Alpha7 nAChR Agonist Research Articles

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.

Repeated administration of α7 nicotinic acetylcholine receptor (nAChR) agonists, but not positive allosteric modulators, increases α7 nAChR levels in the brain

The alpha7 nicotinic acetylcholine receptor (nAChR) is an important target for treatment of cognitive deficits in schizophrenia and Alzheimer's disease. However, the receptor desensitizes rapidly in vitro, which has led to concern regarding its applicability as a clinically relevant drug target. Here we investigate the effects of repeated agonism on alpha7 nAChR receptor levels and responsiveness in vivo in rats. Using [(125)I]-alpha-bungarotoxin (BTX) autoradiography we show that acute or repeated administration with the selective alpha7 nAChR agonist A-582941 increases the number of alpha7 nAChR binding sites in several brain regions, particularly in the prefrontal cortex. The alpha7 nAChR agonists SSR180711 and PNU-282987 also increase [(125)I]-BTX binding, suggesting that this is a general consequence of alpha7 nAChR agonism. Interestingly, the alpha7 nAChR positive allosteric modulators PNU-120596 and NS1738 do not increase [(125)I]-BTX binding. Furthermore, A-582941-induced increase in Arc and c-fos mRNA expression in the prefrontal cortex is enhanced and unaltered, respectively, after repeated administration, demonstrating that the alpha7 nAChRs remain responsive. Contrarily, A-582941-induced phosphorylation of Erk2 in the prefrontal cortex occurs following acute, but not repeated administration. Our results demonstrate that repeated agonist administration increases the number of alpha7 nAChRs in the brain, and leads to coupling versus uncoupling of specific intracellular signaling pathways. Additionally, our data suggest a fundamental difference between the sequelae of repeated administration with agonists and allosteric modulators of the alpha7 nAChR.

Protective effect of alpha7 nAChR: Behavioural and morphological features on neuropathy

Traumatic, toxic or metabolic damage to the nervous system is the etiological foundation of neuropathic pain. Neuropathies are extremely difficult to treat and available drugs rarely joint an anti-hyperalgesic with a neurorestorative effect. From the literature, evidences support the alpha7 nicotinic receptor (nAChR) subtype as having a role in neuropathic pain as well as possessing neuroprotective properties. Aimed to inquire the anti-neuropathic effect of the alpha7 nAChR stimulation, we evaluated the pharmacological profile of the alpha7 nAChR agonist PNU-282987 on pain and on morphological alterations induced in the rat sciatic nerve by loose ligation (CCI). Acute administration of PNU-282987, 10 and 30 mg kg −1 p.o. (15 days after ligation), was able to reduce hyperalgesia in a methyllicaconitine-reversed manner. This alpha7 nAChR agonist exerted no analgesic effects. Chronic PNU-282987 treatments, 30 mg kg −1 once a day for 7 days and 10 mg kg −1 for 28 days, were able to decrease pain perception. The histological studies highlighted that the ligation induces oedema and macrophagic infiltrate. Moreover, osmicated preparations revealed a decrease in axons’ compactness and diameter, together with a significant loss of myelin sheath. Repeated treatment with PNU-282987 reduced the presence of oedema and macrophagic infiltrate and, on the coronal sections of the nerve, a significant higher myelin sheath, axonal diameter and number of fibers were observable. These results strongly suggest the pivotal role of alpha7 nAChR in the neuroprotection during neuropathy.

Whole blood cytokine attenuation by cholinergic agonists ex vivo and relationship to vagus nerve activity in rheumatoid arthritis

The central nervous system regulates innate immunity in part via the cholinergic anti-inflammatory pathway, a neural circuit that transmits signals in the vagus nerve that suppress pro-inflammatory cytokine production by an alpha-7 nicotinic acetylcholine receptors (alpha7nAChR) dependent mechanism. Vagus nerve activity is significantly suppressed in patients with autoimmune diseases, including rheumatoid arthritis (RA). It has been suggested that stimulating the cholinergic anti-inflammatory pathway may be beneficial to patients, but it remains theoretically possible that chronic deficiencies in this pathway will render these approaches ineffective. Here we addressed the hypothesis that inflammatory cells from RA patients can respond to cholinergic agonists with reduced cytokine production in the setting of reduced vagus nerve activity. Measurement of RR interval variability (heart rate variability, HRV), in RA patients (n = 13) and healthy controls (n = 10) revealed that vagus nerve activity was significantly depressed in patients. Whole blood cultures stimulated by exposure to endotoxin produced significantly less tumour necrosis factor in samples from RA patients as compared to healthy controls. Addition of cholinergic agonists (nicotine and GTS-21) to the stimulated whole blood cultures however significantly suppressed cytokine production to a similar extent in patients and healthy controls. These findings suggest that it is possible to pharmacologically target the alpha7nAChR dependent control of cytokine release in RA patients with suppressed vagus nerve activity. As alpha7nAChR agonists ameliorate the clinical course of collagen induced arthritis in animals, it may be possible in the future to explore whether alpha7nAChR agonists can improve clinical activity in RA patients.

In Vitro Pharmacological Characterization of a Novel Selective α7 Neuronal Nicotinic Acetylcholine Receptor Agonist ABT-107

Enhancement of alpha7 nicotinic acetylcholine receptor (nAChR) activity is considered a therapeutic approach for ameliorating cognitive deficits present in Alzheimer's disease and schizophrenia. In this study, we describe the in vitro profile of a novel selective alpha7 nAChR agonist, 5-(6-[(3R)-1-azabicyclo[2,2,2]oct-3-yloxy]pyridazin-3-yl)-1H-indole (ABT-107). ABT-107 displayed high affinity binding to alpha7 nAChRs [rat or human cortex, [(3)H](1S,4S)-2,2-dimethyl-5-(6-phenylpyridazin-3-yl)-5-aza-2-azoniabicyclo[2.2.1]heptane (A-585539), K(i) = 0.2-0.6 nM or [(3)H]methyllycaconitine (MLA), 7 nM] that was at least 100-fold selective versus non-alpha7 nAChRs and other receptors. Functionally, ABT-107 did not evoke detectible currents in Xenopus oocytes expressing human or nonhuman alpha3beta4, chimeric (alpha6/alpha3)beta4, or 5-HT(3A) receptors, and weak or negligible Ca(2+) responses in human neuroblastoma IMR-32 cells (alpha3* function) and human alpha4beta2 and alpha4beta4 nAChRs expressed in human embryonic kidney 293 cells. ABT-107 potently evoked human and rat alpha7 nAChR current responses in oocytes (EC(50), 50-90 nM total charge, approximately 80% normalized to acetylcholine) that were enhanced by the positive allosteric modulator (PAM) 4-[5-(4-chloro-phenyl)-2-methyl-3-propionyl-pyrrol-1-yl]-benzenesulfonamide (A-867744). In rat hippocampus, ABT-107 alone evoked alpha7-like currents, which were inhibited by the alpha7 antagonist MLA. In dentate gyrus granule cells, ABT-107 enhanced spontaneous inhibitory postsynaptic current activity when coapplied with A-867744. In the presence of an alpha7 PAM [A-867744 or N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide hydrochloride (PNU-120596)], the addition of ABT-107 elicited MLA-sensitive alpha7 nAChR-mediated Ca(2+) signals in IMR-32 cells and rat cortical cultures and enhanced extracellular signal-regulated kinase phosphorylation in differentiated PC-12 cells. ABT-107 was also effective in protecting rat cortical cultures against glutamate-induced toxicity. In summary, ABT-107 is a selective high affinity alpha7 nAChR agonist suitable for characterizing the roles of this subtype in pharmacological studies.

Novel Alpha-7 Nicotinic Acetylcholine Receptor Agonists Containing a Urea Moiety: Identification and Characterization of the Potent, Selective, and Orally Efficacious Agonist 1-[6-(4-Fluorophenyl)pyridin-3-yl]-3-(4-piperidin-1-ylbutyl) Urea (SEN34625/WYE-103914)

Alpha-7 nicotinic acetylcholine receptor (alpha7 nAChR) agonists are promising therapeutic candidates for the treatment of cognitive impairment. We report a series of novel, potent small molecule agonists (4-18) of the alpha7 nAChR deriving from our continuing efforts in the areas of Alzheimer's disease and schizophrenia. One of the compounds of the series containing a urea moiety (16) was further shown to be a selective agonist of the alpha7 nAChR with excellent in vitro and in vivo profiles, brain penetration, and oral bioavailability and demonstrated in vivo efficacy in multiple behavioral cognition models. Structural modifications leading to the improved selectivity profile and the biological evaluation of this series of compounds are discussed.

Selective α7 nicotinic acetylcholine receptor agonists worsen disease in experimental colitis

In various models vagus nerve activation has been shown to ameliorate intestinal inflammation, via nicotinic acetylcholine receptors (nAChRs) expressed on immune cells. As the alpha7 nAChR has been put forward to mediate this effect, we studied the effect of nicotine and two selective alpha7 nAChR agonists (AR-R17779, (-)-spiro[1-azabicyclo[2.2.2] octane-3,5'-oxazolidin-2'-one and GSK1345038A) on disease severity in two mouse models of experimental colitis. Colitis was induced by administration of 1.5% dextran sodium sulphate (DSS) in drinking water or 2 mg 2,4,6-trinitrobenzene sulphonic acid (TNBS) intrarectally. Nicotine (0.25 and 2.50 micromol.kg(-1)), AR-R17779 (0.6-30 micromol.kg(-1)) or GSK1345038A (6-120 micromol.kg(-1)) was administered daily by i.p. injection. After 7 (DSS) or 5 (TNBS) days clinical parameters and colonic inflammation were scored. Nicotine and both alpha7 nAChR agonists reduced the activation of NF-kappaB and pro-inflammatory cytokines in whole blood and macrophage cultures. In DSS colitis, nicotine treatment reduced colonic cytokine production, but failed to reduce disease parameters. Reciprocally, treatment with AR-R17779 or GSK1345038A worsened disease and led to increased colonic pro-inflammatory cytokine levels in DSS colitis. The highest doses of GSK1345038A (120 micromol.kg(-1)) and AR-R17779 (30 micromol.kg(-1)) ameliorated clinical parameters, without affecting colonic inflammation. Neither agonist ameliorated TNBS-induced colitis. Although nicotine reduced cytokine responses in vitro, both selective alpha7 nAChR agonists worsened the effects of DSS-induced colitis or were ineffective in those of TNBS-induced colitis. Our data indicate the need for caution in evaluating alpha7 nAChR as a drug target in colitis.

Prefrontal beta2 subunit-containing and alpha7 nicotinic acetylcholine receptors differentially control glutamatergic and cholinergic signaling.

One-second-long increases in prefrontal cholinergic activity ("transients") were demonstrated previously to be necessary for the incorporation of cues into ongoing cognitive processes ("cue detection"). Nicotine and, more robustly, selective agonists at alpha4beta2* nicotinic acetylcholine receptors (nAChRs) enhance cue detection and attentional performance by augmenting prefrontal cholinergic activity. The present experiments determined the role of beta2-containing and alpha7 nAChRs in the generation of prefrontal cholinergic and glutamatergic transients in vivo. Transients were evoked by nicotine, the alpha4beta2* nAChR agonist ABT-089 [2-methyl-3-(2-(S)-pyrrolindinylmethoxy) pyridine dihydrochloride], or the alpha7 nAChR agonist A-582941 [2-methyl-5-(6-phenyl-pyridazin-3-yl)-octahydro-pyrrolo[3,4-c]pyrrole]. Transients were recorded in mice lacking beta2 or alpha7 nAChRs and in rats after removal of thalamic glutamatergic or midbrain dopaminergic inputs to prefrontal cortex. The main results indicate that stimulation of alpha4beta2* nAChRs evokes glutamate release and that the presence of thalamic afferents is necessary for the generation of cholinergic transients. ABT-089-evoked transients were completely abolished in mice lacking beta2* nAChRs. The amplitude, but not the decay rate, of nicotine-evoked transients was reduced by beta2* knock-out. Conversely, in mice lacking the alpha7 nAChR, the decay rate, but not the amplitude, of nicotine-evoked cholinergic and glutamatergic transients was attenuated. Substantiating the role of alpha7 nAChR in controlling the duration of release events, stimulation of alpha7 nAChR produced cholinergic transients that lasted 10- to 15-fold longer than those evoked by nicotine. alpha7 nAChR-evoked cholinergic transients are mediated in part by dopaminergic activity. Prefrontal alpha4beta2* nAChRs play a key role in evoking and facilitating the transient glutamatergic-cholinergic interactions that are necessary for cue detection and attentional performance.

In Vivo Evaluation of α7 Nicotinic Acetylcholine Receptor Agonists [11C]A-582941 and [11C]A-844606 in Mice and Conscious Monkeys

BackgroundThe α7 nicotinic acetylcholine receptors (nAChRs) play an important role in the pathophysiology of neuropsychiatric diseases such as schizophrenia and Alzheimer's disease. The goal of this study was to evaluate the two carbon-11-labeled α7 nAChR agonists [11C]A-582941 and [11C]A-844606 for their potential as novel positron emission tomography (PET) tracers.Methodology/Principal FindingsThe two tracers were synthesized by methylation of the corresponding desmethyl precursors using [11C]methyl triflate. Effects of receptor blockade in mice were determined by coinjection of either tracer along with a carrier or an excess amount of a selective α7 nAChR agonist (SSR180711). Metabolic stability was investigated using radio-HPLC. Dynamic PET scans were performed in conscious monkeys with/without SSR180711-treatment. [11C]A-582941 and [11C]A-844606 showed high uptake in the mouse brain. Most radioactive compounds in the brain were detected as an unchanged form. However, regional selectivity and selective receptor blockade were not clearly observed for either compound in the mouse brain. On the other hand, the total distribution volume of [11C]A-582941 and [11C]A-844606 was high in the hippocampus and thalamus but low in the cerebellum in the conscious monkey brain, and reduced by pretreatment with SSR180711.Conclusions/SignificanceA nonhuman primate study suggests that [11C]A-582941 and [11C]A-844606 would be potential PET ligands for imaging α7 nAChRs in the human brain.

DMXB (GTS‐21) ameliorates the cognitive deficits in beta amyloid injected mice through preventing the dysfunction of alpha7 nicotinic receptor

Intracerebroventricular injection of beta-amyloid(25-35) (Abeta(25-35)) in mice leads to cognitive deficits with the dysfunction of alpha7 nicotinic acetylcholine receptor (alpha7nAChR) within 1-2 weeks in a dose-dependent manner. The present study focused on the effect of DMXB, a selective alpha7nAChR agonist, on Abeta(25-35) (3 nmol)-impaired spatial memory and alpha7nAChR function. We found that the treatment with DMXB on days 1-10 after Abeta(25-35) injection dose-dependently prevented Abeta(25-35)-induced impairment of acquisition performance and probe trail test in Morris water maze. Importantly, the treatment with DMXB (1 mg/kg) perfectly prevented Abeta(25-35)-induced depression of alpha7nAChR response, which was associated with improving the probability of presynaptic glutamate release and the induction of high-frequency stimulation (HFS)-dependent long-term potentiation (LTP) in hippocampal Schaffer collaterale-CA1 synapse. Furthermore, although either the basal level of extracellular signal-regulated kinase 2 (ERK2) or its phosphorylation in the hippocampus had no difference between control and Abeta(25-35) mice, the Abeta(25-35) injection significantly attenuated HFS-triggered increase in ERK2 phosphorylation. The treatment with DMXB also rescued the ERK2 phosphorylation triggered by HFS in Abeta(25-35) mice that is required for LTP induction. This study firstly provides in vivo evidence that the anti-amnesic effect of DMXB is likely due to preventing the Abeta(25-35)-induced dysfunction of alpha7nAChR.

Novel Insights for Systemic Inflammation in Sepsis and Hemorrhage

The inflammatory responses in sepsis and hemorrhage remain a major cause of death. Clinically, it is generally accepted that shock in sepsis or hemorrhage differs in its mechanisms. However, the recognition of inflammatory cytokines as a common lethal pathway has become consent. Proinflammatory cytokines such as tumor necrosis factor (TNF) or high-mobility group box1 (HMGB1) are fanatically released and cause lethal multiorgan dysfunction. Inhibition of these cytokines can prevent the inflammatory responses and organ damage. In seeking potential anti-inflammatory strategies, we reported that ethyl pyruvate and alpha7 nicotinic acetylcholine receptor (alpha7nAChR) agonists effectively restrained cytokine production to provide therapeutic benefits in both experimental sepsis and hemorrhage. Here, we review the inflammatory responses and the anti-inflammatory strategies in experimental models of sepsis and hemorrhage, as they may have a consistent inflammatory pathway in spite of their different pathophysiological processes.

Positive allosteric modulation of α7 neuronal nicotinic acetylcholine receptors: lack of cytotoxicity in PC12 cells and rat primary cortical neurons

alpha7-Nicotinic acetylcholine receptors (alpha7 nAChRs) play an important role in cognitive function. Positive allosteric modulators (PAMs) amplify effects of alpha7 nAChR agonist and could provide an approach for treatment of cognitive deficits in neuropsychiatric diseases. PAMs can either predominantly affect the apparent peak current response (type I) or increase both the apparent peak current response and duration of channel opening, due to prolonged desensitization (type II). The delay of receptor desensitization by type II PAMs raises the possibility of Ca2+-induced toxicity through prolonged activation of alpha7 nAChRs. The present study addresses whether type I and II PAMs exhibit different cytotoxicity profiles. The present studies evaluated cytotoxic effects of type I PAM [N-(4-chlorophenyl)]-alpha-[(4-chlorophenyl)-aminomethylene]-3-methyl-5-isoxazoleacet-amide (CCMI) and type II PAM 1-[5-chloro-2,4-dimethoxy-phenyl]-3-[5-methyl-isoxazol-3-yl]-urea (PNU-120596), or 4-[5-(4chloro-phenyl)-2-methyl-3-propionyl-pyrrol-1-yl]-benzenesulphonamide (A-867744). The studies used cultures of PC12 cells and primary cultures of rat cortical neuronal cells. Our results showed that neither type I nor type II PAMs had any detrimental effect on cell integrity or cell viability. In particular, type II PAMs did not affect neuron number and neurite outgrowth under conditions when alpha7 nAChR activity was measured by Ca2+ influx and extracellular signal-regulated kinases 1 and 2 phosphorylation, following exposure to alpha7 nAChR agonists. This study demonstrated that both type I and type II alpha7 nAChR selective PAMs, although exhibiting differential electrophysiological profiles, did not exert cytotoxic effects in cells endogenously expressing alpha7 nAChRs.

Role of channel activation in cognitive enhancement mediated by α7 nicotinic acetylcholine receptors

Several agonists of the alpha7 nicotinic acetylcholine receptor (nAChR) have been developed for treatment of cognitive deficits. However, agonist efficacy in vivo is difficult to reconcile with rapid alpha7 nAChR desensitization in vitro; and furthermore, the correlation between in vitro receptor efficacy and in vivo behavioural efficacy is not well delineated. The possibility that agonists of this receptor actually function in vivo as inhibitors via desensitization has not been finally resolved. Two structurally related alpha7 nAChR agonists were characterized and used to assess the degree of efficacy required in a behavioural paradigm. NS6784 activated human and rat alpha7 nAChR with EC(50)s of 0.72 and 0.88 microM, and apparent efficacies of 77 and 97% respectively. NS6740, in contrast, displayed little efficacy at alpha7 nAChR (<2% in oocytes, < or =8% in GH4C1 cells), although its agonist-like properties were revealed by adding a positive allosteric modulator of alpha7 nAChRs or using the slowly desensitizing alpha7V274T receptor. In mouse inhibitory avoidance (IA) memory retention, NS6784 enhanced performance as did the 60% partial agonist A-582941. In contrast, NS6740 did not enhance performance, but blocked effects of A-582941. Collectively, these findings suggest that a degree of alpha7 nAChR agonist efficacy is required for behavioural effects in the IA paradigm, and that such behavioural efficacy is not due to alpha7 nAChR desensitization. Also, a partial agonist of very low efficacy for this receptor could be used as an inhibitor, in the absence of alpha7 nAChR antagonists with favourable CNS penetration.

Overexpressed α7 nicotinic acetylcholine receptor inhibited proinflammatory cytokine release in NIH3T3 cells

Alpha-7 nicotinic acetylcholine receptor (alpha7nAChR) played an important role during the process of inflammation, and was a potential therapeutic target for many diseases, including Alzheimer's disease, endotoxaemia, hypertension etc. However, it was difficult to express alpha7nAChR on commonly used host cells because of the lack of the chaperone protein RIC-3, which was indispensable to promote folding, assembly, and surface expression of alpha7nAChR. This work was designed to develop a cell line, which not only expressed alpha7nAChR highly and stably, but possessed biological activity. Full-length alpha7nAChR gene was extracted from rat peritoneal macrophages, recombinant plasmid pIRES2-EGFP-alpha7nAChR was constructed, and then transfected to the mouse fibroblast cell line NIH3T3, a cell line expressing RIC-3. Compared with the NIH3T3-pIRES2-EGFP, expression of alpha7nAChR was significantly higher in NIH3T3-pIRES2-EGFP-alpha7nAChR on both mRNA and protein levels. To evaluate the anti-inflammation activity of the over-expressed alpha7nAChR, TNF-alpha, IL-6, and IL-1beta release induced by lipopolysaccharide was determined. No changes were found between NIH3T3-pIRES2-EGFP and NIH3T3-pIRES2-EGFP-alpha7nAChR culture supernatant under LPS challenge, however under nicotine pre-stimulation, pro-inflammatory cytokine release was significantly attenuated in alpha7nAChR transfected cell line. Moreover, in this established cell line, PNU-282987, a selective alpha7nAChR agonist, exerted a stronger ability to reduce TNF-alpha release than nicotine. We concluded that a functional NIH3T3-pIRES2-EGFP-alpha7nAChR cell line was developed, which might be useful for agonist screen and biological research.

The α7 nicotinic acetylcholine receptor on fibroblast‐like synoviocytes and in synovial tissue from rheumatoid arthritis patients: A possible role for a key neurotransmitter in synovial inflammation

Recent studies have suggested an important role for neurotransmitters as modulators of inflammation. Therefore, we undertook this study to investigate the expression of the alpha7 subunit of the nicotinic acetylcholine receptor (alpha7nAChR) and its function in rheumatoid arthritis (RA). The potential role of the alpha7nAChR in modulating proinflammatory cytokine expression in fibroblast-like synoviocytes (FLS) was identified by screening an adenoviral short hairpin RNA (Ad.shRNA) library. An alpha7-specific antibody was used for immunohistochemistry, and fluorescein isothiocyanate-labeled alpha-bungarotoxin, which binds specifically to the alpha7nAChR, was used for immunofluorescence. Gene expression in FLS was determined by quantitative polymerase chain reaction with primers specific for the alpha7nAChR. In addition, we analyzed messenger RNA (mRNA) expression of dupalpha7, a variant alpha7 transcript. Next, we studied the functional role of the alpha7nAChR in RA FLS by examining the effects of alpha7-specific agonists on the production of interleukin-6 (IL-6) and IL-8 by activated FLS. A screen using an Ad.shRNA library against 807 transcripts revealed that a specific alpha7nAChR shRNA potently modulated IL-8 and matrix metalloproteinase expression in FLS. The alpha7nAChR was expressed in the inflamed synovium from RA patients, predominantly in the intimal lining layer. We found alpha7nAChR expression at both the mRNA and protein level in cultured RA FLS. FLS also constitutively expressed dupalpha7 mRNA. Specific alpha7nAChR agonists reduced tumor necrosis factor alpha-induced IL-6 and IL-8 production by FLS. The alpha7nAChR and its dupalpha7 variant are expressed in RA synovium, where they may play a critical role in regulating inflammation. Targeting the alpha7nAChR could provide a novel antiinflammatory approach to the treatment of RA.

Procognitive and Neuroprotective Activity of a Novel α7 Nicotinic Acetylcholine Receptor Agonist for Treatment of Neurodegenerative and Cognitive Disorders

The alpha7 nicotinic acetylcholine receptor (nAChR) is a promising target for treatment of cognitive dysfunction associated with Alzheimer's disease and schizophrenia. Here, we report the pharmacological properties of 5-morpholin-4-yl-pentanoic acid (4-pyridin-3-yl-phenyl)-amide [SEN12333 (WAY-317538)], a novel selective agonist of alpha7 nAChR. SEN12333 shows high affinity for the rat alpha7 receptor expressed in GH4C1 cells (K(i) = 260 nM) and acts as full agonist in functional Ca(2+) flux studies (EC(50) = 1.6 microM). In whole-cell patch-clamp recordings, SEN12333 activated peak currents and maximal total charges similar to acetylcholine (EC(50) = 12 microM). The compound did not show agonist activity at other nicotinic receptors tested and acted as a weak antagonist at alpha3-containing receptors. SEN12333 treatment (3 mg/kg i.p.) improved episodic memory in a novel object recognition task in rats in conditions of spontaneous forgetting as well as cognitive disruptions induced via glutamatergic [5H-dibenzo[a,d]cyclohepten-5,10-imine (dizocilpine maleate); MK-801] or cholinergic (scopolamine) mechanisms. This improvement was blocked by the alpha7-selective antagonist methyllycaconitine, indicating that it is mediated by alpha7 activation. SEN12333 also prevented a scopolamine-induced deficit in a passive avoidance task. In models targeting other cognitive domains, including attention and perceptual processing, SEN12333 normalized the apomorphine-induced deficit of prepulse inhibition. Neuroprotection of SEN12333 was demonstrated in quisqualate-lesioned animals in which treatment with SEN12333 (3 mg/kg/day i.p.) resulted in a significant protection of choline acetyltransferase-positive neurons in the lesioned hemisphere. Cumulatively, our results demonstrate that the novel alpha7 nAChR agonist SEN12333 has procognitive and neuroprotective properties, further demonstrating utility of alpha7 agonists for treatment of neurodegenerative and cognitive disorders.

Pharmacology of α7 nicotinic acetylcholine receptor mediated extracellular signal‐regulated kinase signalling in PC12 cells

Neuronal nicotinic acetylcholine receptors (nAChR) can modulate cell survival and memory processing. The involvement of specific nAChR subtypes in downstream signalling events has been ill defined thus far, because of a lack of subtype-selective ligands. In this study, we investigated activation and modulation of alpha7 nAChR-mediated phosphorylation of extracellular signal-regulated kinases (ERK1/2) in PC12 cells, using selective agonists and positive allosteric modulators. We used undifferentiated PC12 cells endogenously expressing alpha7 nAChR for both biochemical and functional studies. ERK phosphorylation changes were measured by using a novel In-Cell Western procedure. alpha7 nAChR-mediated Ca(2+) signalling was determined by using the fluorometric imaging plate reader assay. Robust induction of ERK phosphorylation followed exposure of PC12 cells to the selective agonist PNU-282987 in the presence of the alpha7 nAChR modulator PNU-120596. ERK phosphorylation was transient and was attenuated by the selective antagonist methyllycaconitine. Consistent with allosteric modulation of alpha7 nAChRs, PNU-120596 enhanced both the agonist potency and efficacy in activating ERK. Moreover, alpha7 nAChR agonists could be quantitatively differentiated based on their potency in activating ERK signalling. The rank order of potencies correlated fairly well with the corresponding binding K(i) values of these alpha7 nAChR agonists. The present work extends previous observations demonstrating the involvement of alpha7 nAChRs in ERK1/2 phosphorylation in PC12 cells. The In-Cell Western procedure allowed a detailed investigation of alpha7 nAChR function and downstream ERK signalling in response to agonist and allosteric modulators.

Pro-Cognitive and Antipsychotic Efficacy of the α7 Nicotinic Partial Agonist SSR180711 in Pharmacological and Neurodevelopmental Latent Inhibition Models of Schizophrenia

Schizophrenia symptoms can be segregated into positive, negative and cognitive, which exhibit differential sensitivity to drug treatments. Accumulating evidence points to efficacy of alpha7 nicotinic receptor (nAChR) agonists for cognitive deficits in schizophrenia but their activity against positive symptoms is thought to be minimal. The present study examined potential pro-cognitive and antipsychotic activity of the novel selective alpha7 nAChR partial agonist SSR180711 using the latent inhibition (LI) model. LI is the reduced efficacy of a previously non-reinforced stimulus to gain behavioral control when paired with reinforcement, compared with a novel stimulus. Here, no-drug controls displayed LI if non-reinforced pre-exposure to a tone was followed by weak but not strong conditioning (2 vs 5 tone-shock pairings). MK801 (0.05 mg/kg, i.p.) -treated rats as well as rats neonatally treated with nitric oxide synthase inhibitor L-NoArg (10 mg/kg, s.c.) on postnatal days 4-5, persisted in displaying LI with strong conditioning, whereas amphetamine (1 mg/kg) -treated rats failed to show LI with weak conditioning. SSR180711 (0.3, 1, 3 mg/kg, i.p.) was able to alleviate abnormally persistent LI produced by acute MK801 and neonatal L-NoArg; these models are believed to model cognitive aspects of schizophrenia and activity here was consistent with previous findings with alpha7-nAChR agonists. In addition, unexpectedly, SSR180711 (1, 3 mg/kg, i.p.) potentiated LI with strong conditioning in no-drug controls and reversed amphetamine-induced LI disruption, two effects considered predictive of activity against positive symptoms of schizophrenia. These findings suggest that SSR180711 may be beneficial not only for the treatment of cognitive symptoms in schizophrenia, as reported multiple times previously, but also positive symptoms.

Molecular imaging of α7 nicotinic acetylcholine receptors: design and evaluation of the potent radioligand [18F]NS10743

The outstanding diversity of cellular properties mediated by neuronal and nonneuronal alpha7 nicotinic acetylcholine receptors (alpha7 nAChR) points to the diagnostic potential of quantitative nuclear molecular imaging of alpha7 nAChR in neurology and oncology. It was our goal to radiolabel the alpha7 nAChR agonist 4-[5-(4-fluoro-phenyl)-[1,3,4]oxadiazol-2-yl]-1,4-diaza-bicyclo[3.2.2]nonane (NS10743) and to assess the selectivity of [(18)F]NS10743 binding site occupancy in animal experiments. [(18)F]NS10743 was synthesized by nucleophilic substitution of the nitro precursor. In vitro receptor affinity and selectivity were assessed by radioligand competition and autoradiography. The radiotracer properties were evaluated in female CD-1 mice by brain autoradiography and organ distribution. Target specificity was validated after treatment with SSR180711 (10 mg/kg, intraperitoneal), and metabolic stability was investigated using radio-HPLC. The specific activity of [(18)F]NS10743 exceeded 150 GBq/micromol at a radiochemical purity >99%. In vitro, NS10743 and [(18)F]NS10743 showed high affinity and specificity towards alpha7 nAChR. The brain permeation of [(18)F]NS10743 was fast and sufficient with values of 4.83 and 1.60% injected dose per gram and brain to plasma ratios of 3.83 and 2.05 at 5 and 60 min after radiotracer administration. Brain autoradiography and organ distribution showed target-specific accumulation of [(18)F]NS10743 in brain substructures and various alpha7 nAChR-expressing organs. The radiotracer showed a high metabolic stability in vivo with a single polar radiometabolite, which did not cross the blood-brain barrier. The good in vitro and in vivo features of [(18)F]NS10743 make this radioligand a promising candidate for quantitative in vivo imaging of alpha7 nAChR expression and encourage further investigations.

Stimulation of nicotinic acetylcholine receptors attenuates collagen‐induced arthritis in mice

The parasympathetic nervous system, through the vagus nerve, can down-regulate inflammation in vivo by decreasing the release of cytokines, including tumor necrosis factor alpha (TNFalpha), by activated macrophages. The vagus nerve may exert antiinflammatory actions via a specific effect of its principal neurotransmitter, acetylcholine, on the alpha7 subunit of nicotinic acetylcholine receptors (alpha7nAChR) on macrophages. The present study was undertaken to obtain insight into the role of the cholinergic antiinflammatory pathway in arthritis. To inhibit the cholinergic antiinflammatory pathway, mice were subjected to unilateral cervical vagotomy or sham surgery, after which arthritis was induced with type II collagen. In a separate study, nicotine was added to the drinking water of mice with collagen-induced arthritis (CIA). In addition, we investigated the effects of intraperitoneally (IP)-injected nicotine and the specific alpha7nAChR agonist AR-R17779. Clinical arthritis was exacerbated by vagotomy and ameliorated by oral nicotine administration. Moreover, oral nicotine inhibited bone degradation and reduced TNFalpha expression in synovial tissue. Both IP-injected nicotine and AR-R17779 ameliorated clinical arthritis and reduced synovial inflammation. This was accompanied by a reduction of TNFalpha levels in both plasma and synovial tissue. The effect of AR-R17779 was more potent compared with that of nicotine and was associated with delayed onset of the disease as well as with protection against joint destruction. These data provide the first evidence of a role of the cholinergic antiinflammatory pathway in the murine CIA model of rheumatoid arthritis.