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Recently, alpha 7 nicotinic acetylcholine receptors (nAChRs), primarily activated by binding of orthosteric agonists, represent a target for anti-inflammatory and analgesic drug development. These receptors may also be modulated by positive allosteric modulators (PAMs), ago-allosteric ligands (ago-PAMs), and alpha 7-silent agonists. Activation of 00 nAChRs has been reported to increase the brain levels of endogenous ligands for nuclear peroxisome proliferator-activated receptors type-alpha (PPAR-alpha), palmitoylethanolamide (PEA) and oleoylethanolamide (OEA), in a Ca2+-dependent manner. Here, we investigated potential crosstalk between alpha 7 nAChR and PPAR-alpha, using the formalin test, a mouse model of tonic pain. Using pharmacological and genetic approaches, we found that PNU282987, a full alpha 7 agonist, attenuated formalin-induced nociceptive behavior in alpha 7 -dependent manner. Interestingly, the selective PPAR-alpha antagonist GW6471 blocked the antinociceptive effects of PNU282987, but did not alter the antinociceptive responses evoked by the alpha 7 nAChR PAM PNU120596, ago-PAM GAT107, and silent agonist NS6740. Moreover, GW6471 administered systemically or spinally, but not via the intraplantar surface of the formalin-injected paw blocked PNU282987-induced antinociception. Conversely, exogenous administration of the naturally occurring PPAR-alpha agonist PEA potentiated the antinociceptive effects of PNU282987. In contrast, the cannabinoid 031 antagonist rimonabant and the CB2 antagonist SR144528 failed to reverse the antinociceptive effects of PNU282987. These findings suggest that PPAR-alpha plays a key role in a putative antinociceptive alpha 7 nicotinic signaling pathway. United States Department of Health & Human Services National Institutes of Health (NIH) - USA - GM57481 - R01 CA206028 United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Cancer Institute (NCI) - R01CA206028 United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Institute of General Medical Sciences (NIGMS) - R01GM057481 United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Institute on Drug Abuse (NIDA) European Commission - T32DA007027

Introduction: α7 nicotinic acetylcholine receptors (nAChRs) play an important role in vagus nerve-based cholinergic anti-inflammatory effects. This study was designed to assess the role of α7 nAChRs in dextran sodium sulfate (DSS)-induced colitis in male and female mouse. We first compared disease activity and pathogenesis of colitis in α7 knockout and wild-type mice. We then evaluated the effect of several α7 direct and indirect agonists on the severity of disease in the DSS-induced colitis. Methods: Male and female adult mice were administered 2.5% DSS solution freely in the drinking water for 7 consecutive days and the colitis severity (disease activity index) was evaluated as well as colon length, colon histology, and levels of tumor necrosis factor-alpha colonic levels. Results: Male, but not female, α7 knockout mice displayed a significantly increased colitis severity and higher tumor necrosis factor-alpha levels as compared with their littermate wild-type mice. Moreover, pretreatment with selective α7 ligands PHA-543613, choline, and PNU-120596 decreased colitis severity in male but not female mice. The anti-colitis effects of these α7 compounds dissipated when administered at higher doses. Conclusions: Our results suggest the presence of a α7-dependent anti-colitis endogenous tone in male mice. Finally, our results show for the first time that female mice are less sensitive to the anti-colitis activity of α7 agonists. Ovarian hormones may play a key role in the sex difference effect of α7 nAChRs modulation of colitis in the mouse. Implications: Our collective results suggest that targeting α7 nAChRs could represent a viable therapeutic approach for intestinal inflammation diseases such as ulcerative colitis with the consideration of sex differences.

Background and PurposeOrthosteric agonists and positive allosteric modulators (PAMs) of the 7 nicotinic ACh receptor (nAChR) represent novel therapeutic approaches for pain modulation. Moreover, compounds with dual function as allosteric agonists and PAMs, known as ago-PAMs, add further regulation of receptor function. Experimental ApproachInitial studies examined the 7 ago-PAM, GAT107, in the formalin, complete Freund's adjuvant (CFA), LPS inflammatory pain models, the chronic constriction injury neuropathic pain model and the tail flick and hot plate acute thermal nociceptive assays. Additional studies examined the locus of action of GAT107 and immunohistochemical markers in the dorsal horn of the spinal cord in the CFA model. Key ResultsComplementary pharmacological and genetic approaches confirmed that the dose-dependent antinociceptive effects of GAT107 were mediated through 7 nAChR. However, GAT107 was inactive in the tail flick and hot plate assays. In addition, GAT107 blocked conditioned place aversion elicited by acetic acid injection. Furthermore, intrathecal, but not intraplantar, injections of GAT107 reversed nociception in the CFA model, suggesting a spinal component of action. Immunohistochemical evaluation revealed an increase in the expression of astrocyte-specific glial fibrillary acidic protein and phosphorylated p38MAPK within the spinal cords of mice treated with CFA, which was attenuated by intrathecal GAT107 treatment. Importantly, GAT107 did not elicit motor impairment and continued to produce antinociceptive effects after subchronic administration in both phases of the formalin test. Conclusions and ImplicationsCollectively, these results provide the first proof of principle that 7 ago-PAMs represent an effective pharmacological strategy for treating inflammatory and neuropathic pain. Pilot Project from VCU Massey Cancer Center United States Department of Health & Human Services National Institutes of Health (NIH) - USA - DA032246 NIH National Institute on Drug Abuse (NIDA) European Commission United States Department of Health & Human Services National Institutes of Health (NIH) - USA - GM57481 - EY024717 - DA038493-01A1 R01DA032246 P30DA033934 F32DA038493

Chronic pain remains one of the most challenging of all neurologic diseases and, currently approved drugs have only a modest efficacy in several patient groups and numerous side effects. In recent years, a variety of structurally distinct α7 nicotinic acetylcholine receptor (nAChR) agonists have been developed and profiled for a variety of neurologic diseases. In particular, some of these agonists have been shown to have therapeutic significance in reducing inflammation and nociception, as well as in providing neuroprotection in animal models.1-8 The α7 nAChRs were targeted because they are expressed on both supraspinal and spinal pain transmission pathways.9-14 These receptors are also found on immune and nonimmune cytokine-producing cells, such as macrophages and keratinocytes.15 However, there are still a number of uncertainties in the development of α7 nAChR agonists for the treatment of pain, including receptor selectivity (namely cross-reactivity with 5-HT3 receptors, which have high homology with α7 nAChRs) and possible adverse effects.16,17 In addition, although α7 nAChR agonists have shown beneficial effects in chronic pain models in some studies, this effect was not consistently seen in other studies.18 An alternative therapeutic approach has been the development of positive allosteric modulators (PAMs), which can synergize and augment orthosteric-site–mediated signaling of endogenous neurotransmitters, including acetylcholine and choline, without, in most cases, directly activating or desensitizing the receptor. Several selective α7 nAChR PAMs have been reported and classified as type I and type II. Type I PAMs increase peak agonist-evoked responses, but have little or no effect on the decay rate of macroscopic currents or the equilibrium desensitization of α7 nAChRs, whereas type II PAMs both increase peak currents and slow down the apparent desensitization profile of the agonist response and/or recover nAChRs from the desensitized state.19,20 Both PAM types have been recently tested in vivo for their efficacy in animal models of inflammation and neuropathic pain; however, type II but not type I PAMs were shown to be effective in neuropathic pain models.21,22 Most studies with α7 nAChR PAMs in chronic pain models were conducted with PNU-120596,21-24 and more recently with the newly reported 2,4,20,50-tetrahydroxychalcone.25 Hence, additional studies are needed to fully explore and investigate the analgesic-like properties of α7 nAChR PAMs in animal models of chronic pain. Therefore, in this study, we sought to evaluate the anti-nociceptive and antiinflammatory effects of 3-furan-2-yl-N-p-tolyl-acrylamide (PAM-2), a novel and putative selective α7 nAChR type II PAM,26,27 using different mouse models of chronic pain. PAM-2 has recently been found to enhance α7 nAChR activity in vitro26 and to produce antidepressant activity in mice in vivo.27 The first aim of this study was to evaluate whether PAM-2 produces antiallodynic or antihyperalgesic activities in several mouse inflammatory and neuropathic pain models. We also assessed the effects of PAM-2 on aversion, an important affective component of pain.

The alpha 7 nicotinic acetylcholine receptor (nAChR) is a promising drug target for a number of neurological disorders including chronic pain and inflammatory diseases. Since alpha 7 can function as a ligand-gated ion channel, drug development initially focused on ligands that were selective activators of the alpha 7 ion channel. However, the best alpha 7 drugs for chronic pain and inflammation indications may not be ion channel activators but rather "silent agonists", which bind to the receptor but preferentially induce non-conducting states that modulate signal transduction in non-neuronal cells. One such compound is NS6740. We show that NS6740 selectively induces prolonged desensitization of alpha 7 nAChRs. There are two forms of alpha 7 desensitization that can be distinguished by their sensitivity to the positive allosteric modulators (PAMs). At high concentrations, NS6740 preferentially induces PAM-insensitive desensitization, which over the course of several minutes reverts to the sensitive form. NS6740 was tested in several pain models after in vivo administration in the mouse. Although it had no effects in acute thermal pain, NS6740 induced significant dose- and time-dependent antinociceptive activity in formalin- and acetic acid-induced nociceptive behaviors as well as in the chronic constrictive nerve injury (CCI) model for neuropathic pain. The antinociceptive activity of NS6740 in these models was alpha 7-dependent. In addition, NS6740 administration reversed pain-induced aversion, an important affective component of pain. The time and concentration dependence of the effects were consistent with NS6740 induction of PAM-insensitive non-conducting states, suggesting that signal transduction required for analgesia is accomplished by alpha 7 receptors in that conformation. VCU Massey Cancer Center (A-35337) United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Institute on Drug Abuse (NIDA) European Commission (DA032246) United States Department of Health & Human Services National Institutes of Health (NIH) - USA (GM57481) United States Department of Health & Human Services National Institutes of Health (NIH) - USA (DA027113) United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Institute of General Medical Sciences (NIGMS) (R01GM057481) United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Institute on Drug Abuse (NIDA) European Commission (R01DA032246) United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Institute on Drug Abuse (NIDA) European Commission (R01DA012610) United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Institute on Drug Abuse (NIDA) European Commission (R03DA027113)