A CB(2) receptor agonist, A-836339, modulates wide dynamic range neuronal activity in neuropathic rats: contributions of spinal and peripheral CB(2) receptors.

We investigated the systemic and site-specific actions of a selective CB(2) receptor agonist, A-836339 on mechanically evoked (10 g von Frey hair) and spontaneous firing of spinal wide dynamic range (WDR) neurons in neuropathic (L5 and L6 ligations) and sham rats. Systemic administration of A-836339 (0.3-3 micromol/kg, i.v.) reduced both evoked and spontaneous WDR neuronal activity in neuropathic, but not sham rats. The effects in neuropathic rats were blocked by pre-administration of a CB(2), but not a CB(1), receptor antagonist. Similar to systemic delivery, intra-spinal injection of A-836339 (0.3 and 1 nmol) also attenuated both von Frey-evoked and spontaneous firing of WDR neurons in neuropathic rats. Intra-spinal injections of A-836339 were ineffective in sham rats. Application of A-836339 (3-30 nmol) onto the ipsilateral L5 dorsal root ganglion (DRG) of neuropathic rats reduced the von Frey-evoked activity of WDR neurons, but spontaneous firing was unaltered. All effects of A-836339 on WDR neuronal activity following either intra-spinal or intra-DRG administration were blocked by pre-administration of a CB(2) receptor antagonist. Pre-administration of a CB(1) receptor antagonist did not alter the site-specific effects of A-836339. Injection of A-836339 (300 nmol) into the neuronal receptive field on the ipsilateral hind paw did not affect evoked or spontaneous firing of WDR neurons. Thus, the current data demonstrate that modulation of spinal neuronal activity by a CB(2) receptor agonist is enhanced following peripheral nerve injury, and further delineate the contribution of spinal and peripheral CB(2) receptors to this modulation.

In vitro and in vivo characterization of A-796260: a selective cannabinoid CB2 receptor agonist exhibiting analgesic activity in rodent pain models.

BACKGROUND AND PURPOSE: Selective cannabinoid CB2 receptor agonists have demonstrated analgesic activity across multiple preclinical pain models. AM1241 is an indole derivative that exhibits high affinity and selectivity for the CB2 binding site and broad spectrum analgesic activity in rodent models, but is not an antagonist of CB2 in vitro functional assays. Additionally, its analgesic effects are mu-opioid receptor-dependent. Herein, we describe the in vitro and in vivo pharmacological properties of A-796260, a novel CB2 agonist. EXPERIMENTAL APPROACH: A-796260 was characterized in radioligand binding and in vitro functional assays at rat and human CB1 and CB2 receptors. The behavioural profile of A-796260 was assessed in models of inflammatory, post-operative, neuropathic, and osteoarthritic (OA) pain, as well as its effects on motor activity. The receptor specificity was confirmed using selective CB1, CB2 and mu-opioid receptor antagonists. KEY RESULTS: A-796260 exhibited high affinity and agonist efficacy at human and rat CB2 receptors, and was selective for the CB2 vs CB1 subtype. Efficacy in models of inflammatory, post-operative, neuropathic and OA pain was demonstrated, and these activities were selectively blocked by CB2, but not CB1 or mu-opioid receptor-selective antagonists. Efficacy was achieved at doses that had no significant effects on motor activity. CONCLUSIONS AND IMPLICATIONS: These results further confirm the therapeutic potential of CB2 receptor-selective agonists for the treatment of pain. In addition, they demonstrate that A-796260 may be a useful new pharmacological compound for further studying CB2 receptor pharmacology and for evaluating its role in the modulation of pain.

Differential effects of cannabinoid receptor agonists on regional brain activity using pharmacological MRI.

BACKGROUND AND PURPOSE: Activation of cannabinoid CB1 and/or CB2 receptors mediates analgesic effects across a broad spectrum of preclinical pain models. Selective activation of CB2 receptors may produce analgesia without the undesirable psychotropic side effects associated with modulation of CB1 receptors. To address selectivity in vivo, we describe non-invasive, non-ionizing, functional data that distinguish CB1 from CB2 receptor neural activity using pharmacological MRI (phMRI) in awake rats. EXPERIMENTAL APPROACH: Using a high field (7 T) MRI scanner, we examined and quantified the effects of non-selective CB1/CB2 (A-834735) and selective CB2 (AM1241) agonists on neural activity in awake rats. Pharmacological specificity was determined using selective CB1 (rimonabant) or CB2 (AM630) antagonists. Behavioural studies, plasma and brain exposures were used as benchmarks for activity in vivo. KEY RESULTS: The non-selective CB1/CB2 agonist produced a dose-related, region-specific activation of brain structures that agrees well with published autoradiographic CB1 receptor density binding maps. Pretreatment with a CB1 antagonist but not with a CB2 antagonist, abolished these activation patterns, suggesting an effect mediated by CB1 receptors alone. In contrast, no significant changes in brain activity were found with relevant doses of the CB2 selective agonist. CONCLUSION AND IMPLICATIONS: These results provide the first clear evidence for quantifying in vivo functional selectivity between CB1 and CB2 receptors using phMRI. Further, as the presence of CB2 receptors in the brain remains controversial, our data suggest that if CB2 receptors are expressed, they are not functional under normal physiological conditions.

In vitro pharmacological characterization of AM1241: a protean agonist at the cannabinoid CB2 receptor?

BACKGROUND AND PURPOSE: The CB2 receptor has been proposed as a novel target for the treatment of pain, and CB2 receptor agonists defined in in vitro assays have demonstrated analgesic activity in animal models. Based on its in vivo analgesic efficacy, AM1241 has been classified as a CB2-selective agonist. However, in vitro characterization of AM1241 in functional assays has not been reported. EXPERIMENTAL APPROACH: In this study, AM1241 was characterized across multiple in vitro assays employing heterologous recombinant receptor expression systems to assess its binding potencies at the human CB2 and CB1 receptors and its functional efficacies at the human CB2 receptor. KEY RESULTS: AM1241 exhibited distinct functional properties depending on the assay conditions employed, a unique profile in contrast to those of the agonist CP 55,940 and the inverse agonist SR144528. AM1241 displayed neutral antagonist activities in FLIPR and cyclase assays. However, when cyclase assays were performed using lower forskolin concentrations for stimulation, AM1241 exhibited partial agonist efficacy. In addition, it behaved as a partial agonist in ERK (or MAP) kinase assays. CONCLUSIONS AND IMPLICATIONS: The unusual phenomenon of inconsistent functional efficacies suggests that AM1241 is a protean agonist at the CB2 receptor. We postulate that functional efficacies displayed by protean agonists in various assay systems may depend on the levels of receptor constitutive activities exhibited in the assay systems, and therefore, efficacies observed in in vitro assays may not predict in vivo activities.

A selective α2 B adrenoceptor agonist (A-1262543) and duloxetine modulate nociceptive neurones in the medial prefrontal cortex, but not in the spinal cord of neuropathic rats.

BACKGROUND: The noradrenergic system contributes to pain modulation, but the roles of its specific adrenoceptors are still being defined. We have identified a novel, potent (rat EC50 = 4.3 nM) and selective α2B receptor agonist, A-1262543, to further explore this adrenoceptor subtype's contribution to pathological nociception. METHODS: Systemic administration of A-1262543 (1-10 mg/kg, intraperitoneal) dose-dependently attenuated mechanical allodynia in animals with a spinal nerve ligation injury. To further explore its mechanism of action, the activity of nociceptive neurones in the spinal cord and medial prefrontal cortex (mPFC) were examined after injection of 3 mg/kg of A-1262543 (intravenous, i.v.). These effects were compared with duloxetine (3 mg/kg, i.v.), a dual noradrenaline (NA) and serotonin (5-HT) reuptake inhibitor. RESULTS: Systemic administration of A-1262543 or duloxetine did not alter the spontaneous or evoked firing of spinal wide dynamic range and nociceptive-specific neurones in the neuropathic rats, indicating that neither compound engaged spinal, peripheral or descending pathways. In contrast to the lack of effect on spinal neurones, both A-1262543 and duloxetine reduced the evoked and spontaneous firing of 'pain-responsive' (PR) neurones in the mPFC. Duloxetine, but not A-1262543, also inhibited the firing of pain non-responsive (nPR) neurones in the mPFC probably reflecting duloxetine's contribution to modulating non-pain endpoints. CONCLUSIONS: These data highlight that activation of the α2B adrenoceptor as well as inhibiting NA and 5-HT reuptake can result in modulating the ascending nociceptive system, and in particular, dampening the firing of PR neurones in the mPFC.

The identification of novel structural compound classes exhibiting high affinity for neuronal nicotinic acetylcholine receptors and analgesic efficacy in preclinical models of pain.

Neuronal nicotinic acetylcholine receptors represent a new and potentially useful target for the development of novel non-opioid, non-NSAID (nonsteroidal antiinflammatory drug) analgesic agents. A variety of nicotinic acetylcholine receptor agonists such as nicotine, epibatidine and the azetidinyl ether, (R)-5-(2-azetidinylmethoxy-2-chloropyridine (ABT-594) possesses significant efficacy in preclinical models of pain. A preponderance of evidence suggests that nicotinic acetylcholine receptor agonists produce their analgesic effects predominantly via activation of descending inhibitory pain pathways originating in the key brainstem regions of the nucleus raphe magnus, dorsal raphe, and locus coeruleus, and that alpha4-containing nicotinic acetylcholine receptor subunits mediate these effects. Although these studies may provide a pharmacological target for the development of nicotinic acetylcholine receptor analgesics, the rational design of selective ligands based on the protein structure of the binding site is hampered by insufficient structural information. Using an approach based upon homology to known high-affinity ligands for the alpha4beta2 binding site, a four-point model is proposed which defines distance and directionality parameters common to this set of nicotinic acetylcholine receptor ligands.

Peripheral and central sites of action for the non-selective cannabinoid agonist WIN 55,212-2 in a rat model of post-operative pain.

BACKGROUND AND PURPOSE: Activation of cannabinoid (CB) receptors decreases nociceptive transmission in inflammatory or neuropathic pain states. However, the effects of CB receptor agonists in post-operative pain remain to be investigated. Here, we characterized the anti-allodynic effects of WIN 55,212-2 (WIN) in a rat model of post-operative pain. EXPERIMENTAL APPROACH: WIN 55,212-2 was characterized in radioligand binding and in vitro functional assays at rat and human CB(1) and CB(2) receptors. Analgesic activity and site(s) of action of WIN were assessed in the skin incision-induced post-operative pain model in rats; receptor specificity was investigated using selective CB(1) and CB(2) receptor antagonists. KEY RESULTS: WIN 55,212-2 exhibited non-selective affinity and agonist efficacy at human and rat CB(1) versus CB(2) receptors. Systemic administration of WIN decreased injury-induced mechanical allodynia and these effects were reversed by pretreatment with a CB(1) receptor antagonist, but not with a CB(2) receptor antagonist, given by systemic, intrathecal and supraspinal routes. In addition, peripheral administration of both CB(1) and CB(2) antagonists blocked systemic WIN-induced analgesic activity. CONCLUSIONS AND IMPLICATIONS: Both CB(1) and CB(2) receptors were involved in the peripheral anti-allodynic effect of systemic WIN in a pre-clinical model of post-operative pain. In contrast, the centrally mediated anti-allodynic activity of systemic WIN is mostly due to the activation of CB(1) but not CB(2) receptors at both the spinal cord and brain levels. However, the increased potency of WIN following i.c.v. administration suggests that its main site of action is at CB(1) receptors in the brain.

Structural aspects of high affinity ligands for the alpha 4 beta 2 neuronal nicotinic receptor.

Neuronal nicotinic acetylcholine receptors (nAChRs) are a heterogeneous family of related ion channels that are widely distributed throughout the central and peripheral nervous systems. They all share a common architecture of five subunit proteins that combine at the cell surface to create a ligand-gated cation permeable pore. Significant effort is currently being expended by medicinal chemistry teams to synthesize ligands that exhibit selectivity for central over peripheral nAChR subtypes. Within the CNS, multiple nAChR subtypes are recognized, and the discovery of ligands exhibiting selectivity among these subtypes offers an opportunity for the development of novel therapeutic agents. The alpha 4 beta 2 subtype is one of the most abundant nAChR subtypes within the CNS, and has been the primary focus of high affinity ligand design. Nicotine (1), and more recently, epibatidine (2) have served as structural templates for the design of the majority of active compounds. Although the diversity of nAChR ligands is growing, the structural requirements necessary for high affinity binding with the alpha 4 beta 2 receptor remain poorly understood. The putative pharmacophoric elements common to all potent alpha 4 beta 2 ligands include (1) a basic or quaternized nitrogen atom, and (2) a less basic nitrogen or a carbonyl oxygen that presumably interact with electron rich and electron deficient sites on the receptor, respectively. The family of currently known high affinity analogs consists of a diverse array of azacycles containing a basic amine. Several additional basic amine fragments have been identified, including the pyrrolizidine nucleus (exemplified by 8) and the 2-azabicyclo[2.2.1]heptane skeleton (exemplified by 9). In addition, we have found that the furo[2,3-b]pyridine heterocycle (compound 10) serves as useful bioisosteric replacement for the pyridyl substituent of nicotine. A preliminary pharmacophore model is proposed in which a reasonable superposition of the putative pharmacophoric elements of the diverse array of high affinity ligands for the alpha 4 beta 2 nAChR reported herein may be accommodated.