of the side chain was modified by adding different substituents. Several such analogs were synthesized and tested for their CB1 and CB2 binding affinities and in vivo activity (tetrad tests). The details of the synthesis and the biological activity of these compounds are described.
Subject(s)
Arachidonic Acids/chemistry , Cannabinoid Receptor Agonists , Dronabinol/analogs & derivatives , Dronabinol/chemistry , Polyunsaturated Alkamides/chemistry , Animals , Arachidonic Acids/pharmacology , Cell Line , Cells, Cultured , Dronabinol/pharmacology , Drug Evaluation, Preclinical , Endocannabinoids , Humans , Mice , Models, Molecular , Molecular Structure , Polyunsaturated Alkamides/pharmacology , Structure-Activity RelationshipABSTRACT
A facile synthesis of 1-fluoro-1-deoxy-Delta(8)-THC analogs with side chains seven carbons in length, in the alkane/ene/yne- series (6, 5, and 4), was achieved from 1-fluoro-3,5-dimethoxybenzene (1). In vitro studies show that substitution by a fluorine has a significant detrimental effect on CB1 binding which is supported by in vivo testing. The implications of these results on the SAR of classical cannabinoids are discussed.
Subject(s)
Cannabinoids/chemistry , Cannabinoids/pharmacology , Fluorine/chemistry , Animals , Body Temperature/drug effects , Cannabinoids/chemical synthesis , Indicators and Reagents , Mice , Motor Activity/drug effects , Reaction Time/drug effects , Receptor, Cannabinoid, CB1/drug effects , Structure-Activity RelationshipABSTRACT
Presently, there are numerous structural classes of cannabinoid receptor agonists, all of which require solubilization for experimental purposes. One strategy for solubilizing water-insoluble tetrahydrocannabinols is conversion of the phenolic hydroxyl to a morpholinobutyryloxy substituent. The hydrochloride salts of these analogs are water-soluble and active in vivo when administered in saline. The present investigation demonstrated that hydrochloride salts of numerous substituted butyryloxy esters are water-soluble and highly potent. The substitutions include piperidine, piperazine, and alkyl-substituted amino moieties. It was also discovered that incorporation of a nitrogenous moiety in the alkyl side chain increased the pharmacological potency of tetrahydrocannabinol. For example, an analog containing a pyrazole in the side chain (O-2545) was found to have high affinity and efficacy at cannabinoid 1 (CB(1)) and CB(2) receptors, and when dissolved in saline, it was highly efficacious when administered either intravenously or intracerebroventricularly to mice. A series of carboxamido and carboxylic acid amide analogs exhibited high pharmacological potency, but their hydrochloride salts were not water-soluble. On the other hand, incorporation of imidazoles into the terminus of the side chain led to water-soluble hydrochloride salts that were highly potent when administered in saline to laboratory animals. It is now possible to conduct cannabinoid research with agonists that are water-soluble and thus obviating the need of solubilizing agents.