Merouane Bencherif

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.

Convergence of alpha 7 nicotinic acetylcholine receptor-activated pathways for anti-apoptosis and anti-inflammation: Central role for JAK2 activation of STAT3 and NF-κB

Our laboratories have previously identified the alpha7 nAChR-JAK2 pathway as playing a central role in nicotine-induced neuroprotection. We have also reported that the angiotensin II (Ang II) AT(2) receptor induced activation of SHP-1 induces the tyrosine dephosphorylation of JAK2 that results in a complete neutralization of the alpha7 nAChR-JAK2 pro-survival cascade. In this study, we investigated the effects of inhibiting the alpha7 nAChR-JAK2 pro-survival cascade on the nicotine-induced production of the survival factor Bcl-2 and the transcriptional activation of NF-kappaB, AP-1, STAT1, STAT3, and STAT5. We report that nicotine induced the production of Bcl-2 and increased the transcriptional activation of NF-kappaB, AP-1, STAT1, and STAT3, and with the exception of AP-1, the other transcription factors (NF-kappaB, STAT1, and STAT3) were significantly reduced by JAK2 inhibition. We also demonstrate that, via transfection of either Bcl-2 antisense or NF-kappaB, STAT1 and STAT3 transcription factor decoys oligodeoxyribonucleotides into PC12 cells, nicotine induces its neuroprotection in PC12 cells via activation of the alpha7 nAChR-JAK2-(NF-kappaB; STAT3)-Bcl-2 pro-survival pathway. Finally, the neuroprotective nicotine-induced production of Bcl-2 appears to fully counteract the Abeta (1-42)-induced apoptosis of PC12 cells by blocking Abeta (1-42)-induced mitochondrial release of cytosolic cytochrome C.

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.

Regulation by nicotine of its own receptors

Nicotinic acetylcholine receptors (nAChR) are diverse members of the ligand‐gated ion channel superfamily of neurotransmitter receptors and principal biological targets of nicotine action. Different nAChR subtypes play critical roles in chemical signaling throughout the brain and body and represent ideal targets for the modulation of nervous system function. Available evidence suggests that acute nicotine exposure activates function of all nAChR subtypes. However, more chronic exposure to nicotine, as would occur during long‐term use of tobacco products, might have different effects on nAChR that could contribute to other changes in nervous system function. Our studies suggest that chronic nicotine exposure has some common effects on numbers and function of all nAChR subtypes examined. In model cell lines, numbers of muscle‐type (α1β1γδ‐nAChR), ganglionic (α3β4‐nAChR), neuronal curaremimetic neurotoxin‐binding (α7‐nAChR), and central nervous system nicotine‐binding (α4β2‐nAChR) nicotinic receptors are increased on chronic exposure to nicotine. However, functional activity of muscle‐type and ganglionic nAChR is lost through a process that is distinct from reversible desensitization of nAChR. Nevertheless, there is diversity in the magnitude, rate of onset, and nicotine dose dependence of these effects across nAChR subtypes. Moreover, the ability of other nicotinic ligands to mimic or block effects of chronic nicotine exposure differs across nAChR subtypes. These findings suggest that distinct nAChR subtypes, perhaps expressed in different quantities and/or sites of the nervous system across human individuals or animal strains, have unique sensitivities to chronic nicotine treatment, possibly helping to explain individual differences in responses to, and during recovery from, chronic nicotine exposure. Drug Dev. Res. 38:136–148

TC-2559: a novel orally active ligand selective at neuronal acetylcholine receptors.

TC-2559 [(E)-N-Methyl-4-[3-(5-ethoxypyridin)yl]-3-buten-1-amine] is a novel nicotinic agonist markedly more selective than recently reported novel nicotinic receptor ligands (selectivity ratio for central nervous system (CNS) to peripheral nervous system (PNS)>4000). TC-2559 competes effectively with [3H]-nicotine binding (K(i)=5 nM) but not with [125I]-bungarotoxin (>50,000 nM). Dopamine release from striatal synaptosomes and ion flux from thalamic synaptosomes indicate that TC-2559 is potent and efficacious in the activation of CNS receptors and significantly reduced glutamate-induced neurotoxicity in vitro. TC-2559 has no detectable effects on muscle and ganglion-type nicotinic acetylcholine receptors at concentrations up to 1 mM. TC-2559 significantly attenuates scopolamine-induced cognitive deficits in a step-through passive avoidance task. Acute and repeated oral dosing of TC-2559 enhances performance in a radial arm maze task. In contrast to the effects of equimolar concentrations of (-) nicotine, TC-2559 does not induce hypothermia and locomotor activity is not enhanced following repeated daily administration of 14 days. TC-2559 has a markedly enhanced CNS-PNS selectivity ratio and an intra-CNS selectivity as evidenced by the improved cognition without increased locomotor activity. The in vitro and in vivo studies in the present study suggest that TC-2559 has the desired profile to be further evaluated as a potential therapeutic agent for neurodegenerative diseases.

Heterologous Expression of Human α6β4β3α5 Nicotinic Acetylcholine Receptors: Binding Properties Consistent with Their Natural Expression Require Quaternary Subunit Assembly Including the α5 Subunit

Heterologous expression and lesioning studies were conducted to identify possible subunit assembly partners in nicotinic acetylcholine receptors (nAChR) containing α6 subunits (α6* nAChR). SH-EP1 human epithelial cells were transfected with the requisite subunits to achieve stable expression of human α6β2, α6β4, α6β2β3, α6β4β3, or α6β4β3α5 nAChR. Cells expressing subunits needed to form α6β4β3α5 nAChR exhibited saturable [3H]epibatidine binding (Kd = 95.9 ± 8.3 pM and Bmax = 84.5 ± 1.6 fmol/mg of protein). The rank order of binding competition potency (Ki) for prototypical nicotinic compounds was α-conotoxin MII (6 nM) > nicotine (156 nM) ∼ methyllycaconitine (200 nM) > α-bungarotoxin (>10 μM), similar to that for nAChR in dopamine neurons displaying a distinctive pharmacology. 6-Hydroxydopamine lesioning studies indicated that β3 and α5 subunits are likely partners of the α6 subunits in nAChR expressed in dopaminergic cell bodies. Similar to findings in rodents, quantitative real-time reverse transcription-polymerase chain reactions of human brain indicated that α6 subunit mRNA expression was 13-fold higher in the substantia nigra than in the cortex or the rest of the brain. Thus, heterologous expression studies suggest that the human α5 subunit makes a critical contribution to α6β4β3α5 nAChR assembly into a ligand-binding form with native α6*-nAChR-like pharmacology and of potential physiological and pathophysiological relevance.