Patrick M. Lippiello

An evaluation of neuronal nicotinic acetylcholine receptor activation by quaternary nitrogen compounds indicates that choline is selective for the α7 subtype

The agonist properties of acetylcholine (ACh), tetramethylammonium, ethyl-trimethylammonium and choline were evaluated for muscle and neuronal nicotinic receptors in Xenopus oocytes. The only essential feature for a neuronal receptor agonist appears to be the charged nitrogen. For specific receptor subtypes, other structural elements appear permissive (neither increasing nor decreasing activity) or non-permissive (decreasing activity). Choline was a full agonist for alpha 7, but a hydroxyl group was strongly non-permissive for other receptor subtypes (alpha 1 beta 1 gamma delta, alpha 3 beta 4, alpha 3 beta 2, and alpha 4 beta 2). The binding of these ligands to brain membranes is consistent with the electrophysiological results. Physiological concentrations of choline desensitize alpha 7 receptors to ACh suggesting that, in vivo, choline may regulate both the activation and inactivation of this receptor.

TC-5619: an alpha7 neuronal nicotinic receptor-selective agonist that demonstrates efficacy in animal models of the positive and negative symptoms and cognitive dysfunction of schizophrenia.

A growing body of evidence suggests that the alpha7 neuronal nicotinic receptor (NNR) subtype is an important target for the development of novel therapies to treat schizophrenia, offering the possibility to address not only the positive but also the cognitive and negative symptoms associated with the disease. In order to probe the relationship of alpha7 function to relevant behavioral correlates we employed TC-5619, a novel selective agonist for the alpha7 NNR subtype. TC-5619 binds with very high affinity to the alpha7 subtype and is a potent full agonist. TC-5619 has little or no activity at other nicotinic receptors, including the alpha4beta2, ganglionic (alpha3beta4) and muscle subtypes. The transgenic th(tk-)/th(tk-) mouse model that reflects many of the developmental, anatomical, and multi-transmitter biochemical aspects of schizophrenia was used to assess the antipsychotic effects of TC-5619. In these mice TC-5619 acted both alone and synergistically with the antipsychotic clozapine to correct impaired pre-pulse inhibition (PPI) and social behavior which model positive and negative symptoms, respectively. Antipsychotic and cognitive effects of TC-5619 were also assessed in rats. Similar to the results in the transgenic mice, TC-5619 significantly reversed apomorphine-induced PPI deficits. In a novel object recognition paradigm in rats TC-5619 demonstrated long-lasting enhancement of memory over a wide dose range. These results suggest that alpha7-selective agonists such as TC-5619, either alone or in combination with antipsychotics, could offer a new approach to treating the constellation of symptoms associated with schizophrenia, including cognitive dysfunction.

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.

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 α4β2 NNRs. This is supported by the observation of similar effects with α4β2‐selective partial agonists such as cytisine and with α4β2‐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.

An α7 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 α7 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-α. These changes were reversed by the α7-selective antagonist methyllycaconitine, confirming the involvement of α7 nAChRs. Prevention of weight gain, decreased food intake, and normalization of glucose levels were also blocked by the Janus kinase 2 (JAK2) inhibitor α-cyano-(3,4-dihydroxy)-N-benzylcinnamide (AG-490), suggesting that these effects involve linkage of α7 nAChRs to the JAK2-signal transducer and activator of transcription 3 signaling pathway. The results show that α7 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.

The Activation and Inhibition of Human Nicotinic Acetylcholine Receptor by RJR‐2403 Indicate a Selectivity for the α4β2 Receptor Subtype

Abstract : Human nicotinic acetylcholine (ACh) receptor subtypes expressed in Xenopus oocytes were characterized in terms of their activation by the experimental agonist RJR‐2403. Responses to RJR‐2403 were compared with those evoked by ACh and nicotine. These agonists were also characterized in terms of whether application of the drugs had the effect of producing a residual inhibition that was manifest as a decrease in subsequent control responses to ACh measured 5 min after the washout of the drug. For the activation of α4β2 receptors, RJR‐2403 had an efficacy equivalent to that of ACh and was more potent than ACh. RJR‐2403 was less efficacious than ACh for other human receptor subtypes, suggesting that it is a partial agonist for all these receptors. Nicotine activated peak currents in human α4β2 and α3β2 receptors that were 85 and 50% of the respective ACh maximum responses. Nicotine was an efficacious activator of human α7 receptors, with a potency similar to ACh, whereas RJR‐2403 had very low potency and efficacy for these receptors. At concentrations of <1 mM, RJR‐2403 did not produce any residual inhibition of subsequent ACh responses for any receptor subtype. In contrast, nicotine produced profound residual inhibition of human α4β2, α3β2, and α7 receptors with IC50 values of 150, 200, and 150 μM, respectively. Co‐expression of the human α5 subunit with α3 and β2 subunits had the effect of producing protracted responses to ACh and increasing residual inhibition by ACh and nicotine but not RJR‐2403. In conclusion, our results, presented in the context of the complex pharmacology of nicotine for both activating and inhibiting neuronal nicotinic receptor subtypes, suggest that RJR‐2403 will be a potent and relatively selective activator of human α4β2 receptors.

Desensitization of Nicotine-Stimulated 86Rb+ Efflux from Mouse Brain Synaptosomes

Abstract: The desensitization of nicotine‐stimulated 86Rb+ efflux from synaptosomes prepared from C57BL/6 mouse brain was investigated. Nicotine stimulated a saturable, concentration‐dependent efflux of 86Rb+ from synaptosomes (EC50 = 0.60 µM), but the response decreased with time of exposure to nicotine. The rate of decrease of the response (desensitization) increased as the nicotine concentration was increased (EC50 = 0.35 µM; maximal rate of desensitization = 1.1 min−1). Desensitization of nicotine‐stimulated 86Rb+ efflux was also observed when synaptosomes were exposed to low (1–200 nM) concentrations of nicotine that caused little or no stimulation of efflux (EC50 = 13 nM). The rate of desensitization observed with low nicotine concentrations (0.30 min−1) was less than that measured at stimulating concentrations. Desensitization was not fully reversible for synaptosomes exposed to nicotine concentrations between 10 nM and 10 µM: Only 60‐40% of the control response was regained after a 10‐min washout period. The kinetics of functional desensitization were compared with the kinetics of [3H]nicotine binding. [3H]Nicotine binding to midbrain particulate fractions displayed both a fast and a slow phase. The EC50 values for these two phases were 2.6 and 14 nM, respectively. Data obtained from functional desensitization and ligand binding experiments were analyzed using a two‐state model. The kinetic constants obtained from the analyses of these two processes were very similar. Overall, the results suggest that nicotinic receptor function measured with ion flux desensitizes when exposed to either stimulating or nonstimulating concentrations of nicotine. In addition, the kinetic properties calculated for the functional desensitization are comparable to those for [3H]nicotine binding.

A microdialysis study of the effects of the nicotinic agonist RJR-2403 on cortical release of acetylcholine and biogenic amines

Transcortical dialysis was employed to investigate the effects of subcutaneous (s.c.) injections of RJR-2403 (1.2–7.2 μmol/kg) on extracellular levels of acetylcholine (ACh), norepinephrine (NE), dopamine (DA), and serotonin (5-HT) in rat. Systemic administration of RJR-2403 produced a 90% increase of cortical extracellular ACh levels that persisted for up to 90 minutes after injection. Norepinephrine and DA release were increased 124% and 131% above basal values, respectively. Serotonin (5-HT) levels in the dialysate were also significantly elevated by RJR-2403 (3.6 μmol/kg, s.c.) 70% above baseline at 90 minutes post-injection. Comparison of these responses to those of (−)nicotine from a previous study reveals little difference between the two compounds in their ability to influence cortical neurotransmitter release following systemic administration.

Purification of L-[3H] nicotine eliminates low affinity binding

Some studies of L-[3H]nicotine binding to rodent and human brain tissue have detected two binding sites as evidenced by nonlinear Scatchard plots. Evidence presented here indicates that the low affinity binding site is not stereospecific, is not inhibited by low concentrations of cholinergic agonists and is probably due to breakdown products of nicotine since purification of the L-[3H]nicotine eliminates the low affinity site.

Application of Alpha7 Nicotinic Acetylcholine Receptor Agonists in Inflammatory Diseases: An Overview

ABSTRACTInflammatory 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.