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RATIONALE

Nicotinic acetylcholine receptor (nAChR) agonists, partial agonists and antagonists have antidepressant-like effects in rodent models and reduce symptoms of depression in humans.

OBJECTIVE

The aim of this study was to determine if the β2* partial agonist sazetidine-A (sazetidine) showed an antidepressant-like effect in the forced swim test that was mediated by β2* nAChRs activation or desensitization.

RESULTS

Sazetidine, the less selective β2* partial agonist varenicline and the full β2* agonist 5-I-A8350, exhibited acute antidepressant-like effects in the forced swim test. The role of β2* nAChRs was confirmed by results showing 1) reversal of sazetidine’s antidepressant-like effects in the forced swim test by nAChR antagonists mecamylamine and dihydro-β-erythroidine (DHβE); 2) no effect of sazetidine in mice lacking the β2 subunit of the nAChR; and 3) a high correspondence between behaviorally active doses of sazetidine and β2* receptor occupancy. β2* receptor occupancy following acute sazetidine, varenicline, and 5-I-A8350 extended beyond the duration of action in the forced swim test. The long lasting receptor occupancy of sazetidine did not diminish behavioral efficacy in the forced swim test following repeated dosing.

CONCLUSIONS

These results demonstrate that activation of β2* nAChRs mediate sazetidine’s antidepressant-like actions and suggest that ligands that activate β2* nAChRs would be promising targets for the development of a new class of antidepressant.

In order to advance therapeutic applications of nicotinic ligands, continuing research efforts are being directed toward the identification and characterization of novel nicotinic acetylcholine receptor (nAChR) ligands that are both potent and subtype selective. Herein we report the synthesis and pharmacological evaluation of members of a new series of 3-alkoxy-5-aminopyridine derivatives that display good selectivity for the α4β2-nAChR subtype based on ligand binding and functional evaluations. The most potent ligand in this series, compound 64, showed high radioligand binding affinity and selectivity for rat α4β2-nAChR with a Ki value of 1.2 nM and 4700-fold selectivity for α4β2-over α3β4-nAChR, and ~100-fold selectivity for functional, high-sensitivity, human α4β2-nAChR over α3β4*-nAChR. In the mouse forced swim test, compound 64 exhibited antidepressant-like effects. Structure-activity relationship (SAR) analyses suggest that the introduction of additional substituents to the amino group present on the pyridine ring of the N-demethylated analogue of compound 17 can provide potent α4β2-nAChR-selective ligands for possible use in treatment of neurological and psychiatric disorders including depression.

Despite their discovery in the early 20th century and intensive study over the last twenty years, nicotinic acetylcholine receptors (nAChRs) are still far from being well understood. Only a few chemical entities targeting nAChRs are currently undergoing clinical trials, and even fewer have reached the marketplace. In our efforts to discover novel and truly selective nAChR ligands, we designed and synthesized a series of chiral cyclopropane-containing α4β2-specific ligands that display low nanomolar binding affinities and excellent subtype selectivity, while acting as partial agonists at α4β2-nAChRs. Their favorable antidepressant-like properties were demonstrated in the classical mouse forced swim test. Preliminary ADMET studies and broad screening towards other common neurotransmitter receptors were also carried out to further evaluate their safety profile and eliminate their potential off-target activity. These highly potent cyclopropane ligands possess superior subtype selectivity compared to other α4β2-nAChR agonists reported to date, including the marketed drug varenicline, and therefore may fully satisfy the crucial prerequisite for avoiding adverse side effects. These novel chemical entities could potentially be advanced to the clinic as new drug candidates for treating depression.

There is considerable evidence to support the hypothesis that the blockade of nAChR is responsible for the antidepressant action of nicotinic ligands. The nicotinic acetylcholine receptor (nAChR) antagonist, mecamylamine, has been shown to be an effective add-on in patients that do not respond to selective serotonin reuptake inhibitors. This suggests that nAChR ligands may address an unmet clinical need by providing relief from depressive symptoms in refractory patients. In this study, a new series of nAChR ligands based on an isoxazole-ether scaffold have been designed and synthesized for binding and functional assays. Preliminary structure-activity relationship (SAR) efforts identified a lead compound 43, which possesses potent antidepressant-like activity (1 mg/kg, IP; 5 mg/kg, PO) in the classical mouse forced swim test. Early stage absorption, distribution, metabolism, excretion, and toxicity (ADME-Tox) studies also suggested favorable drug-like properties, and broad screening towards other common neurotransmitter receptors indicated that compound 43 is highly selective for nAChRs over the other 45 neurotransmitter receptors and transporters tested.

AMOP-H-OH (6-[5-(azetidin-2-ylmethoxy)pyridin-3-yl]hex-5-yn-1-ol) and some of its sulfur-bearing analogs were tested for their actions in vitro at human α4β2-, α4β4-, α3β4*- and α1*-nicotinic acetylcholine receptors (nAChRs). AMOP-H-OH also was assessed in a model of antidepressant efficacy. AMOP-H-OH and some of its analogs have high potency and selectivity for α4β2-nAChRs over other nAChR subtypes. Effects are manifest as partial agonism, perhaps reflecting selectivity for high sensitivity (α4)3(β2)2-nAChRs. More prolonged exposure to AMOP-H-OH and its analogs produces inhibition of subsequent responses to acute challenges with nicotinic full agonists, again selectively for α4β2-nAChRs over other nAChR subtypes. The inhibition is mediated either via antagonism or desensitization of nAChR function, but the degree of inhibition of α4β2-nAChRs is limited by the drugs’ activities as partial agonists. Certain aspects of the in vitro pharmacology suggest that AMOP-H-OH and some of its analogs have a set of binding sites on α4β2-nAChRs that are distinct from those for full agonists. The in vitro pharmacological profile suggests that peripheral side effects of AMOP-H-OH or its analogs would be minimal and that their behavioral effects would be dominated by central nAChR actions. AMOP-H-OH also has profound and high potency antidepressant-like effects in the forced swim test. The net action of prolonged exposure to AMOP-H-OH or its analogs, as for nicotine, seems to be a selective decrease in α4β2-nAChR function. Inactivation of nAChRs may be a common neurochemical endpoint for nicotine dependence, its treatment, and some of its manifestations, including relief from depression.

Neuronal nicotinic acetylcholine receptors (nAChRs) can regulate the activity of many neurotransmitter pathways throughout the central nervous system and are considered important modulators of cognition and emotion. nAChRs also are the primary site of action in the brain for nicotine, the major addictive component of tobacco smoke. nAChRs consist of five membrane-spanning subunits (α and β isoforms) that can associate in various combinations to form functional nAChR ion channels. Because of a dearth of nAChR subtype-selective ligands, the precise subunit composition of the nAChRs that regulate the rewarding effects of nicotine and the development of nicotine dependence are unknown. However, the advent of mice with genetic nAChR subunit modifications has provided a useful experimental approach to assess the contribution of individual subunits in vivo. Here we review data generated from nAChR subunit knockout and genetically modified mice supporting a role for discrete nAChR subunits in nicotine reinforcement and dependence processes. Importantly, the rates of tobacco dependence are far higher in patients suffering from comorbid psychiatric illnesses compared with the general population, which may at least partly reflect disease-associated alterations in nAChR signaling. An understanding of the role of nAChRs in psychiatric disorders associated with high rates of tobacco addiction, therefore, may reveal novel insights into mechanisms of nicotine dependence. Thus, we also briefly review data generated from genetically modified mice to support a role for discrete nAChR subunits in anxiety disorders, depression, and schizophrenia.

Subtype-selective ligands are important tools for the pharmacological characterisation of neurotransmitter receptors. This is particularly the case for nicotinic acetylcholine receptors (nAChRs), given the heterogeneity of their subunit composition. In addition to agonists and antagonists that interact with the extracellular orthosteric nAChR binding site, a series of nAChR allosteric modulators have been identified that interact with a distinct transmembrane site. Here we report studies conducted with three pharmacologically distinct nicotinic ligands, an orthosteric agonist (compound B), a positive allosteric modulator (TQS) and an allosteric agonist (4BP-TQS). The primary focus of the work described in this study is to examine the suitability of these compounds for the characterisation of native neuronal receptors (both rat and human). However, initial experiments were conducted on recombinant nAChRs demonstrating the selectivity of these three compounds for α7 nAChRs. In patch-clamp recordings on rat primary hippocampal neurons we found that all these compounds displayed pharmacological properties that mimicked closely those observed on recombinant α7 nAChRs. However, it was not possible to detect functional responses with compound B, an orthosteric agonist, using a fluorescent intracellular calcium assay on either rat hippocampal neurons or with human induced pluripotent stem cell-derived neurons (iCell neurons). This is, presumably, due to the rapid desensitisation of α7 nAChR that is induced by orthosteric agonists. In contrast, clear agonist-evoked responses were observed in fluorescence-based assays with the non-desensitising allosteric agonist 4BP-TQS and also when compound B was co-applied with the non-desensitising positive allosteric modulator TQS. In summary, we have demonstrated the suitability of subtype-selective orthosteric and allosteric ligands for the pharmacological identification and characterisation of native nAChRs and the usefulness of ligands that minimise receptor desensitisation for the characterisation of α7 nAChRs in fluorescence-based assays.

Recently, the smoking cessation therapeutic varenicline, a nicotinic acetylcholine receptor (nAChR) partial agonist, has been shown to reduce alcohol consumption. However, the mechanism and nAChR subtype(s) involved are unknown. Here we demonstrate that varenicline and alcohol exposure, either alone or in combination, selectively activates dopaminergic (DAergic) neurons within the posterior, but not the anterior, ventral tegmental area (VTA). To gain insight into which nAChR subtypes may be involved in the response to alcohol, we analyzed nAChR subunit gene expression in posterior VTA DAergic neurons. Ethanol-activated DAergic neurons expressed higher levels of α4, α6, and β3 subunit genes compared to non-activated neurons. To examine the role of nicotinic receptors containing the α4 subunit (α4* nAChRs) in varenicline-induced reduction of alcohol consumption, we examined the effect of the drug in two complementary mouse models, a knockout line that does not express the α4 subunit (α4 KO) and another line that expresses α4* nAChRs hypersensitive to agonist (Leu9′Ala). While varenicline (0.1 – 0.3 mg/kg, i.p.) reduced 2 % and 20 % alcohol consumption in wildtype (WT) mice, the drug did not significantly reduce consumption in α4 KO animals. Conversely, low doses of varenicline (0.0125 – 0.05 mg/kg, i.p.) that had little effect in WT mice dramatically reduced ethanol intake in Leu9′fAla mice. Infusion of varenicline into the posterior, but not the anterior VTA was sufficient to reduce alcohol consumption. Together, our data indicate that activation of α4* nAChRs is necessary and sufficient for varenicline reduction of alcohol consumption.

Alcohol use disorders (AUDs) impact millions of individuals and there remain few effective treatment strategies. Despite evidence that neuronal nicotinic acetylcholine receptors (nAChRs) play a role in AUDs, it has not been established which subtypes of the nAChR are involved. Recent human genetic association studies have implicated the gene cluster CHRNA3-CHRNA5-CHRNB4 encoding the α3, α5 and β4 subunits of the nAChR in susceptibility to develop nicotine and alcohol dependence; however, their role in ethanol-mediated behaviors is unknown due to the lack of suitable and selective research tools. To determine the role of the α3, and β4 subunits of the nAChR in ethanol self-administration, we developed and characterized high affinity partial agonists at α3β4 nAChRs, CP-601932 and PF-4575180. Both CP-601932 and PF-4575180 selectively decrease ethanol but not sucrose consumption and operant self-administration following long-term exposure. We show that the functional potencies of CP-601932 and PF-4575180 at α3β4 nAChRs correlate with their unbound rat brain concentrations suggesting that the effects on ethanol self-administration are mediated via interaction with α3β4 nAChRs. Also varenicline, an approved smoking cessation aid previously shown to decrease ethanol consumption and seeking in rats and mice, reduces ethanol intake at unbound brain concentrations that allow functional interactions with α3β4 nAChRs. Furthermore, the selective α4β2* nAChR antagonist, DHβE, did not reduce ethanol intake. Together, these data provide further support for the human genetic association studies implicating CHRNA3 and CHRNB4 genes in ethanol-mediated behaviors. CP-601932 has been shown to be safe in humans and may represent a potential novel treatment for AUDs.

Alcohol use disorders (AUDs) impact millions of individuals and there remain few effective treatment strategies. Despite evidence that neuronal nicotinic acetylcholine receptors (nAChRs) have a role in AUDs, it has not been established which subtypes of the nAChR are involved. Recent human genetic association studies have implicated the gene cluster CHRNA3–CHRNA5–CHRNB4 encoding the α3, α5, and β4 subunits of the nAChR in susceptibility to develop nicotine and alcohol dependence; however, their role in ethanol-mediated behaviors is unknown due to the lack of suitable and selective research tools. To determine the role of the α3, and β4 subunits of the nAChR in ethanol self-administration, we developed and characterized high-affinity partial agonists at α3β4 nAChRs, CP-601932, and PF-4575180. Both CP-601932 and PF-4575180 selectively decrease ethanol but not sucrose consumption and operant self-administration following long-term exposure. We show that the functional potencies of CP-601932 and PF-4575180 at α3β4 nAChRs correlate with their unbound rat brain concentrations, suggesting that the effects on ethanol self-administration are mediated via interaction with α3β4 nAChRs. Also varenicline, an approved smoking cessation aid previously shown to decrease ethanol consumption and seeking in rats and mice, reduces ethanol intake at unbound brain concentrations that allow functional interactions with α3β4 nAChRs. Furthermore, the selective α4β2* nAChR antagonist, DHβE, did not reduce ethanol intake. Together, these data provide further support for the human genetic association studies, implicating CHRNA3 and CHRNB4 genes in ethanol-mediated behaviors. CP-601932 has been shown to be safe in humans and may represent a potential novel treatment for AUDs.

Previous studies have suggested that treatment with antagonists or partial agonists of nicotinic acetylcholine receptors containing the β2 subunit (β2* nAChRs) results in antidepressant-like effects. In the current study we tested 3 novel compounds with different affinity and functional efficacy at α4β2* nAChRs, which were synthesized as part of nAChR discovery projects at Pfizer in the tail suspension, forced swim and novelty-suppressed feeding tests of antidepressant efficacy. All compounds tested reduced immobility in the forced swim test and one of the compounds also reduced immobility in the tail suspension test. All the compounds appeared to affect food intake on their own, with 2 compounds reducing feeding significantly in the home cage, precluding a clear interpretation of the results in the novelty-suppressed feeding test. None of the compounds altered locomotor activity at the doses and time points used here. Therefore, a subset of these compounds has pharmacological and behavioral properties that demonstrate the potential of nicotinic compounds as a treatment of mood disorders. Further development of nicotinic-based antidepressants should focus on increasing nAChR subtype selectivity to obtain consistent antidepressant properties with an acceptable side effect profile.

The nicotinic acetylcholine receptor (nAChR) β3 subunit is thought to serve an accessory role in nAChR subtypes expressed in dopaminergic regions implicated in drug dependence and reward. When β3 subunits are expressed in excess, they have a dominant-negative effect on function of selected nAChR subtypes. In this study, we show, in Xenopus oocytes expressing α2, α3 or α4 plus either β2 or β4 subunits, that in the presumed presence of similar amounts of each nAChR subunit, co-expression with wild-type β3 subunits generally (except for α3*-nAChR) lowers amplitudes of agonist-evoked, inward peak currents by 20–50% without having dramatic effects (≤ 2-fold) on agonist potencies. By contrast, co-expression with mutant β3V9'S subunits generally (except for α4β2*-nAChR) increases agonist potencies, consistent with an expected gain-of-function effect. This most dramatically demonstrates formation of complexes containing three kinds of subunit. Moreover, for oocytes expressing nAChR containing any α subunit plus β4 and β3V9'S subunits, there is spontaneous channel opening sensitive to blockade by the open channel blocker, atropine. Collectively, the results indicate that β3 subunits integrate into all of the studied receptor assemblies and suggest that natural co-expression with β3 subunits can influence levels of expression and agonist sensitivities of several nAChR subtypes.

An important new area of antidepressant drug development involves targeting the nicotinic acetylcholine receptor (nAChR). This receptor, which is distributed widely in regions of the brain associated with depression, is also implicated in other important processes that are relevant to depression, such as stress and inflammation. The two classes of drugs that target nAChRs can be broadly divided into mecamylamine- and cytisine-based compounds. These drugs probably exert their effects via antagonism at α4β2 nAChRs, and strong preclinical data support the antidepressant efficacy of both classes when used in conjunction with other primary antidepressants (e.g., monoamine reuptake inhibitors). Although clinical data remain limited, preliminary results in this area constitute a compelling argument for further evaluation of the nAChR as a target for future antidepressant drug development.

This review summarizes studies that attempted to determine the subtypes of nicotinic acetylcholine receptors (nAChR) expressed in the dopaminergic nerve terminals in the mouse. A variety of experimental approaches has been necessary to reach current knowledge of these subtypes, including in situ hybridization, agonist and antagonist binding, function measured by neurotransmitter release from synaptosomal preparations, and immunoprecipitation by selective antibodies. Early developments that facilitated this effort include the radioactive labeling of selective binding agents, such as [125I]-α-bungarotoxin and [3H]-nicotine, advances in cloning the subunits, and expression and evaluation of function of combinations of subunits in Xenopus oocytes. The discovery of epibatidine and α-conotoxin MII (α-CtxMII), and the development of nAChR subunit null mutant mice have been invaluable in determining which nAChR subunits are important for expression and function in mice, as well as allowing validation of the specificity of subunit specific antibodies. These approaches have identified five nAChR subtypes of nAChR that are expressed on dopaminergic nerve terminals. Three of these contain the α6 subunit (α4α6β2β3, α6β2β3, α6β2) and bind α-CtxMII with high affinity. One of these three subtypes (α4α6β2β3) also has the highest sensitivity to nicotine of any native nAChR that has been studied, to date. The two subtypes that do not have high affinity for α-CtxMII (α4β2, α4α5β2) are somewhat more numerous than the α6* subtypes, but do bind nicotine with high affinity. Given that our first studies detected readily measured differences in sensitivity to agonists and antagonists among these five nAChR subtypes, it seems likely that subtype selective compounds could be developed that would allow therapeutic manipulation of diverse nAChRs that have been implicated in a number of human conditions.

The nicotine in tobacco is thought to modulate neuronal systems regulating mood. Moreover, it appears possible that blockade rather than activation of β2-containing (β2*) nicotinic acetylcholine receptors (nAChRs) may lead to antidepressant-like effects. We used cytisine, a partial agonist of α4/β2* nAChRs and a full agonist at α3/β4* nAChRs, in several tests of antidepressant efficacy. Further, we used c-fos expression to identify potential neurobiological correlates of the antidepressant-like effects of cytisine. Cytisine had antidepressant-like effects in several animal models of antidepressant efficacy. In addition, immunohistochemical analyses indicated that cytisine could reduce c-fos immunoreactivity in the basolateral amygdala by ~ 50%. These data show that cytisine acts like classical antidepressants in rodent models of antidepressant efficacy. In addition, cytisine’s ability to block α4/β2* nAChRs may be responsible for its antidepressant-like properties, and these may be mediated through a reduction of neuronal activity in the basolateral amygdala. These studies also suggest that both antagonists and partial agonists of α4/β2* nAChRs would be interesting targets for the development of novel antidepressant drugs.

Recently, we investigated the molecular mechanisms of the smoking cessation drug varenicline, a nicotinic acetylcholine receptor (nAChR) partial agonist, in its ability to decrease voluntary ethanol intake in mice. Previous to our study, other labs had shown that this drug can decrease ethanol consumption and seeking in rat models of ethanol intake. Although varenicline was designed to be a high affinity partial agonist of nAChRs containing the α4 and β2 subunits (designated as α4β2*), at higher concentrations it can also act upon α3β2*, α6*, α3β4* and α7 nAChRs. Therefore, to further elucidate the nAChR subtype responsible for varenicline-induced reduction of ethanol consumption, we utilized a pharmacological approach in combination with two complimentary nAChR genetic mouse models, a knock-out line that does not express the α4 subunit (α4 KO) and another line that expresses α4* nAChRs hypersensitive to agonist (the Leu9′Ala line). We found that activation of α4* nAChRs was necessary and sufficient for varenicline-induced reduction of alcohol consumption. Consistent with this result, here we show that a more efficacious nAChR agonist, nicotine, also decreased voluntary ethanol intake, and that α4* nAChRs are critical for this reduction.

Muscarinic and nicotinic acetylcholine (ACh) receptors (mAChRs and nAChRs) are emerging as important targets for the development of novel treatments for the symptoms associated with schizophrenia. Preclinical and early proof-of-concept clinical studies have provided strong evidence that activators of specific mAChR (M1 and M4) and nAChR (α7 and α2β4) subtypes are effective in animal models of antipsychotic-like activity and/or cognitive enhancement, and in the treatment of positive and cognitive symptoms in patients with schizophrenia. While early attempts to develop selective mAChR and nAChR agonists provided important preliminary findings, these compounds have ultimately failed in clinical development due to a lack of true subtype selectivity and subsequent dose-limiting adverse effects. In recent years, there have been major advances in the discovery of highly selective activators for the different mAChR and nAChR subtypes with suitable properties for optimization as potential candidates for clinical trials. One novel strategy has been to identify ligands that activate a specific receptor subtype through actions at sites that are distinct from the highly conserved ACh-binding site, termed allosteric sites. These allosteric activators, both allosteric agonists and positive allosteric modulators, of mAChR and nAChR subtypes demonstrate unique mechanisms of action and high selectivity in vivo, and may provide innovative treatment strategies for schizophrenia.

Acetylcholine (ACh) is the major neurotransmitter released from vestibular efferent terminals onto hair cells and afferents. Previous studies indicate that the two classes of acetylcholine receptors, nicotinic (nAChRs) and muscarinic receptors (mAChRs), are expressed by vestibular hair cells (VHCs). To identify if both classes of receptors are present in VHCs, whole cell, voltage-clamp- and current-clamp- patch recordings were performed on isolated pigeon vestibular type I and type II HCs during the application of the cholinergic agonists, acetylcholine and carbachol, and the cholinergic antagonists, d-tubocurarine and atropine. By applying in different combinations, these compounds were used to selectively activate either nAChRs or mAChRs. The effects of nAChR and mAChR activation on HC currents and zero electrode current potential (Vz) were monitored. It was found that presumed mAChR activation decreased both inward and outward currents in both type I and type II HCs, resulting in either a depolarization or hyperpolarization. Conversely, nAChR activation mainly increased both inward and outward currents in type II HCs, resulting in a hyperpolarization of their Vz. nAChR activation also increased outward currents in type I HCs resulting in either a depolarization or hyperpolarization of their Vz. The decrease of inward and outward currents and the depolarization of the Vz in type I pigeon HCs by activation of mAChRs represents a new finding. Ion channel candidates in pigeon vestibular HCs that might underlie the modulation of the macroscopic ionic currents and Vz by different AChR activation are discussed.

The most abundant subtype of cerebral nicotinic acetylcholine receptors (nAChR), α4β2, plays a critical role in various brain functions and pathological states. Imaging agents suitable for visualization and quantification of α4β2 nAChRs by positron emission tomography (PET) would present unique opportunities to define the function and pharmacology of the nAChRs in the living human brain. In this study, we report the synthesis, nAChR binding affinity, and pharmacological properties of several novel 3-pyridyl ether compounds. Most of these derivatives displayed a high affinity to the nAChR and a high subtype selectivity for α4β2-nAChR. Three of these novel nAChR ligands were radiolabeled with the positron-emitting isotope 11C and evaluated in animal studies as potential PET radiotracers for imaging of cerebral nAChRs with improved brain kinetics.

Background

Although converging evidence has suggested that nicotinic acetylcholine receptors (nAChR) play a role in the modulation of GABA release in rat hippocampus, the specific involvement of different nAChR subtypes at presynaptic level is still a matter of debate. In the present work we investigated, using selective α7 and α4β2 nAChR agonists, the presence of different nAChR subtypes on hippocampal GABA nerve endings to assess to what extent and through which mechanisms they stimulate endogenous GABA release.

Methodology/Findings

All agonists elicited GABA overflow. Choline (Ch)-evoked GABA overflow was dependent to external Ca2+, but unaltered in the presence of Cd2+, tetrodotoxin (TTX), dihydro-β-erythroidine (DHβE) and 1-(4,4-Diphenyl-3-butenyl)-3-piperidinecarboxylic acid hydrochloride SKF 89976A. The effect of Ch was blocked by methyllycaconitine (MLA), α-bungarotoxin (α-BTX), dantrolene, thapsigargin and xestospongin C, suggesting that GABA release might be triggered by Ca2+ entry into synaptosomes through the α7 nAChR channel with the involvement of calcium from intracellular stores. Additionally, 5-Iodo-A-85380 dihydrochloride (5IA85380) elicited GABA overflow, which was Ca2+ dependent, blocked by Cd2+, and significantly inhibited by TTX and DHβE, but unaffected by MLA, SKF 89976A, thapsigargin and xestospongin C and dantrolene. These findings confirm the involvement of α4β2 nAChR in 5IA85380-induced GABA release that seems to occur following membrane depolarization and opening calcium channels.

Conclusions/Significance

Rat hippocampal synaptosomes possess both α7 and α4β2 nAChR subtypes, which can modulate GABA release via two distinct mechanisms of action. The finding that GABA release evoked by the mixture of sub-maximal concentration of 5IA85380 plus sub-threshold concentrations of Ch was significantly larger than that elicited by the sum of the effects of the two agonists is compatible with the possibility that they coexist on the same nerve terminals. These findings would provide the basis for possible selective pharmacological strategies to treat neuronal disorders that involve the dysfunction of hippocampal cholinergic system.

Structure-based drug design can potentially accelerate the development of new therapeutics. In this study, a co-crystal structure of the acetylcholine binding protein (AChBP) from Capitella teleta (Ct) in complex with a cyclopropane-containing, selective α4β2-nicotinic acetylcholine receptor (nAChR) partial agonist (compound 5) was acquired. The structural determinants required for ligand binding obtained from this AChBP X-ray structure were used to refine our previous model of the human α4β2-nAChR, thus possibly providing a better understanding of the structure of the human receptor. In order to validate the potential application of the structure of the Ct-AChBP in the engineering of new α4β2-nAChR ligands, homology modeling methods, combined with in silico ADME calculations, were used to design analogs of compound 5. The most promising compound 12, exhibited an improved metabolic stability in comparison to the parent compound 5 while retaining favorable pharmacological parameters together with appropriate behavioral endpoints in the rodent studies.

Small airway-derived pulmonary adenocarcinoma (PAC) and pancreatic ductal adenocarcinoma (PDAC) are among the most common human cancers and smoking is a risk factor for both. Emerging research has identified cAMP signaling stimulated by the stress neurotransmitters adrenaline and noradrenaline as important stimulators of several adenocarcinomas, including PAC and PDAC. The nicotine-derived nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent mutagen and the most powerful tobacco carcinogen. NNK is also an agonist for nicotinic acetylcholine receptors (nAChRs). Using hamster models of NNK-induced PAC and PDAC, we have tested the hypothesis that in analogy to chronic effects of nicotine in the brain, NNK may modulate the α7- and α4β2nAChRs, causing an increase in stress neurotransmitters and decrease in the inhibitory neurotransmitter γ-aminobutyric acid (GABA). In support of our hypothesis, immunoassays showed a significant increase in serum adrenaline/noradrenaline and increased intracellular cAMP in the cellular fraction of blood of NNK treated hamsters. Western blots were done with cells harvested by laser capture microcopy from control small airway epithelia, alveolar epithelia, pancreatic islet and pancreatic duct epithelia and from NNK-induced PACs and PDACs. The GABA synthesizing enzyme glutamate decarboxylase 65 (GAD65) and GABA were suppressed in NNK-induced PACs and PDACs whereas protein expression of the α7nAChR, α4nAChR as well as p-CREB and p-ERK1/2 were upregulated. These findings suggest, for the first time, that NNK-induced alterations in regulatory nAChRs may contribute to the development of smoking-associated PAC and PDAC by disturbing the balance between cancer stimulating and inhibiting neurotransmitters.

Nicotinic acetylcholine receptors (nAchR) are key receptors in the autonomic nervous system, but also are present on immune cells. The alpha seven subunit of nAchR (α7nAchR) suppresses pro-inflammation in peripheral monocytes by decreasing proinflammatory cytokine production. In spinal cord, α7nAchR are found on microglia, which are known to induce and maintain pain. We predicted that α7nAchR agonists might attenuate intrathecal HIV-1 gp120-induced, proinflammatory cytokine- and microglia-dependent mechanical allodynia. Choline, a precursor for acetylcholine and selective agonist for α7nAchR, was administered intrathecally either with, or 30 min after, intrathecal gp120. Choline significantly blocked and reversed gp120 induced mechanical allodynia for at least 4 hr after drug administration. In addition, intrathecal choline, delivered either with or 30 min after gp120, reduced gp120-induced IL-1β protein and pro-inflammatory cytokine mRNAs within the lumbar spinal cord. A second α7nAchR agonist, GTS-21, also significantly reversed gp120-induced mechanical allodynia and lumbar spinal cord levels of proinflammatory cytokine mRNAs and IL-1β protein. A role of microglia is suggested by the observation that intrathecal choline suppressed the gp120-induced expression of, cd11b, a macrophage/microglial activation marker. Taken together, the data support that α7nAchR may be a novel target for treating pain where microglia maintain the proinflammatory state within the spinal cord.

The α7 nAChR subtype is of particular interest as a potential therapeutic target since it has been implicated as a mediator of both cognitive and neuroprotective activity. The rigid nicotine analog ACME and the N-cyanoborane conjugate ACME-B are selective partial agonists of rat α7 receptors expressed in Xenopus oocytes, with no significant activation of either α3β4 or α4β2 receptors. ACME-B is both more potent and efficacious than ACME. The efficacies of ACME-B and ACME are approximately 26% and 10% of the efficacy of ACh, respectively. Similar N-conjugation of S(−)nicotine with cyanoborane decreased efficacy for α3β4 and α4β2 receptors, as well as for α7 nAChR. Structural comparison of ACME with the benzylidene anabaseines, another class of previously identified α7-selective agonists, suggests that they share a similar structural motif that may be applicable to other α7-selective agonists.

Limitations in efficacy and high relapse rates of currently available smoking cessation agents reveal the need for more efficacious pharmacotherapies. One strategy is to develop subtype-selective nicotinic receptor (nAChR) antagonists that inhibit nicotine-evoked dopamine (DA) release, the primary neurotransmitter involved in nicotine reward. Simple alkylation of the pyridino N-atom converts nicotine from a potent agonist into a potent antagonist. The classical antagonists, hexamethonium and decamethonium, differentiate between peripheral nAChR subtypes. Using a similar approach, we interconnected varying quaternary ammonium moieties with a lipophilic linker to provide N,N′- bis-nicotinium analogs, affording a lead compound, N,N′-dodecyl-1,12-diyl-bis-3-picolinium dibromide (bPiDDB), which inhibited nicotine-evoked DA release and decreased nicotine self-administration. The current work describes a novel compound, 1-(3-picolinium)-12-triethylammonium-dodecane dibromide (TMPD), a hybrid of bPiDDB and decamethonium. TMPD completely inhibited (IC50 = 500 nM) nicotine-evoked DA release from superfused rat striatal slices, suggesting that TMPD acts as a nAChR antagonist at more than one subtype. TMPD (1 μM) inhibited the response to acetylcholine at α3β4, α4β4, α4β2, and α1β1εδ receptors expressed in Xenopus oocytes. TMPD had a 2-fold higher affinity for the blood-brain barrier choline transporter, suggesting that is brain bioavailable. TMPD did not inhibit the hyperactivity in nicotine sensitized rats, but significantly and specifically decreased nicotine self-administration. Together, the results suggest that TMPD may have the ability to reduce the rewarding effect of nicotine with minimal side effects, a pharmacological profile indicative of potential clinical utility for the treatment of tobacco dependence.