ABSTRACT
The synthetic forms of delta-9-tetrahydrocannabinol (Δ9-THC), dronabinol or nabilone, have been approved to treat several indications. However, due to safety concerns their clinical utility remains limited. Consequently, there is a need for developing cannabinoid (CB) ligands that display better behavioral pharmacological profiles than Δ9-THC. Here, we utilized drug discrimination methods to compare the interoceptive effects of CB ligands that vary in potency, efficacy, and selectivity at the CB receptors, including two ligands, AM411 and AM4089, that show CB1 partial agonist-like actions in vitro. Male rats were trained to discriminate 0.1 mg/kg AM2201 from saline under a fixed-ratio (FR) 10 response schedule of food reinforcement. After establishing AM2201's discriminative-stimulus effects, pretreatment tests with the CB1 antagonist/inverse agonist rimonabant blocked AM2201's effects, whereas the peripherally-restricted antagonist AM6545 had no effect. Next, the generalization profiles of AM411 and AM4089 with CB1 full agonists (JWH-018, CP-55,940, AM8936), partial agonist (Δ9-THC), and non-cannabinoids (fentanyl, atropine) were compared. The CBs either fully (AM2201, CP-55,940, JWH-018, AM8936, Δ9-THC) or partially (AM411, AM4089) substituted for AM2201, whereas fentanyl and atropine did not produce AM2201-like effects. All CB drugs were more potent than Δ9-THC and correlation analysis confirmed that the relative behavioral potencies of CBs corresponded strongly with their relative affinities at the CB1 but not CB2 receptors. Together, our results further demonstrate that AM411 and AM4089 exhibit better pharmacological profiles compared to Δ9-THC, in that they are more potent and display in vivo partial agonist-like actions that are centrally mediated via CB1 receptors.
Subject(s)
Cannabinoids , Dronabinol , Rats , Male , Animals , Dronabinol/pharmacology , Cannabinoid Receptor Agonists/pharmacology , Drug Inverse Agonism , Cannabinoids/pharmacology , Fentanyl , Atropine Derivatives , Receptor, Cannabinoid, CB1 , Dose-Response Relationship, DrugABSTRACT
[This corrects the article DOI: 10.3389/fphar.2020.575691.].
ABSTRACT
N-Acylethanolamines are signaling lipid molecules implicated in pathophysiological conditions associated with inflammation and pain. N-Acylethanolamine acid amidase (NAAA) favorably hydrolyzes lipid palmitoylethanolamide, which plays a key role in the regulation of inflammatory and pain processes. The synthesis and structure-activity relationship studies encompassing the isothiocyanate pharmacophore have produced potent low nanomolar inhibitors for hNAAA, while exhibiting high selectivity (>100-fold) against other serine hydrolases and cysteine peptidases. We have followed a target-based structure-activity relationship approach, supported by computational methods and known cocrystals of hNAAA. We have identified systemically active inhibitors with good plasma stability (t1/2 > 2 h) and microsomal stability (t1/2 â¼ 15-30 min) as pharmacological tools to investigate the role of NAAA in inflammation, pain, and drug addiction.
Subject(s)
Amidohydrolases/antagonists & inhibitors , Drug Design , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Isothiocyanates/chemistry , Isothiocyanates/pharmacology , Amidohydrolases/metabolism , Drug Stability , Humans , Hydrolysis , Structure-Activity RelationshipABSTRACT
Adamantyl groups are key structural subunit commonly used in many marketed drugs targeting diseases ranging from viral infections to neurological disorders. The metabolic disposition of adamantyl compounds has been mostly studied using LC-MS based approaches. However, metabolite quantities isolated from biological preparations are often insufficient for unambiguous structural characterization by NMR. In this work, we utilized microcoil NMR in conjunction with LC-MS to characterize liver microsomal metabolites of an adamantyl based CB2 agonist AM9338, 1-(3-(1H-1,2,3-triazol-1-yl) propyl)-N-(adamantan-1-yl)-1H-indazole-3-carboxamide, a candidate compound for potential multiple sclerosis treatment. We have identified a total of 9 oxidative metabolites of AM9338 whereas mono- or di-hydroxylation of the adamantyl moiety is the primary metabolic pathway. While it is generally believed that the tertiary adamantyl carbons are the preferred sites of CYP450 oxidation, both the mono- and di-hydroxyl metabolites of AM9338 show that the primary oxidative sites are located on the secondary adamantyl carbons. To our knowledge this di-hydroxylated metabolite is a novel adamantyl metabolite that has not been reported before. Further, the stereochemistry of both mono- and di-hydroxyl adamantyl metabolites has been determined using NOE correlations. Furthermore, docking of AM9338 into the CYP3A4 metabolic enzyme corroborates with our experimental findings, and the modelling results also provide a possible mechanism for the unusual susceptibility of adamantyl secondary carbons to metabolic oxidations. The novel dihydroxylated AM9338 metabolite identified in this study, along with the previously known adamantyl metabolites, gives a more complete picture of the metabolic disposition for adamantyl compounds.
ABSTRACT
Human alpha/beta hydrolase domain 6 (hABHD6) is an enzyme that hydrolyzes 2-arachidonoylglycerol (2-AG), a potent agonist at both cannabinoid CB1 and CB2 receptors. In vivo modulation of ABHD6 expression has been shown to have potential therapeutic applications, making the enzyme a promising drug target. However, the lack of structural information on hABHD6 limits the discovery and design of selective inhibitors. We have performed E. coli expression, purification and activity profiling screening of different hABHD6 constructs and identified a truncated variant without N-terminal transmembrane (TM) domain, hΔ29-3-ABHD6, as the most promising protein for further characterization. The elimination of the TM domain did not affect 2-AG or fluorogenic arachidonoyl, 7-hydroxy-6-methoxy-4-methylcoumarin ester (AHMMCE) substrates hydrolysis, suggesting that the TM is not essential for enzyme catalytic activity. The hΔ29-3-ABHD6 variant was purified in a single step using Immobilized Metal Affinity Chromatography (IMAC), in-solution trypsin digested, and proteomically characterized by Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS). The N-terminal peptide without methionine was identified indicating on a post-translational modification of the recombinant protein. The mechanism of inhibition of hABHD6 with AM6701 and WWL70 covalent probes was elucidated based on MS analysis of trypsin digested hABHD6 following the Ligand Assisted Protein Structure (LAPS) approach. We identified the carbamylated peptides containing catalytic serine (Ser148) suggesting a selective carbamylation of the enzyme by AM6701 or WWL70 and confirming an essential role of this residue in catalysis. The ability to produce substantial quantities of functional, pure hABHD6 will aid in the downstream structural characterization, and development of potent, selective inhibitors.
Subject(s)
Monoacylglycerol Lipases/chemistry , Monoacylglycerol Lipases/metabolism , Recombinant Proteins , Amino Acid Sequence , Chromatography, Affinity , Enzyme Activation , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Humans , Monoacylglycerol Lipases/genetics , Monoacylglycerol Lipases/isolation & purification , Recombinant Fusion Proteins , Solubility , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Substrate SpecificityABSTRACT
Monoacylglycerol lipase (MGL) inhibition provides a potential treatment approach to glaucoma through the regulation of ocular 2-arachidonoylglycerol (2-AG) levels and the activation of CB1 receptors. Herein, we report the discovery of new series of carbamates as highly potent and selective MGL inhibitors. The new inhibitors showed potent nanomolar inhibitory activity against recombinant human and purified rat MGL, were selective (>1000-fold) against serine hydrolases FAAH and ABHD6 and lacked any affinity for the cannabinoid receptors CB1 and CB2. Protein-based 1H NMR experiments indicated that inhibitor 2 rapidly formed a covalent adduct with MGL with a residence time of about 6â¯h. This interconversion process "intrinsic reversibility" was exploited by modifications of the ligand's size (length and bulkiness) to generate analogs with "tunable' adduct residence time (τ). Inhibitor 2 was evaluated in a normotensive murine model for assessing intraocular pressure (IOP), which could lead to glaucoma, a major cause of blindness. Inhibitor 2 was found to decrease ocular pressure by â¼4.5â¯mmHg in a sustained manner for at least 12â¯h after a single ocular application, underscoring the potential for topically-administered MGL inhibitors as a novel therapeutic target for the treatment of glaucoma.
Subject(s)
Carbamates/therapeutic use , Enzyme Inhibitors/therapeutic use , Glaucoma/drug therapy , Monoacylglycerol Lipases/antagonists & inhibitors , Animals , Carbamates/chemical synthesis , Carbamates/chemistry , Carbamates/pharmacokinetics , Catalytic Domain , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacokinetics , Humans , Male , Mice, Inbred C57BL , Microsomes, Liver/metabolism , Molecular Docking Simulation , Molecular Structure , Monoacylglycerol Lipases/chemistry , Piperazines/chemical synthesis , Piperazines/chemistry , Piperazines/pharmacokinetics , Piperazines/therapeutic use , Piperidines/chemical synthesis , Piperidines/chemistry , Piperidines/pharmacokinetics , Piperidines/therapeutic use , Rats , Structure-Activity RelationshipABSTRACT
The recent recreational use of synthetic cannabinoid ligands, collectively referred to as 'Spice', has raised concerns about their safety and possible differences in their biological effect(s) from marijuana/Δ-tetrahydrocannabinol (THC). AM2201, a highly efficacious, potent cannabinoid receptor 1 (CB1R) agonist, is a recently detected compound in 'Spice' preparations. Furthermore, structural analogs of AM2201 are now being found in 'Spice'. The present studies were conducted to investigate their Δ-THC-like effects using drug (Δ-THC) discrimination in rats. Results show that the tested compounds were potent cannabinergics that generalized to the response to Δ-THC, with AM2201 being most potent, exhibiting a 14-fold potency difference over Δ-THC. The other analogs were between 2.5-fold and 4-fold more potent than THC. Surmountable antagonism of AM2201 with the selective CB1R antagonist/inverse agonist rimonabant also established that the discrimination is CB1R dependent. Time-course data reveal that AM2201 likely peaks rapidly with an in-vivo functional half-life of only 60 min. The present data confirm and extend previous observations regarding Δ-THC-like effects of 'Spice' components.
Subject(s)
Discrimination Learning/drug effects , Dronabinol/pharmacology , Indoles/pharmacology , Psychotropic Drugs/pharmacology , Animals , Conditioning, Operant/drug effects , Dose-Response Relationship, Drug , Male , Rats , Rats, Sprague-DawleyABSTRACT
Several lines of evidence have shown that the endogenous cannabinoids are implicated in several neuropsychiatric diseases. Notably, preclinical and human clinical studies have shown a pivotal role of the cannabinoid system in nicotine addiction. The CB1 receptor inverse agonist/antagonist rimonabant (also known as SR141716) was effective to decrease nicotine-taking and nicotine-seeking in rodents, as well as the elevation of dopamine induced by nicotine in brain reward area. Rimonabant has been shown to improve the ability of smokers to quit smoking in randomized clinical trials. However, rimonabant was removed from the market due to increased risk of psychiatric side-effects observed in humans. Recently, other components of the endogenous cannabinoid system have been explored. Here, we present the recent advances on the understanding of the role of the different components of the cannabinoid system on nicotine's effects. Those recent findings suggest possible alternative ways of modulating the cannabinoid system that could have implication for nicotine dependence treatment.
ABSTRACT
BACKGROUND: Chemotherapeutic treatment results in chronic pain in an estimated 30-40 percent of patients. Limited and often ineffective treatments make the need for new therapeutics an urgent one. We compared the effects of prophylactic cannabinoids as a preventative strategy for suppressing development of paclitaxel-induced nociception. The mixed CB1/CB2 agonist WIN55,212-2 was compared with the cannabilactone CB2-selective agonist AM1710, administered subcutaneously (s.c.), via osmotic mini pumps before, during, and after paclitaxel treatment. Pharmacological specificity was assessed using CB1 (AM251) and CB2 (AM630) antagonists. The impact of chronic drug infusion on transcriptional regulation of mRNA markers of astrocytes (GFAP), microglia (CD11b) and cannabinoid receptors (CB1, CB2) was assessed in lumbar spinal cords of paclitaxel and vehicle-treated rats. RESULTS: Both WIN55,212-2 and AM1710 blocked the development of paclitaxel-induced mechanical and cold allodynia; anti-allodynic efficacy persisted for approximately two to three weeks following cessation of drug delivery. WIN55,212-2 (0.1 and 0.5 mg/kg/day s.c.) suppressed the development of both paclitaxel-induced mechanical and cold allodynia. WIN55,212-2-mediated suppression of mechanical hypersensitivity was dominated by CB1 activation whereas suppression of cold allodynia was relatively insensitive to blockade by either CB1 (AM251; 3 mg/kg/day s.c.) or CB2 (AM630; 3 mg/kg/day s.c.) antagonists. AM1710 (0.032 and 3.2 mg/kg /day) suppressed development of mechanical allodynia whereas only the highest dose (3.2 mg/kg/day s.c.) suppressed cold allodynia. Anti-allodynic effects of AM1710 (3.2 mg/kg/day s.c.) were mediated by CB2. Anti-allodynic efficacy of AM1710 outlasted that produced by chronic WIN55,212-2 infusion. mRNA expression levels of the astrocytic marker GFAP was marginally increased by paclitaxel treatment whereas expression of the microglial marker CD11b was unchanged. Both WIN55,212-2 (0.5 mg/kg/day s.c.) and AM1710 (3.2 mg/kg/day s.c.) increased CB1 and CB2 mRNA expression in lumbar spinal cord of paclitaxel-treated rats in a manner blocked by AM630. CONCLUSIONS AND IMPLICATIONS: Cannabinoids block development of paclitaxel-induced neuropathy and protect against neuropathic allodynia following cessation of drug delivery. Chronic treatment with both mixed CB1/CB2 and CB2 selective cannabinoids increased mRNA expression of cannabinoid receptors (CB1, CB2) in a CB2-dependent fashion. Our results support the therapeutic potential of cannabinoids for suppressing chemotherapy-induced neuropathy in humans.
Subject(s)
Analgesics/administration & dosage , Antineoplastic Agents, Phytogenic/toxicity , Cannabinoids/administration & dosage , Neuralgia/chemically induced , Neuralgia/prevention & control , Paclitaxel/toxicity , Animals , Disease Models, Animal , Dose-Response Relationship, Drug , Drug Delivery Systems , Gene Expression Regulation/drug effects , Glial Fibrillary Acidic Protein/genetics , Glial Fibrillary Acidic Protein/metabolism , Hyperalgesia/etiology , Hyperalgesia/prevention & control , Male , Motor Activity/drug effects , Neuralgia/complications , Pain Measurement , Pain Threshold/drug effects , Rats , Rats, Sprague-Dawley , Spinal Cord/drug effects , Spinal Cord/metabolismABSTRACT
BACKGROUND AND PURPOSE: HIV-1 glycoprotein Gp120 induces apoptosis in rodent and human neurons in vitro and in vivo.â HIV-1/Gp120 is involved in the pathogenesis of HIV-associated dementia (HAD) and inhibits proliferation of adult neural progenitor cells (NPCs) in glial fibrillary acidic protein (GFAP)/Gp120 transgenic (Tg) mice. As cannabinoids exert neuroprotective effects in several model systems, we examined the protective effects of the CB2 receptor agonist AM1241 on Gp120-mediated insults on neurogenesis. EXPERIMENTAL APPROACH: We assessed the effects of AM1241 on survival and apoptosis in cultures of human and murine NPCs with immunohistochemical and TUNEL techniques. Neurogenesis in the hippocampus of GFAP/Gp120 transgenic mice in vivo was also assessed by immunohistochemistry. KEY RESULTS: AM1241 inhibited in vitroâ Gp120-mediated neurotoxicity and apoptosis of primary human and murine NPCs and increased their survival. AM1241 also promoted differentiation of NPCs to neuronal cells. While GFAP/Gp120 Tg mice exhibited impaired neurogenesis, as indicated by reduction in BrdU⺠cells and doublecortin⺠(DCXâº) cells, and a decrease in cells with proliferating cell nuclear antigen (PCNA), administration of AM1241 to GFAP/Gp120 Tg mice resulted in enhanced in vivo neurogenesis in the hippocampus as indicated by increase in neuroblasts, neuronal cells, BrdU⺠cells and PCNA⺠cells. Astrogliosis and gliogenesis were decreased in GFAP/Gp120 Tg mice treated with AM1241, compared with those treated with vehicle. CONCLUSIONS AND IMPLICATIONS: The CB2 receptor agonist rescued impaired neurogenesis caused by HIV-1/Gp120 insult. Thus, CB2 receptor agonists may act as neuroprotective agents, restoring impaired neurogenesis in patients with HAD.
Subject(s)
Glial Fibrillary Acidic Protein/genetics , HIV Envelope Protein gp120/genetics , Neurogenesis/drug effects , Receptor, Cannabinoid, CB2/agonists , Animals , Apoptosis/drug effects , Cannabinoids/pharmacology , Cell Differentiation/drug effects , Cell Survival/drug effects , Doublecortin Protein , Hippocampus/cytology , Hippocampus/drug effects , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Neural Stem Cells/drug effects , Neurogenesis/genetics , Neurogenesis/physiology , Primary Cell Culture , Real-Time Polymerase Chain Reaction , Receptor, Cannabinoid, CB2/biosynthesisABSTRACT
RATIONALE: The "subjective high" from marijuana ingestion is likely due to Δ(9)-tetrahydrocannabinol (THC) activating the central cannabinoid receptor type 1 (CB1R) of the endocannabinoid signaling system. THC is a weak partial agonist according to in vitro assays, yet THC mimics the behavioral effects induced by more efficacious cannabinergics. This distinction may be important for understanding similarities and differences in the dose-effect spectra produced by marijuana/THC and designer cannabimimetics ("synthetic marijuana"). OBJECTIVE: We evaluated if drug discrimination is able to functionally detect/differentiate between a full, high-efficacy CB1R agonist [(±)AM5983] and the low-efficacy agonist THC in vivo. MATERIALS AND METHODS: Rats were trained to discriminate between four different doses of AM5983 (0.10 to 0.56 mg/kg), and vehicle and dose generalization curves were determined for both ligands at all four training doses of AM5983. The high-efficacy WIN55,212-2 and the lower-efficacy (R)-(+)-methanandamide were examined at some AM5983 training conditions. Antagonism tests involved rimonabant and WIN55,212-2 and AM5983. The separate (S)- and (R)-isomers of (±)AM5983 were tested at one AM5983 training dose (0.30 mg/kg). The in vitro cyclic adenosine monophosphate (cAMP) assay examined AM5983 and the known CB1R agonist CP55,940. RESULTS: Dose generalization ed50 values increased as a function of the training dose of AM5983, but more so for the partial agonists. The order of potency was (R)-isomer > (±)AM5983 > (S)-isomer and AM5983 > WIN55,212-2 ≥ THC > (R)-(+)-methanandamide. Surmountable antagonism of AM5983 and WIN55,212-2 occurred with rimonabant. The cAMP assay confirmed the cannabinergic nature of AM5983 and CP55,940. CONCLUSIONS: Drug discrimination using different training doses of a high-efficacy, full CB1R agonist differentiated between low- and high-efficacy CB1R agonists.
Subject(s)
Arachidonic Acids/pharmacology , Benzoxazines/pharmacology , Discrimination, Psychological/drug effects , Dronabinol/pharmacology , Indoles/pharmacology , Morpholines/pharmacology , Naphthalenes/pharmacology , Piperidines/pharmacology , Receptor, Cannabinoid, CB1/agonists , Animals , Cyclic AMP/chemistry , Cyclohexanols/chemistry , Cyclohexanols/pharmacology , Dose-Response Relationship, Drug , Dronabinol/chemistry , Indoles/chemistry , Isomerism , Male , Molecular Structure , Piperidines/chemistry , Pyrazoles/pharmacology , Random Allocation , Rats, Sprague-Dawley , RimonabantABSTRACT
AIMS: AM-1241, a novel, racemic cannabinoid-2 receptor (CB2) ligand, is the primary experimental agonist used to characterize the role of CB2-mediated lipid signaling in health and disease, including substance abuse disorders. In vivo pharmacological effects have been used as indirect proxies for AM-1241 biotransformation processes that could modulate CB2 activity. We report the initial pre-clinical characterization of AM-1241 biotransformation and in vivo distribution. MAIN METHODS: AM-1241 metabolism was characterized in a variety of predictive in vitro systems (Caco-2 cells; mouse, rat and human microsomes) and in the mouse in vivo. Liquid chromatography and mass spectrometry techniques were used to quantify AM-1241 tissue distribution and metabolic conversion. KEY FINDINGS: AM-1241 bound extensively to plasma protein/albumin. A pharmacological AM-1241 dose (25mg/kg, i.v.) was administered to mice for direct determination of its plasma half-life (37 min), following which AM-1241 was quantified in brain, spleen, liver, and kidney. After p.o. administration, AM-1241 was detected in plasma, spleen, and kidney; its oral bioavailability was ~21%. From Caco-2 permeability studies and microsomal-based hepatic clearance estimates, in vivo AM-1241 absorption was moderate. Hepatic microsomal metabolism of AM-1241 in vitro generated hydroxylation and demethylation metabolites. Species-dependent differences were discovered in AM-1241's predicted hepatic clearance. Our data demonstrate that AM-1241 has the following characteristics: a) short plasma half-life; b) limited oral bioavailability; c) extensive plasma/albumin binding; d) metabolic substrate for hepatic hydroxylation and demethylation; e) moderate hepatic clearance. SIGNIFICANCE: These results should help inform the design, optimization, and pre-clinical profiling of CB2 ligands as pharmacological tools and medicines.
Subject(s)
Analgesics/pharmacology , Endocannabinoids/physiology , Lipids/physiology , Receptor, Cannabinoid, CB2/agonists , Animals , Biotransformation , Blood Proteins/metabolism , Caco-2 Cells , Cannabinoids/chemistry , Cannabinoids/pharmacokinetics , Cannabinoids/pharmacology , Chromatography, High Pressure Liquid , Humans , Indicators and Reagents , Injections, Intraperitoneal , Injections, Subcutaneous , Liver/metabolism , Male , Mice , Protein Binding , Signal Transduction/drug effects , Spectrum Analysis , Tissue DistributionABSTRACT
During pathological pain, the actions of the endocannabinoid system, including the cannabinoid 2 receptor (CB(2)R), leads to effective anti-allodynia and modifies a variety of spinal microglial and astrocyte responses. Here, following spinal administration of the CB(2)R compound, AM1241, we examined immunoreactive alterations in markers for activated p38 mitogen-activated protein kinase, interleukin-1ß (IL-1ß), the anti-inflammatory cytokine, interleukin-10 (IL-10) as well as degradative endocannabinoid enzymes, and markers for altered glial responses in neuropathic rats. In these studies, the dorsal horn of the spinal cord and dorsal root ganglia were examined. AM1241 produced profound anti-allodynia with corresponding immunoreactive levels of p38 mitogen-activated kinase, IL-1ß, IL-10, the endocannabinoid enzyme monoacylglycerol lipase, and astrocyte activation markers that were similar to nonneuropathic controls. In contrast, spinal AM1241 did not suppress the increased microglial responses observed in neuropathic rats. The differences in fluorescent markers were determined within discrete anatomical regions by applying spectral analysis methods, which virtually eliminated nonspecific signal during the quantification of specific immunofluorescent intensity. These data reveal expression profiles that support the actions of intrathecal AM1241 control pathological pain through anti-inflammatory mechanisms by modulating critical glial factors, and additionally decrease expression levels of endocannabinoid degradative enzymes.
ABSTRACT
Over the last decade there have been significant advances in the discovery and understanding of the cannabinoid system along with the development of pharmacologic tools that modulate its function. Characterization of the crosstalk between nicotine addiction and the cannabinoid system may have significant implications on our understanding of the neurobiological mechanisms underlying nicotine dependence. Two types of cannabinoid receptors (CB1 and CB2) have been identified. CB1 receptors are expressed in the brain and modulate drug taking and drug seeking for various drugs of abuse, including nicotine. CB2 receptors have been recently identified in the brain and have been proposed to play a functional role in mental disorders and drug addiction. Our objective was to explore the role of CB2 receptors on intravenous nicotine self administration under two schedules of reinforcement (fixed and progressive ratio) and on nicotine seeking induced by nicotine priming or by nicotine associated cues. For this, we evaluated the effects of various doses of the selective CB2 antagonist AM630 (1.25 to 5 mg/kg) and CB2 agonist AM1241 (1 to 10 mg/kg) on these behavioral responses in rats. Different groups of male Long Evans rats were trained to lever press for nicotine at a unit dose of 30 µg/kg/infusion. Subsequently, animals were randomized using a Latin-square design and injected with either AM1241 or AM630 using a counterbalanced within subject design. Administration of the CB2 ligands did not affect either nicotine-taking nicotine-seeking behavior. Our results do not support the involvement of CB2 receptors in nicotine-taking or nicotine-seeking behavior.
Subject(s)
Drug-Seeking Behavior/drug effects , Indoles/pharmacology , Nicotine/administration & dosage , Tobacco Use Disorder/psychology , Animals , Behavior, Animal/drug effects , Cannabinoids/pharmacology , Conditioning, Operant , Drug-Seeking Behavior/physiology , Injections, Intravenous , Male , Rats , Rats, Long-Evans , Receptor, Cannabinoid, CB2/agonists , Receptor, Cannabinoid, CB2/antagonists & inhibitors , Recurrence , Reinforcement, Psychology , Self Administration , Substrate Specificity/drug effects , Tobacco Use Disorder/pathologyABSTRACT
RATIONALE: The endocannabinoid signaling system (ECS) has been targeted for developing novel therapeutics since ECS dysfunction has been implicated in various pathologies. Current focus is on chemical modifications of the hexahydrocannabinol (HHC) nabilone (Cesamet(®)). OBJECTIVE: To characterize the novel, high-affinity cannabinoid receptor 1 (CB(1)R) HHC-ligand AM2389 [9ß-hydroxy-3-(1-hexyl-cyclobut-1-yl)-hexahydrocannabinol in two rodent pre-clinical assays. MATERIALS AND METHODS: CB(1)R mediation of AM2389-induced hypothermia in mice was evaluated with AM251, a CB(1)R-selective antagonist/inverse agonist. Additionally, two groups of rats discriminated the full cannabinergic aminoalkylindole AM5983 (0.18 and 0.56 mg/kg) from vehicle 20 min post-injection in a two-choice operant conditioning task motivated by 0.1% saccharin/water. Generalization/substitution tests were conducted with AM2389, AM5983, and Δ(9)-tetrahydrocannabinol (Δ(9)-THC). RESULTS: Δ(9)-THC (30 mg/kg)-induced hypothermia exhibited a faster onset and shorter duration of action compared with AM2389 (0.1 and 0.3 mg/kg). AM251 (3 and 10 mg/kg) attenuated/blocked hypothermia induced by 0.3 mg/kg AM2389. In drug discrimination, the order of potency was AM2389 > AM5983 > Δ(9)-THC with ED(50) values of 0.0025, 0.0571, and 0.2635 mg/kg, respectively, in the low-dose condition. The corresponding ED(50) values in the high-dose condition were 0.0069, 0.1246, and 0.8438 mg/kg, respectively. Onset of the effects of AM2389 was slow with a protracted time-course; the functional, perceptual in vivo half-life was approximately 17 h. CONCLUSIONS: This potent cannabinergic HHC exhibited a slow onset of action with a protracted time-course. The AM2389 chemotype appears well suited for further drug development, and AM2389 currently is used to probe behavioral consequences of sustained ECS activation.
Subject(s)
Benzopyrans/pharmacology , Hypothermia/chemically induced , Receptor, Cannabinoid, CB1/agonists , Animals , Benzopyrans/antagonists & inhibitors , Conditioning, Operant/drug effects , Discrimination, Psychological/drug effects , Dose-Response Relationship, Drug , Dronabinol/pharmacology , Drug Interactions , Indoles/pharmacology , Male , Mice , Mice, Inbred C57BL , Piperidines/pharmacology , Pyrazoles/pharmacology , Rats , Rats, Sprague-Dawley , Receptor, Cannabinoid, CB1/antagonists & inhibitors , Time FactorsABSTRACT
Drug self-administration methods were used to test the hypothesis that rats would self-medicate with a cannabinoid CB(2) agonist to attenuate a neuropathic pain state. Self-medication of the CB(2) agonist (R,S)-AM1241, but not vehicle, attenuated mechanical hypersensitivity produced by spared nerve injury. Switching rats from (R,S)-AM1241 to vehicle self-administration also decreased lever responding in an extinction paradigm. (R,S)-AM1241 self-administration did not alter paw withdrawal thresholds in sham-operated or naive animals. The percentage of active lever responding was similar in naive groups self-administering vehicle or (R,S)-AM1241. The CB(2) antagonist SR144528 blocked both antiallodynic effects of (R,S)-AM1241 self-medication and the percentage of active lever responding in neuropathic (but not naive) rats. Neuropathic and sham groups exhibited similar percentages of active lever responding for (R,S)-AM1241 on a fixed ratio 1 (FR1) schedule. However, neuropathic animals worked harder than shams to obtain (R,S)-AM1241 when the schedule of reinforcement was increased (to FR6). (R,S)-AM1241 self-medication on FR1, FR3, or FR6 schedules attenuated nerve injury-induced mechanical allodynia. (R,S)-AM1241 (900µg intravenously) failed to produce motor ataxia observed after administration of the mixed CB(1)/CB(2) agonist WIN55,212-2 (0.5mg/kg intravenously). Our results suggest that cannabinoid CB(2) agonists may be exploited to treat neuropathic pain with limited drug abuse liability and central nervous system side effects. These studies validate the use of drug self-administration methods for identifying nonpsychotropic analgesics possessing limited abuse potential. These methods offer potential to elucidate novel analgesics that suppress spontaneous neuropathic pain that is not measured by traditional assessments of evoked pain.
Subject(s)
Disease Models, Animal , Neuralgia/drug therapy , Receptor, Cannabinoid, CB2/agonists , Self Medication , Animals , Cannabinoids/administration & dosage , Conditioning, Operant/drug effects , Conditioning, Operant/physiology , Male , Neuralgia/physiopathology , Rats , Rats, Sprague-Dawley , Receptor, Cannabinoid, CB2/physiology , Self Medication/methodsABSTRACT
Cannabinoid CB(2) agonists produce antinociception without central nervous system (CNS) side-effects. This study was designed to characterize the pharmacological and antinociceptive profile of AM1710, a CB(2) agonist from the cannabilactone class of cannabinoids. AM1710 did not exhibit off-target activity at 63 sites evaluated. AM1710 also exhibited limited blood brain barrier penetration. AM1710 was evaluated in tests of antinociception and CNS activity. CNS side-effects were evaluated in a modified tetrad (tail flick, rectal temperature, locomotor activity and rota-rod). Pharmacological specificity was established using CB(1) (SR141716) and CB(2) (SR144528) antagonists. AM1710 (0.1-10mg/kg i.p.) produced antinociception to thermal but not mechanical stimulation of the hindpaw. AM1710 (5mg/kg i.p.) produced a longer duration of antinociceptive action than the aminoalkylindole CB(2) agonist (R,S)-AM1241 (1mg/kg i.p.) at maximally antinociceptive doses. Antinociception produced by the low (0.1mg/kg i.p.) dose of AM1710 was blocked selectively by the CB(2) antagonist SR144528 (6mg/kg i.p.), whereas antinociception produced by the high dose of AM1710 (5mg/kg i.p.) was blocked by either SR144528 (6mg/kg i.p.) or SR141716 (6mg/kg i.p.). AM1710 did not produce hypoactivity, hypothermia, tail flick antinociception, or motor ataxia when evaluated in the tetrad at any dose. In conclusion, AM1710, a CB(2)-preferring cannabilactone, produced antinociception in the absence of CNS side-effects. Thus, any CB(1)-mediated antinociceptive effects of this compound may be attributable to peripheral CB(1) activity. The observed pattern of pharmacological specificity produced by AM1710 is consistent with limited blood brain barrier penetration of this compound and absence of CNS side-effects.
Subject(s)
Chromones/pharmacology , Receptor, Cannabinoid, CB2/agonists , Analgesics/pharmacokinetics , Analgesics/pharmacology , Analgesics/toxicity , Animals , Behavior, Animal/drug effects , Binding, Competitive , Blood-Brain Barrier , Camphanes/pharmacology , Cannabinoids/pharmacokinetics , Cannabinoids/pharmacology , Cannabinoids/toxicity , Central Nervous System/drug effects , Chromones/pharmacokinetics , Chromones/toxicity , HEK293 Cells , Humans , Male , Mice , Pain Threshold/drug effects , Piperidines/pharmacology , Pyrazoles/pharmacology , Rats , Rats, Sprague-Dawley , Receptor, Cannabinoid, CB1/antagonists & inhibitors , Receptor, Cannabinoid, CB1/genetics , Receptor, Cannabinoid, CB2/antagonists & inhibitors , Receptor, Cannabinoid, CB2/genetics , Recombinant Proteins/agonists , Recombinant Proteins/genetics , RimonabantABSTRACT
RATIONALE: Discovery of an endocannabinoid signaling system launched the development of the blocker rimonabant, a cannabinoid CB1 receptor (CB(1)R) antagonist/inverse agonist. Due to untoward effects, this medication was withdrawn and efforts have been directed towards discovering chemicals with more benign profiles. OBJECTIVE: This study aims to comparatively evaluate new ligands using a rimonabant discriminated drinking aversion procedure. METHODS: Rats discriminated between rimonabant (5.6 mg/kg) and vehicle. The 30 min saccharin (0.1%) drinking after rimonabant pretreatment was followed by injection of lithium chloride (120 mg/kg) in the experimental animals. After vehicle pretreatment, experimental animals were given i.p. NaCl (10 ml/kg). Postdrinking treatment for controls was NaCl, irrespective of pretreatment condition (rimonabant or vehicle). RESULTS: The centrally acting neutral CB(1)R antagonist AM4113, but not the limited brain penetrating CB(1)R neutral antagonist AM6545, substituted for rimonabant. The CB(1)R agonists THC (1-10 mg/kg), AM1346 (1-10 mg/kg) did not substitute. The rimonabant-induced conditioned suppression of saccharin drinking was attenuated when CB(1)R agonists AM5983 (0.01-1 mg/kg) and THC (10 mg/kg), but not the CB(1)R agonist AM1346 (0.1-18 mg/kg), were combined with rimonabant (5.6 mg/kg). By varying the injection-to-test interval, we gauged the relative duration of the cueing effects of rimonabant, and the in vivo functional half-life was estimated to be approximately 1.5 h. CONCLUSION: A neutral CB(1)R antagonist (AM4113) produced cueing effects similar to those of rimonabant and generalization likely was centrally mediated. The functional cueing effects of rimonabant are relatively short-acting, pharmacologically selective, and differentially blocked by cannabinergics.
Subject(s)
Cannabinoids/pharmacology , Discrimination, Psychological/drug effects , Indoles/pharmacology , Piperidines/pharmacology , Pyrazoles/pharmacology , Receptor, Cannabinoid, CB1/agonists , Receptor, Cannabinoid, CB1/antagonists & inhibitors , Animals , Arachidonic Acids/pharmacology , Cannabinoids/chemistry , Dose-Response Relationship, Drug , Drinking/drug effects , Dronabinol/pharmacology , Ligands , Male , Morpholines/pharmacology , Polyunsaturated Alkamides/pharmacology , Rats , Rats, Sprague-Dawley , Rimonabant , Time FactorsABSTRACT
Cannabinoid CB(2) receptors represent a therapeutic target that circumvents unwanted central side effects (e.g., psychoactivity and/or addiction) associated with activation of CB(1) receptors. One of the primary investigative tools used to study functions of the CB(2) receptor is the aminoalkylindole (R,S)-AM1241. However, (R,S)-AM1241 has been described as an atypical CB(2) agonist which produces antinociception mediated indirectly by opioid receptors. (R,S)-AM1241 and its enantiomers, (R)-AM1241 and (S)-AM1241, were evaluated for antinociception in response to thermal (Hargreaves) and mechanical (von Frey) stimulation. Pharmacological specificity was established using antagonists for CB(1) (rimonabant [SR141716]) and CB(2) (SR144528). The opioid antagonist naloxone was administered locally in the paw or systemically to evaluate the contribution of opioid receptors to CB(2)-mediated antinociception produced by (R,S)-AM1241, (R)-AM1241, and (S)-AM1241. Comparisons were made with the opioid analgesic morphine. (R,S)-AM1241, (R)-AM1241, and (S)-AM1241 (0.033-10 mg/kg i.p.) produced antinociception to thermal, but not mechanical, stimulation of the hindpaw in naive rats. Antinociception produced by (R,S)-AM1241 and (S)-AM1241 exhibited an inverted U-shaped dose response curve. (R)-AM1241 produced greater antinociception than either (S)-AM1241 or (R,S)-AM1241 at the lowest (0.033 and 0.1 mg/kg i.p.) and highest (10 mg/kg i.p.) doses. Similar levels of antinociception were observed at intermediate doses. (R,S)-AM1241, (R)-AM1241, and (S)-AM1241 each produced CB(2)-mediated antinociception that was blocked by SR144528 but not by rimonabant. Local and systemic naloxone blocked morphine-induced antinociception but did not block antinociceptive effects of (R,S)-AM1241, (R)-AM1241, or (S)-AM1241. The antinociceptive effects of the CB(2)-selective cannabinoid (R,S)-AM1241 and its enantiomers, (R)-AM1241 and (S)-AM1241, are not dependent upon opioid receptors.
Subject(s)
Analgesics/pharmacology , Receptors, Opioid/drug effects , Analgesics/chemistry , Analgesics, Opioid/pharmacology , Animals , Cannabinoids/chemistry , Cannabinoids/pharmacology , Hot Temperature , Isomerism , Male , Morphine/pharmacology , Naloxone/pharmacology , Narcotic Antagonists/pharmacology , Pain Measurement/drug effects , Pain Threshold/drug effects , Physical Stimulation , Rats , Rats, Sprague-Dawley , Receptor, Cannabinoid, CB1/antagonists & inhibitors , Receptor, Cannabinoid, CB2/agonists , Receptor, Cannabinoid, CB2/antagonists & inhibitors , Structure-Activity RelationshipABSTRACT
The extensive physiological influence of transmission through the CB2 cannabinoid receptor makes this G protein-coupled receptor (GPCR) a promising therapeutic target for treating neuropathic pain, inflammation, and immune disorders. However, there is little direct structural information pertaining to either GPCR or CB2-receptor ligand recognition and activation. The present work helps characterize experimentally the ligand-binding interactions of the human CB2 (hCB2) receptor. This study illustrates how our overall experimental approach, "ligand-assisted protein structure" (LAPS), affords direct determination of the requirements for ligand binding to the hCB2 receptor and discrimination among the binding motifs for ligands that activate therapeutically relevant GPCRs.