Immunofluorescent spectral analysis reveals the intrathecal cannabinoid agonist, AM1241, produces spinal anti-inflammatory cytokine responses in neuropathic rats exhibiting relief from allodynia

During pathological pain, the actions of the endocannabinoid system, including the cannabinoid 2 receptor (CB2R), leads to effective anti-allodynia and modifies a variety of spinal microglial and astrocyte responses. Here, following spinal administration of the CB2R compound, AM1241, we examined immunoreactive alterations in markers for activated p38 mitogen-activated protein kinase, interleukin-1β (IL-1β), 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-1β, 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

Effects of a selective cannabinoid CB2 agonist and antagonist on intravenous nicotine self administration and reinstatement of nicotine seeking.

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

Effects of AM1241 on nicotine self-administration under FR5 and PR schedules of reinforcement.

<p><b>A</b>. Effects of pretreatment with AM1241 (1, 3 and 10 mg/kg, IP H 30) on nicotine (30 µg/kg/infusion) self-administration under the FR5 schedule. Data are expressed as means (±SEM) of the number of nicotine infusions obtained during the 60-min session. All doses of AM1241 did not affect responding vs. vehicle (0 mg/kg) pretreatment (N = 10); P = 0.35. <b>B</b>. Effects of pretreatment with AM1241 (1, 3 and 10 mg/kg) on nicotine (30 ug/kg/infusion) self- administration under PR schedule. <b>A</b>, Data are expressed as means (±SEM) of the number of nicotine infusions obtained during the 4-hr sessions. AM1241 did not affect break point P>0.05 compared to vehicle (0 mg/kg) pretreatment. (N = 8) P = 0.89.</p

Effects of AM630 on reinstatement of nicotine-seeking behavior induced by presentation of nicotine associated cues and by Nicotine priming.

<p><b>A</b>. Effects of pretreatment with AM630 (1.25, 2.5 and 5 mg/kg, IP H 30 min) on cue-induced reinstatement of nicotine-seeking behavior. A significant reinstatement of nicotine-seeking behavior was produced by presentation of nicotine-associated cues alone (* P<0.001). ANOVA showed that pretreatment with AM630 (1.25, 2.5 and 5 mg/kg, IP, H 30 min) did not modify cue induced reinstatement of nicotine-seeking behavior compared to vehicle (0 mg/kg) pretreatment (P>0.05). Data are expressed as means (±SEM) of the number of active and inactive lever presses during extinction (Ext); vehicle (0 mg/kg) pre-treatment and after pretreatment with AM630 (1.25, 2.5 and 5 mg). <b>B</b>. A significant reinstatement of nicotine-seeking was also produced by pretreatment with nicotine (0.15 mg/kg) (* P<0.001). ANOVA showed that AM630 (1.25, 2.5, 5 mg/kg, IP, H 30 min) did not modify reinstatement of nicotine-seeking behavior induced by a priming injection of 0.15 mg/kg nicotine administered 1 min before the session (P>0.05). Data are expressed as means (±SEM) of the number of active and inactive lever presses during extinction (Ext); vehicle (0 mg/kg) pre-treatment and after pretreatment with AM630 (1.25, 2.5 and 5 mg).</p

Effect of AM630 on nicotine self administration under FR5 and PR schedules of reinforcement.

<p><b>A</b>. Effects of pretreatment with AM630 (1.25, 2.5 and 5 mg/kg, IP, H 30) on nicotine (30 µg/kg/infusion) self administration under the FR5 schedule. Data are expressed as means (±SEM) of the number of nicotine infusions obtained during the 60-min session. AM630 did not affect responding vs. vehicle (0 mg/kg) pretreatment (N = 12); P = 0.67. <b>B</b>. Effects of pretreatment with AM630 (5 mg/kg, IP) on nicotine (30 ug/kg/infusion) self administration under PR schedule. <b>A</b>, Data are expressed as means (±SEM) of the number of nicotine infusions obtained during the 4-hr sessions. AM630 did not affect break point P>0.05 vs. vehicle (0 mg/kg) pretreatment. (N = 7) P = 0.73.</p

Pattern of respondinng during acquisition and extinction phases.

<p>A. Acquisition of nicotine self-administration (30 µg/kg/infusion). The total number of active (•) and inactive(▪) lever presses (means ± SEM) received in each session (during time in and time out periods) under the different schedules of reinforcement (FR- 1, FR-2, FR-5,). B. Number of nicotine infusions (means ± SEM) earned during acquisition phase in the same group of animals represented as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029900#pone-0029900-g001" target="_blank">fig. 1A</a>. C. The number of active (•) and inactive(▪) lever presses (means ± SEM) received in each extinction session in the same group of animals represented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029900#pone-0029900-g001" target="_blank">figures 1A & 1B</a>.</p

Binding Site Characterization of AM1336, a Novel Covalent Inverse Agonist at Human Cannabinoid 2 Receptor, Using Mass Spectrometric Analysis

Cannabinoid 2 receptor (CB2R), a Class-A G-protein coupled receptor (GPCR), is a promising drug target under a wide array of pathological conditions. Rational drug design has been hindered due to our poor understanding of the structural features involved in ligand binding. Binding of a high-affinity biarylpyrazole inverse agonist AM1336 to a library of the human CB2 receptor (hCB2R) cysteine-substituted mutants provided indirect evidence that two cysteines in transmembrane helix-7 (H7) were critical for the covalent attachment. We used proteomics analysis of the hCB2R with bound AM1336 to directly identify peptides with covalently attached ligand and applied in silico modeling for visualization of the ligand–receptor interactions. The hCB2R, with affinity tags (FlaghCB2His6), was produced in a baculovirus–insect cell expression system and purified as a functional receptor using immunoaffinity chromatography. Using mass spectrometry-based bottom-up proteomic analysis of the hCB2R-AM1336, we identified a peptide with AM1336 attached to the cysteine C284(7.38) in H7. The hCB2R homology model in lipid bilayer accommodated covalent attachment of AM1336 to C284(7.38), supporting both biochemical and mass spectrometric data. This work consolidates proteomics data and in silico modeling and integrates with our ligand-assisted protein structure (LAPS) experimental paradigm to assist in structure-based design of cannabinoid antagonist/inverse agonists