Anticholinergic therapy for overactive bladder: a nicotinic modality?

Until recently the treatment of Overactive Bladder (OAB) has primarily been aimed at mitigating hypercholinergic activity in the bladder via antagonism of muscarinic acetylcholine receptors. However, antimuscarinic therapies have limited efficacy and significant side effects. It is now known that nicotinic acetylcholine receptor (nAChR) subtypes are expressed in the urothelium and on afferent nerve fibers in the bladder, and it is believed that these receptors serve to communicate urgency and facilitate voiding function. This presents the opportunity for an alternative to the antimuscarinic approach, one which involves inhibition of nAChRs in the bladder that are chronically overstimulated by acetylcholine. Specifically, we hypothesize that an orally administered nAChR-selective inhibitor with extensive renal elimination will result in higher local concentrations in the bladder and lower systemic exposure than current therapies, representing a novel targeted approach to the treatment of OAB with a more favorable side effect profile.

Validation of a high-throughput, automated electrophysiology platform for the screening of nicotinic agonists and antagonists.

High-throughput compound screening using electrophysiology-based assays represents an important tool for biomedical research and drug discovery programs. The recent development and availability of devices capable of performing high-throughput electrophysiology-based screening have brought the need to validate these tools by producing data that are consistent with results obtained with conventional electrophysiological methods. In this study, we compared the response properties of hα3ß4 and hα4ß2 nicotinic receptors to their endogenous ligand acetylcholine (ACh) using three separate electrophysiology platforms: Dynaflow (low-throughput, manual system), PatchXpress 7000A (medium-throughput automated platform), and IonWorks Barracuda (high-throughput automated platform). We found that despite the differences in methodological approaches between these technologies, the EC(50) values from the ACh dose-response curves were consistent between all three platforms. In addition, we have validated the IonWorks Barracuda for both competitive and uncompetitive inhibition assays by using the competitive nicotinic antagonist dihydro-beta-erythroidin (DHßE) and uncompetitive nicotinic antagonist mecamylamine. Furthermore, we have demonstrated the utility of a custom-written algorithm for generating dose-response curves from multiple extrapolated current metrics that allows for discriminating between competitive and uncompetitive inhibition while maintaining high-throughput capacity. This study provides validation of the consistency of results using low-, medium-, and high-throughput electrophysiology platforms and supports their use for screening nicotinic compounds.

Functional and structural interaction of (-)-reboxetine with the human α4ß2 nicotinic acetylcholine receptor.

The interaction of the selective norepinephrine reuptake inhibitor (-)-reboxetine with the human α4ß2 nicotinic acetylcholine receptor (nAChR) in different conformational states was studied by several functional and structural approaches. Patch-clamp and Ca(2+)-influx results indicate that (-)-reboxetine does not activate hα4ß2 nAChRs via interaction with the orthosteric sites, but inhibits agonist-induced hα4ß2 activation by a noncompetitive mechanism. Consistently, the results from the electrophysiology-based functional approach suggest that (-)-reboxetine may act via open channel block; therefore, it is capable of producing a use-dependent type of inhibition of the hα4ß2 nAChR function. We tested whether (-)-reboxetine binds to the luminal [(3)H]imipramine site. The results indicate that, although (-)-reboxetine binds with low affinity to this site, it discriminates between the resting and desensitized hα4ß2 nAChR ion channels. Patch-clamp results also indicate that (-)-reboxetine progressively inhibits the hα4ß2 nAChR with two-fold higher potency at the end of one-second application of agonist, compared with the peak current. The molecular docking studies show that (-)-reboxetine blocks the ion channel at the level of the imipramine locus, between M2 rings 6' and 14'. In addition, we found a (-)-reboxetine conformer that docks in the helix bundle of the α4 subunit, near the middle region. According to molecular dynamics simulations, (-)-reboxetine binding is stable for both sites, albeit less stable than imipramine. The interaction of these drugs with the helix bundle might alter allostericaly the functionality of the channel. In conclusion, the clinical action of (-)-reboxetine may be produced (at least partially) by its inhibitory action on hα4ß2 nAChRs.

Slice XVIvo™: a novel electrophysiology system with the capability for 16 independent brain slice recordings.

Here we validate the design and use of a novel, customized electrophysiology system (Slice XVIvo™) that is capable of recording from 16 independent brain slices. The system consists of 16 independent recording chambers in which individual electrodes can be manually manipulated and fixed in order to stimulate and record extracellular responses from 16 brain slices simultaneously. Responses from each brain slice are elicited with individual stimulus isolator units and recorded through separate channels, thus allowing for independent control and analysis of the evoked extracellular activity from each slice. The system was designed to fit on a standard anti-vibration table, thus the Slice XVIvo™ system occupies considerably less space than other currently available multi-slice recording systems. We have demonstrated the utility of the system to obtain stable, extracellular responses from the CA1 region of the hippocampus, as well as induce long-term potentiation. Additionally, we show the utility of the Slice XVIvo™ system to significantly improved throughput for testing compounds in an oxygen and glucose deprivation assay. Overall, we have designed, created and validated a considerably cost- and space-efficient electrophysiology system that greatly improves throughput while minimizing the number of animals used in experiments.

Analgesic effects mediated by neuronal nicotinic acetylcholine receptor agonists: correlation with desensitization of α4ß2* receptors.

Nicotinic α4ß2* agonists are known to be effective in a variety of preclinical pain models, but the underlying mechanisms of analgesic action are not well-understood. In the present study, we characterized activation and desensitization properties for a set of seventeen novel α4ß2*-selective agonists that display druggable physical and pharmacokinetic attributes, and correlated the in vitro pharmacology results to efficacies observed in a mouse formalin model of analgesia. ABT-894 and Sazetidine-A, two compounds known to be effective in the formalin assay, were included for comparison. The set of compounds displayed a range of activities at human (α4ß2)(2)ß2 (HS-α4ß2), (α4ß2)(2)α5 (α4ß2α5) and (α4ß2)(2)α4 (LS-α4ß2) receptors. We report the novel finding that desensitization of α4ß2* receptors may drive part of the antinociceptive outcome. Our molecular modeling approaches revealed that when receptor desensitization rather than activation activitiesat α4ß2* receptors are considered, there is a better correlation between analgesia scores and combined in vitro properties. Our results suggest that although all three α4ß2 subtypes assessed are involved, it is desensitization of α4ß2α5 receptors that plays a more prominent role in the antinociceptive action of nicotinic compounds. For modulation of Phase I responses, correlations are significantly improved from an r(2) value of 0.53 to 0.67 and 0.66 when HS- and LS-α4ß2 DC(50) values are considered, respectively. More profoundly, considering the DC(50) at α4ß2α5 takes the r(2) from 0.53 to 0.70. For Phase II analgesia scores, adding HS- or LS-α4ß2 desensitization potencies did not improve the correlations significantly. Considering the α4ß2α5 DC(50) value significantly increased the r(2) from 0.70 to 0.79 for Phase II, and strongly suggested a more prominent role for α4ß2α5 nAChRs in the modulation of pain in the formalin assay. The present studies demonstrate that compounds which are more potent at desensitization of α4ß2* receptors display better analgesia scores in the formalin test. Consideration of desensitization propertiesat α4ß2* receptors, especially at α4ß2α5, in multiple linear regression analyses significantly improves correlations with efficacies of analgesia. Thus, α4ß2* nicotinic acetylcholine receptor desensitization may contribute to efficacy in the mediation of pain, and represent a mechanism for analgesic effects mediated by nicotinic agonists.

A method for bidirectional solution exchange--"liquid bullet" applications of acetylcholine to α7 nicotinic receptors.

Fast solution exchange techniques have revolutionized the study of synaptic transmission and promise to remain an important neuroscience research tool. Here we provide evidence for the hypothesis that using continuous, rapid transitions through an agonist solution can significantly increase the exchange rate around a cell by reducing the diffusion boundary at the membrane. This novel approach of rapid solution exchange during whole-cell recordings--described as a "liquid bullet" (LB) application--takes advantage of a bidirectional solution flow around the cell, allowing for a full solution exchange within a range of several milliseconds. An exchange rate (10-90% rise time) of about 2 ms could be achieved during both agonist application and washout. We recorded whole-cell currents from cells expressing the rapidly desensitizing α7 neuronal nicotinic receptor (NNR) subtype that exhibited very fast rise times of around 4-5 ms. We further demonstrated the advantages of a LB application over conventional methods by the ability of this method to elicit concentration-dependent responses for rapidly desensitizing compounds that were not measurable with conventional agonist applications. In addition, we illustrate the utility of this approach for frequency-based assays through fast, repeated agonist applications at frequencies of 1 Hz and 30 Hz. This approach could therefore be useful for the study of rapid agonist-receptor interactions that closely mimic the physiological conditions in the synaptic cleft during bursts of neuronal activity.

Alpha7 nicotinic cholinergic neuromodulation may reconcile multiple neurotransmitter hypotheses of schizophrenia.

The long prevailing hypothesis of schizophrenia pathogenesis implicates dopaminergic systems in the mesolimbic pathways as responsible for the positive symptoms of schizophrenia (hallucinations and delusions) and those in the mesocortical pathway as contributing to the negative symptoms (e.g., social disconnection, flattened affect and anhedonia). Several challenges to the dopamine hypothesis and the proposal of an alternative hypothesis implicating glutamate have provided additional support for the development of non-dopaminergic drugs for the management of schizophrenia symptomatology. Furthermore, preclinical and clinical evidence of alpha7 neuronal nicotinic acetylcholine receptor-mediated benefits in the triad of positive symptoms, negative symptoms and cognitive dysfunction in schizophrenia, as well as the genetic linkage of this receptor to the disease, have added another level of complexity. Thus schizophrenia is increasingly believed to involve multi-neurotransmitter deficits, all of which may contribute to altered dopaminergic tone in the mesolimbic, mesocortical and other areas of the brain. In this paper we provide a model that reconciles the dopamine, glutamate and alpha7 cholinergic etiopathogenesis and is consistent with the clinical benefit derived from therapies targeted to these individual pathways.

Application of alpha7 nicotinic acetylcholine receptor agonists in inflammatory diseases: an overview.

Inflammatory disorders are characterized by the influx of immune cells into the vascular wall of veins and/or arteries in response to stimuli such as oxidized-LDL and various pathogens. These factors stimulate the local production of pro-inflammatory cytokines by macrophages and other cells that promote various inflammatory diseases such as atherosclerosis, Crohn's, Alzheimer's and diabetes. Numerous cytokines play a significant role in this process, though tumor necrosis factor (TNF) and various interleukins are thought to be among the most important regulators. These proinflammatory cytokines promote the above-described diseases by inducing endothelial cell dysfunction. In this brief commentary we will discuss some of the latest advances and discoveries in the treatment of these inflammatory diseases, making use of alpha7 nicotinic acetylcholine receptor (alpha7 nAChR) agonists.

Alpha7 nicotinic receptors as novel therapeutic targets for inflammation-based diseases.

In recent years the etiopathology of a number of debilitating diseases such as type 2 diabetes, arthritis, atherosclerosis, psoriasis, asthma, cystic fibrosis, sepsis, and ulcerative colitis has increasingly been linked to runaway cytokine-mediated inflammation. Cytokine-based therapeutic agents play a major role in the treatment of these diseases. However, the temporospatial changes in various cytokines are still poorly understood and attempts to date have focused on the inhibition of specific cytokines such as TNF-α. As an alternative approach, a number of preclinical studies have confirmed the therapeutic potential of targeting alpha7 nicotinic acetylcholine receptor-mediated anti-inflammatory effects through modulation of proinflammatory cytokines. This "cholinergic anti-inflammatory pathway" modulates the immune system through cholinergic mechanisms that act on alpha7 receptors expressed on macrophages and immune cells. If the preclinical findings translate into human efficacy this approach could potentially provide new therapies for treating a broad array of intractable diseases and conditions with inflammatory components.

Alpha7 neuronal nicotinic receptors as targets for novel therapies to treat multiple domains of schizophrenia.

A number of hypotheses have been put forth to explain the underlying abnormalities of schizophrenia. The widely held dopamine hypothesis suggests that positive symptoms are related to elevated subcortical dopamine transmission and that negative symptoms and cognitive impairments are associated with decreased cortical dopamine function. However, recent evidence suggests broader involvement of serotonergic, glutamatergic and other neurotransmitter systems and a growing body of evidence supports a role for nicotinic cholinergic systems. Based on post-mortem studies, there is a decreased density of neuronal nicotinic receptors (NNRs), especially the alpha7 NNR subtype, in the brains of schizophrenics. The alpha7 NNR subtype is the most abundant in the mammalian brain and has been shown to modulate multiple neuronal pathways that are compromised in schizophrenia, including dopaminergic, serotonergic, glutamatergic and GABAergic pathways. Familial linkage studies have associated regions of chromosome 15, which contains the alpha7 NNR gene, with schizophrenia and polymorphisms have been described in the promoter region of the alpha7 NNR gene. Observations from both animal and human studies that alpha7 NNR agonists can improve positive and negative symptoms as well as cognition to varying degrees further support the involvement of this receptor subtype in multiple deficits of schizophrenia and suggest that it may be feasible to develop novel therapies targeting alpha7 NNRs to treat all domains of the disease.

Alpha7 neuronal nicotinic receptors: the missing link to understanding Alzheimer's etiopathology?

Evidence supports the premise that alpha7 nicotinic acetylcholine receptors in the central nervous system, sometimes referred to as neuronal nicotinic receptors (NNRs), play a central role in the development of Alzheimer's disease (AD) pathophysiology. Moreover, these receptors may represent the key to unifying aspects of the cholinergic hypothesis of AD with many of the apparently disparate mechanisms such as beta-amyloid deposition, tau hyperphosphorylation, and ApoE4 abnormalities variously proposed to underlie the progression of the disease. We hypothesize that neuronal degeneration in incipient AD is the result of coincident events involving, at their core, deficits in alpha7 NNR function. The resulting hypocholinergic tone could potentially have serious consequences since alpha7 NNRs are known to modulate fundamental pathways involved in cell survival such as JAK2-STAT3. This hypothesis predicts that any factors that compromise alpha7 function have the potential to negatively impact neuronal viability. For example, such factors could include deficits in the primary neurotransmitter acetylcholine (ACh), underactivity of normal cognitive processes that stimulate alpha7 NNRs (i.e., use-dependency), or the reported binding of beta-amyloid and ApoE4 to alpha7 NNRs, all of which could effectively decouple the receptors from key pro-survival pathways. Since these pathways are known to negatively modulate GSK-3beta, which regulates tau phosphorylation, downstream effects such as tau hyperphosphorylation would be expected to arise. Conversely, the maintenance of normal alpha7 NNR activity by adequate levels of ACh or other NNR agonists would be expected to support normal cholinergic tone, prevent the binding of beta-amyloid and ApoE4 and preserve the integrity of the neurons. We therefore propose that decreased cholinergic tone is at the apex of AD pathophysiology, with factors such as beta-amyloid and ApoE4 playing a contributory role rather that a causal one and hyperphosphorylation of tau representing a detector of concomitant hypocholinergic tone and beta-amyloid deposition. Thus the convergence of beta-amyloid deposition and/or ApoE4 binding and co-localization with alpha7 NNRs, which are favored under conditions of low cholinergic tone, and the downstream cascade of tau hyperphosphorylation through disinhibition of GSK-3beta appear to explain and reconcile many of the discordant findings in this very active area of CNS research.

An alpha7 nicotinic acetylcholine receptor-selective agonist reduces weight gain and metabolic changes in a mouse model of diabetes.

Type 2 diabetes has become a pervasive public health problem. The etiology of the disease has not been fully defined but appears to involve abnormalities in peripheral and central nervous system pathways, as well as prominent inflammatory components. Because nicotinic acetylcholine receptors (nAChRs) are known to interact with anti-inflammatory pathways and have been implicated in control of appetite and body weight, as well as lipid and energy metabolism, we examined their role in modulating biological parameters associated with the disease. In a model of type 2 diabetes, the homozygous leptin-resistant db/db obese mouse, we measured the effects of a novel alpha7 nAChR-selective agonist [5-methyl-N-[2-(pyridin-3-ylmethyl)-1-azabicyclo[2.2.2]oct-3-yl]thiophene-2-carboxamide (TC-7020)] on body mass, glucose and lipid metabolism, and proinflammatory cytokines. Oral administration of TC-7020 reduced weight gain and food intake, reduced elevated glucose and glycated hemoglobin levels, and lowered elevated plasma levels of triglycerides and the proinflammatory cytokine tumor necrosis factor-alpha. These changes were reversed by the alpha7-selective antagonist methyllycaconitine, confirming the involvement of alpha7 nAChRs. Prevention of weight gain, decreased food intake, and normalization of glucose levels were also blocked by the Janus kinase 2 (JAK2) inhibitor alpha-cyano-(3,4-dihydroxy)-N-benzylcinnamide (AG-490), suggesting that these effects involve linkage of alpha7 nAChRs to the JAK2-signal transducer and activator of transcription 3 signaling pathway. The results show that alpha7 nAChRs play a central role in regulating biological parameters associated with diabetes and support the potential of targeting these receptors as a new therapeutic strategy for treatment.

Development and optimization of a high-throughput electrophysiology assay for neuronal alpha4beta2 nicotinic receptors.

Historically, the identification of alpha4beta2 nicotinic acetylcholine receptor ligands has been based on high-throughput radioligand binding, rubidium efflux assays and Ca++ flux assays using a fluorometric imaging plate reader (FLIPR). Among other approaches, low-throughput electrophysiological assays in Xenopus oocytes and two channel application "liquid filament" systems for mammalian cells have been commonly used. More recent technical innovations that have been introduced into the field of electrophysiology allow for automated simultaneous multi-channel operation. Here we report the development and optimization of a high-throughput electrophysiological assay for identifying functionally active alpha4beta2 nicotinic receptor ligands using such a system. Characterization of the test system yielded results comparable to those obtained by other investigators using conventional electrophysiological assays. For example, the concentration-response relationships obtained for alpha4beta2 receptor activation by acetylcholine and nicotine were best described by biphasic Hill equations, and the inhibition of alpha4beta2 receptor currents by the nicotinic antagonist dihydro-beta-erythroidine was consistent with previously published results. Functional up-regulation of alpha4beta2 receptors by prolonged exposure to nicotine or lower temperature was also confirmed. Using this methodology we were able to characterize the activation of alpha4beta2 receptors by multiple compounds in a mammalian cell expression system, exemplifying its utility for rapid identification of novel nicotinic ligands within a screening cascade. Our results demonstrate the utility of this electrophysiological tool for the discovery of alpha4beta2 nicotinic acetylcholine receptor ligands with potential applications in numerous clinical indications.

Atypical antipsychotics as noncompetitive inhibitors of alpha4beta2 and alpha7 neuronal nicotinic receptors.

It has been suggested that the interaction of antipsychotic medications with neuronal nicotinic receptors may increase the cognitive dysfunction associated with schizophrenia and may explain why current therapies only partially address this core feature of the illness. In the present studies we compared the effects of the atypical antipsychotics quetiapine, clozapine and N-desmethylclozapine to those of the typical antipsychotics haloperidol and chlorpromazine on the alpha4beta2 and alpha7 nicotinic receptor subtypes. The binding of [(3)H]-nicotine to rat cortical alpha4beta2 receptors and [(3)H]-methyllycaconitine to rat hippocampal alpha7 receptors was not affected by any of the compounds tested. However, Rb(+) efflux evoked either by nicotine or the selective alpha4beta2 agonist TC-1827 from alpha4beta2 receptors expressed in SH-EP1 cells and nicotine-evoked [(3)H]-dopamine release from rat striatal synaptosomes were non-competitively inhibited by all of the antipsychotics. Similarly, alpha-bungarotoxin-sensitive epibatidine-evoked [(3)H]-norepinephrine release from rat hippocampal slices and acetylcholine-activated currents of alpha7 nicotinic receptors expressed in oocytes were inhibited by haloperidol, chlorpromazine, clozapine and N-desmethylclozapine. The inhibitory effects on nicotinic receptor function produced by the antipsychotics tested occurred at concentrations similar to plasma levels achieved in schizophrenia patients, suggesting that they may lead to clinically relevant effects on cognition.

Differential pharmacologies of mecamylamine enantiomers: positive allosteric modulation and noncompetitive inhibition.

(+/-)-Mecamylamine is a racemic mixture of a widely used brain-permeant noncompetitive inhibitor of muscle-type and neuronal nicotinic receptors (NNRs). The present studies evaluated whether the stereoisomers of this drug show different profiles for inhibition of the high-sensitivity (HS) and low-sensitivity (LS) isoforms of the human alpha4beta2 NNR subtype expressed in subclonal human epithelial 1 cells. We found that at low concentrations (micromolar range), TC-5214 [S-(+)-mecamylamine] was more effective than TC-5213 [R-(-)-mecamylamine] in inhibiting the LS alpha4beta2 NNRs. In addition, we demonstrated that TC-5214 potentiated and TC-5213 inhibited agonist-induced activation of HS alpha4beta2 NNRs. The stereoselectivity of mecamylamine enantiomers at HS and LS alpha4beta2 receptors demonstrates that TC-5214 is the preferred stereoisomer for selective activation of HS, whereas it is more effective in suppressing LS receptor function. This feature could be relevant to therapeutic applications where such a selective mechanism of action is required.

TC-5214 (S-(+)-mecamylamine): a neuronal nicotinic receptor modulator with antidepressant activity.

Both clinical and preclinical data support a potential therapeutic benefit of modulating the activity of CNS neuronal nicotinic receptors (NNRs) to treat depression and anxiety disorders. Based on the notion that the depressive states involve hypercholinergic tone, we have examined the potential palliative role of NNR antagonism in these disorders, using TC-5214 (S-(+) enantiomer of mecamylamine), a noncompetitive NNR antagonist. TC-5214 demonstrated positive effects in a number of animal models of depression and anxiety. TC-5214 was active in the forced swim test in rats (minimum effective dose (MED)=3 mg/kg i.p.), a classical depression model. It was also active in the behavioral despair test in mice (0.1-3.0 mg/kg i.p.), another model of depression. In the social interaction paradigm in rats, a model of generalized anxiety disorder (GAD), TC-5214 was active at a dose of 0.05 mg/kg s.c. In the light/dark chamber paradigm in rats, a model of GAD and phobia, TC-5214 was also active at a dose of 0.05 mg/kg s.c. Although TC-5214 shows modest selectivity among NNR subtypes, the antidepressant and anxiolytic effects seen in these studies are likely attributable to antagonist effects at the alpha4beta2 NNRs. This is supported by the observation of similar effects with alpha4beta2-selective partial agonists such as cytisine and with alpha4beta2-selective antagonists such as TC-2216. TC-5214 was well tolerated in acute and chronic toxicity studies in mice, rats, and dogs, showed no mutagenicity and displayed safety pharmacology, pharmacokinetic and metabolic profiles appropriate for therapeutic development. Overall, the results support a novel nicotinic cholinergic antagonist mechanism for antidepressant and anxiolytic effects and highlight the potential of NNR antagonists such as TC-5214 as therapeutics for the treatment of anxiety and depression.

Long-lasting cognitive improvement with nicotinic receptor agonists: mechanisms of pharmacokinetic-pharmacodynamic discordance.

Agonists of nicotinic acetylcholine receptors (nAChRs) produce long-lasting cognitive effects in animal models and humans. The duration of these cognitive effects can outlast the presence of the agonists in the system, and the persistence of cognitive enhancement is increased further by repeated exposure. The basis for this discrepancy appears be the cellular and systemic mechanisms of learning and memory. Agonists of nAChRs induce long-term potentiation (LTP), which is a strengthening of synaptic connections that is associated with learning and memory formation. Some of the cellular effects of nAChR agonists overlap with the known cellular mechanisms of LTP, including long-lasting increases in intracellular concentrations of Ca2+, activation of second-messenger systems and transcription factors, elevated levels of gene products and enhanced neurotransmitter release. A better understanding of this phenomenon might shed new light on the role of nAChR systems in memory formation and retrieval.