Short-term oral gavage administration of adenine induces a model of fibrotic kidney disease in rats.

INTRODUCTION: The adenine model of kidney disease typically involves dietary delivery of adenine over several weeks. This model can be variable in its disease progression and can result in significant mortality. In the current study, the amount of adenine delivered to rats was controlled by utilizing oral gavage administration over a short period in an attempt to induce robust renal pathology while addressing variability and viability of the animals. METHODS: Adenine (150 or 200 mg/kg) was administered via oral gavage for 10 consecutive days, and assessed over a total of 20 days. RESULTS: Both adenine dose groups manifested pathophysiological features of kidney disease such as proteinuria, elevated serum creatinine and BUN, and tubulointerstitial fibrosis. The animals also displayed a decline in glomerular filtration rate. Renal mRNA expression of genes associated with injury, inflammation, and fibrosis (i.e., Col1a1, Acta2, Serpine1, Timp1, Fn-Eda, Tgfb1, Ccl2, Nlrp3, Aqp1 and Ccnd1) were elevated as were urinary biomarkers that have translational utility (i.e., clusterin, KIM-1, MCP-1, OPN, NGAL, B2M, calbindin, and cystatin C). All disease endpoints were more pronounced in the 200 mg/kg group, however, while measures of tissue fibrosis were sustained, there was partial recovery by day 20 in functional readouts. No mortality was observed in either dose group. DISCUSSION: Short-term delivery of adenine via precise gavage delivery induced a robust model with hallmarks of fibrotic kidney disease, had limited variance between animals, and no animal morbidity within the 20 days studied. This model represents a methodical alternative to long-term dietary dosing of adenine.

Losartan improves renal function and pathology in obese ZSF-1 rats.

BACKGROUND: Losartan, a blocker of the angiotensin II type I receptor, is an important part of the standard of care for diabetic nephropathy (DN). The obese ZSF-1 rats display many aspects of the clinical features of human Type II DN. The current study was designed to examine the treatment effects of losartan on obese ZSF-1 rats and to evaluate the impact of the onset of dosing on efficacy. METHODS: The rats (7-10 weeks) underwent a right uninephrectomy (Unx) or sham surgery. Losartan (3, 10, 30 mg/kg) was dosed 3 or 9 weeks post-Unx and continued for 12 weeks. RESULTS: Treatment with losartan reduced urinary protein excretion and blood lipids (triglyceride and cholesterol) dose-dependently in both studies. The glomerular filtration rate (GFR) was significantly lower in obese ZSF-1 rats compared with those in lean rats, and losartan was efficacious against this endpoint, in particular with the earlier onset of treatment. Losartan also decreased tubulointerstitial fibrosis, and similar to GFR, earlier treatment conferred beneficial actions even at the lowest dose of 3 mg/kg. Several urinary biomarkers were elevated in the obese ZSF-1 rats, but the levels of sTNFR1, TIMP-1, L-FABP and KIM-1 were the only markers decreased by losartan. CONCLUSIONS: Losartan was renoprotective in the ZSF-1 rats with DN, improving both the pathological and functional parameters of the disease. Importantly, the data also highlight the importance of treatment at earlier stages of the disease for protecting against decline in the GFR and the development of fibrosis.

A-306989, an inhibitor of adenosine kinase, is renoprotective in rodent models of podocyte, basement membrane, and obstructive injury.

Adenosine (ADO) is an important regulatory purine nucleoside that accumulates at sites of inflammation and tissue injury including in diseases associated with renal pathology. Endogenous levels of ADO may be increased by inhibiting the ADO-metabolizing enzyme, ADO kinase (AK). AK inhibitors have demonstrated protection in rodent models of diabetic nephropathy. To further investigate AK inhibition as a potential mechanism for renal protection, A-306989, a potent non-nucleoside AK inhibitor, was examined in both in vitro and in vivo assays of renal injury. A-306989 prevented podocyte damage (disruption of actin cytoskeleton) and increased podocyte survival following puromycin aminonucleoside (PAN) application in both mouse and human conditionally immortalized podocytes. Prophylactic oral administration of A-306989 (1.5, 5 and 15mg/kg) reduced proteinuria in a dose-dependent manner and repressed pro-inflammatory/fibrotic gene up-regulation; A-306989 was also efficacious when administered two days following the PAN-insult. A-306989 (10 and 30mg/kg) also significantly reduced proteinuria and macrophage infiltration in a rat model of glomerulonephritis. Finally, A-306989 (15 and 50mg/kg) reduced the expression levels of pro-inflammatory/fibrotic genes, and reduced macrophage infiltration (50mg/kg), but did not affect the deposition of interstitial collagen in fibrotic kidneys from mice with unilateral ureter obstruction. A-306989 also had beneficial actions on "quality of life" measures including improving body weight loss. Thus, these data indicate that enhancement of endogenous ADO levels by A-306989 can positively modulate renal pathology and mimic some of the previously reported beneficial actions of ADO A2A receptor agonists.

Effects of the novel α7 nicotinic acetylcholine receptor agonist ABT-107 on sensory gating in DBA/2 mice: pharmacodynamic characterization.

Nicotinic acetylcholine receptor (nAChR) agonists improve sensory gating deficits in animal models and schizophrenic patients. The aim of this study was to determine whether the novel and selective α7 nAChR full agonist 5-(6-[(3R)-1-azabicyclo[2.2.2]oct-3-yloxy]pyridazin-3-yl)-1H-indole (ABT-107) improves sensory gating deficits in DBA/2 mice. Sensory gating was measured by recording hippocampal-evoked potential P20-N40 waves and determining gating test/conditioning (T/C) ratios in a paired auditory stimulus paradigm. ABT-107 at 0.1 µmol/kg (average plasma concentration of 1.1 ng/ml) significantly improved sensory gating by lowering T/C ratios during a 30-min period after administration in unanesthetized DBA/2 mice. ABT-107 at 1.0 µmol/kg was ineffective at 30 min after administration when average plasma levels were 13.5 ng/ml. However, the 1.0 µmol/kg dose was effective 180 min after administration when plasma concentration had fallen to 1.9 ng/ml. ABT-107 (0.1 µmol/kg) also improved sensory gating in anesthetized DBA/2 mice pretreated with α7 nAChR-desensitizing doses of nicotine (6.2 µmol/kg) or ABT-107 (0.1 µmol/kg) itself. Moreover, repeated b.i.d. dosing of ABT-107 (0.1 µmol/kg) was as efficacious as a single dose. The acute efficacy of ABT-107 (0.1 µmol/kg) was blocked by the nAChR antagonist methyllycaconitine, but not by the α4ß2 nAChR antagonist dihydro-ß-erythroidine. These studies demonstrate that ABT-107 improves sensory gating through the activation of nAChRs, and efficacy is sustained under conditions of repeated dosing or with prior nAChR activation with nicotine.

Structure-activity studies of diazabicyclo[3.3.0]octane-substituted pyrazines and pyridines as potent α4ß2 nicotinic acetylcholine receptor ligands.

A series of diazabicyclo[3.3.0]octane substituted pyridines and pyrazines was synthesized and characterized at the α4ß2 neuronal nicotinic acetylcholine receptor (nAChR). The compounds were designed to mimic the profile of ABT-089, high affinity binding ligand for the α4ß2 nAChR, with limited agonist activity. Carboxamide derivatives of 3-(diazabicyclo[3.3.0]octane)-substituted pyridines or 2-(diazabicyclo[3.3.0]octane)-substituted pyrazines were found to have the desired binding and activity profile. The structure-activity relationship of these compounds is presented.

Role of α7 nicotinic acetylcholine receptors in regulating tumor necrosis factor-α (TNF-α) as revealed by subtype selective agonists.

Immunological responses to protect against excessive inflammation can be regulated by the central nervous system through the cholinergic anti-inflammatory pathway wherein acetylcholine released from vagus nerves can inhibit inflammatory cytokines. Although a role for the α7 nicotinic acetylcholine receptor (α7 nAChR) in mediating this pathway has been suggested, pharmacological modulation of the pathway by selective agonists remains to be further elucidated. In this study, the role of α7 nAChRs in the regulation of TNF-α release was investigated using high affinity and selective α7 nAChR agonists in mouse peritoneal macrophage and human whole blood in vitro, and in mouse serum in vivo. In mouse peritoneal macrophages, LPS-induced TNF-α release in vitro was inhibited by a selective α7 nAChR agonist, A-833834 (5-[6-(5-Methyl-hexahydro-pyrrolo[3,4-c]pyrrol-2-yl)-pyridazin-3-yl]-1H-indole), and that effect was attenuated by α7 nAChR antagonist methyllycaconitine. The inhibitory effect of A-833834 on LPS-induced TNF-α release was also observed in human whole blood in vitro. I.v. LPS-induced TNF-α release in mouse serum was attenuated following i.p. administration of A-833834. Similarly, i.v. LPS-induced TNF-α release in mouse serum was also attenuated following i.p. administration of A-585539, another α7 nAChR agonist with limited brain penetration, suggesting that these effects are mediated by peripheral α7 nAChRs. A-833834 was also efficacious in suppressing TNF-α release in mouse serum following oral administration in zymosan-induced peritonitis. These studies collectively demonstrate that selectively targeting α7 nAChRs could offer a novel therapeutic modality to treat acute and chronic inflammatory disease states.

Functional characterization and high-throughput screening of positive allosteric modulators of α7 nicotinic acetylcholine receptors in IMR-32 neuroblastoma cells.

α7 nicotinic acetylcholine receptors (nAChRs) are characterized by relatively low ACh sensitivity, rapid activation, and fast desensitization kinetics. ACh/agonist evoked currents at the α7 nAChR are transient, and, typically, calcium flux responses are difficult to detect using conventional fluorometric assay techniques. One approach to study interactions of agonists with the α7 nAChR is by utilizing positive allosteric modulators (PAMs). In this study, we demonstrate that inclusion of type II PAMs such as PNU-120596, but not type I, can enable detection of endogenous α7 nAChR-mediated calcium responses in human neuroblastoma (IMR-32) cells. Using this approach, we characterized the pharmacological profile of nicotine, epibatidine, choline, and other nAChR agonists such as PNU-282987, SSR-180711, GTS-21, OH-GTS21, tropisetron, NS6784, and A-582941. The rank order potency of agonists well correlated with α7 nAChR binding affinities measured in brain membranes. Inhibition of calcium response by methyllycaconitine in the presence of increasing concentrations of PNU-282987 or PNU-120596 revealed that the IC(50) value of methyllycaconitine was sensitive to varying concentrations of the agonist, but not that of the PAM. This format demonstrated the feasibility of this approach for high-throughput screening to identify small molecule, PAMs, which were further confirmed in electrophysiological assays of human α7 nAChR expressed in oocytes.

Alpha3* and alpha 7 nAChR-mediated Ca2+ transient generation in IMR-32 neuroblastoma cells.

Alpha3-containing (alpha 3*) and alpha 7 nicotinic acetylcholine receptors (nAChRs) are expressed in human IMR-32 neuroblastoma cells and implicated in Ca(2+) signaling. In this study, we investigated the intracellular Ca(2+) transient generation evoked by selective activation of alpha 3* (agonist potency rank order: epibatidine>varenicline>nicotine approximately cytisine) and alpha 7 (rank order in the presence of alpha 7 positive allosteric modulator or PAM: A-795723>NS6784 approximately PNU-282987) using, respectively, varenicline and NS6784 (+alpha 7 PAM) by Ca(2+) imaging. Effects of inhibitors of nAChRs (MLA and mecamylamine), ER Ca(2+) ATPase pump (CPA and thapsigargin), Ca(2+)-induced Ca(2+) release (ryanodine and dantrolene), Ca(2+) channels (nitrendipine, diltiazem, and Cd(2+)), and removal of extracellular Ca(2+) were examined. alpha 7 PAMs, when tested in the presence of NS6784, were more active when added first, followed by the agonist, than in the reverse order. Removal of extracellular Ca(2+) - but not CPA, thapsigargin, ryanodine, dantrolene, nitrendipine, diltiazem, or Cd(2+) - diminished the alpha 7 agonist-evoked Ca(2+) transients. In contrast, only diltiazem and nitrendipine and removal of extracellular Ca(2+) inhibited the alpha 3*-mediated Ca(2+) transients. The differential effect of diltiazem and nitrendipine versus Cd(2+) was due to direct inhibition of alpha 3* nAChRs as revealed by Ca(2+) imaging in HEK-293 cells expressing human alpha 3 beta 4 nAChRs and patch clamp in IMR-32 cells. In summary, this study provides evidence that alpha 3* and alpha 7 nAChR agonist-evoked global Ca(2+) transient generation in IMR-32 cells does not primarily involve voltage-dependent Ca(2+) channels, intracellular Ca(2+) stores, or Ca(2+)-induced Ca(2+) release. These mechanisms may, however, be still involved in other forms of nAChR-mediated Ca(2+) signaling.

In vitro pharmacological characterization of a novel selective alpha7 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.

Alpha7 nAChR-mediated activation of MAP kinase pathways in PC12 cells.

The alpha7 nicotinic acetylcholine receptor (alpha7 nAChR) plays a fundamental role in Ca(2+)-dependent activation of signaling pathways that can modulate intracellular events involved in learning and memory. Activation of extracellular signal-regulated kinase-1 and -2 (ERK1/2) are well documented Ca(2+) signaling events, but these have not been well characterized in response to alpha7 nAChR-selective ligands. The present study examined activation of ERK1/2 and explored pathways leading to CREB phosphorylation utilizing alpha7 nAChR-selective ligands in PC12 cells endogenously expressing alpha7 nAChRs. Robust concentration-dependent increase in ERK1/2 phosphorylation was triggered by structurally diverse alpha7 nAChR agonists such as nicotine, choline, GTS-21, SSR-180711A and PNU-282987 in the presence of the positive allosteric modulator (PAM) PNU-120596. This effect was attenuated by selective alpha7 nAChR antagonists or by chelation of extracellular Ca(2+). ERK1/2 phosphorylation was also attenuated by inhibitors of calmodulin-dependent protein kinase II (CaMKII), p38 MAP kinase and mitogen-activated protein kinase kinase1/2 (MEK1/2), indicating the involvement of these kinases upstream of ERK1/2. This was confirmed by direct measurement of p38 MAPK and MEK1/2 phosphorylation. These data suggest that alpha7 nAChR agonist-triggered Ca(2+) transient in PC12 cells induces activation of CaMKII, leading to sequential phosphorylation of p38 MAPK, MEK1/2, ERK1/2 and CREB. Such mechanisms may endow the alpha7 nAChRs with roles in modulating Ca(2+)-dependent intracellular second messenger events implicated in diverse aspects of cognition.

Evaluation of alpha7 nicotinic acetylcholine receptor agonists and positive allosteric modulators using the parallel oocyte electrophysiology test station.

Neuronal acetylcholine receptors (nAChRs) of the alpha7 subtype are ligand-gated ion channels that are widely distributed throughout the central nervous system and considered as attractive targets for the treatment of various neuropsychiatric and neurodegenerative diseases. Both agonists and positive allosteric modulators (PAMs) are being developed as means to enhance the function of alpha7 nAChRs. The in vitro characterization of alpha7 ligands, including agonists and PAMs, relies on multiple technologies, but only electrophysiological measurements assess the channel activity directly. Traditional electrophysiological approaches utilizing two-electrode voltage clamp or patch clamp in isolated cells have very low throughput to significantly impact drug discovery. Abbott (Abbott Park, IL) has developed a two-electrode voltage clamp-based system, the Parallel Oocyte Electrophysiology Test Station (POETs()), that allows for the investigation of ligand-gated ion channels such as alpha7 nAChRs in a higher-throughput manner. We describe the utility of this technology in the discovery of selective alpha7 agonists and PAMs. With alpha7 agonists, POETs experiments involved both single- and multiple-point concentration-response testing revealing diverse activation profiles (zero efficacy desensitizing, partial, and full agonists). In the characterization of alpha7 PAMs, POETs testing has served as a reliable primary or secondary screen identifying compounds that fall into distinct functional types depending on the manner in which current potentiation occurred. Type I PAMs (eg, genistein, NS1738, and 5-hydroxyindole) increase predominantly the peak amplitude response, type II PAMs affect the peak current and current decay (eg, PNU-120,596 and 4-(naphthalen-1-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide), and anothertype slowing the current decay kinetics in the absence of increases in the peak current. In summary, POETs technology allows for significant impact on higher throughput in the testing of alpha7 agonists and PAMs and for identification of compounds with unique profiles that could prove valuable in identifying an optimum in vitro profile in the development of therapeutics for which the alpha7 subtype is considered.

Positive allosteric modulation of alpha7 neuronal nicotinic acetylcholine receptors: lack of cytotoxicity in PC12 cells and rat primary cortical neurons.

BACKGROUND AND PURPOSE: 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. EXPERIMENTAL APPROACH: 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. KEY RESULTS: 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. CONCLUSIONS AND IMPLICATIONS: 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 GSK-3beta activation and alpha7 nAChRs in Abeta(1-42)-induced tau phosphorylation in PC12 cells.

Beta-amyloid peptide 1-42 (Abeta(1-42)) and hyperphosphorylated tau are associated with neurodegeneration in Alzheimer's disease. Emerging evidence indicates that Abeta(1-42) can potentiate hyperphosphorylation of tau in cell lines and in transgenic mice, but the underlying mechanism(s) remains unclear. In this study, Abeta(1-42)-induced tau phosphorylation was investigated in differentiated PC12 cells. Treatment of cells with Abeta(1-42) increased phosphorylation of tau at serine-202 as detected by AT8 antibody. This Abeta(1-42)-induced tau phosphorylation paralleled phosphorylation of glycogen synthase kinase-3beta (GSK-3beta) at tyrosine-216 (GSK-3beta-pY216), which was partially inhibited by the GSK-3beta inhibitor, CHIR98023. Abeta(1-42)-induced tau phosphorylation and increase in GSK-3beta-pY216 phosphorylation were also partially attenuated by alpha7 nicotinic acetylcholine receptor (alpha7 nAChR) selective ligands including agonist A-582941 and antagonists methyllycaconitine and alpha-bungarotoxin. The alpha7 nAChR agonist and the GSK-3beta inhibitor had no additive effect. These observations suggest that alpha7 nAChR modulation can influence Abeta(1-42)-induced tau phosphorylation, possibly involving GSK-3beta. This study provides evidence of nAChR mechanisms underlying Abeta(1-42) toxicity and tau phosphorylation, which, if translated in vivo, could provide additional basis for the utility of alpha7 nAChR ligands in the treatment of Alzheimer's disease.

Analysis of gene expression profiles in rat hippocampus following treatment with nicotine and an alpha7 nAChR selective agonist.

The nicotinic acetylcholine receptors (nAChRs) play critical roles in neuronal transmission and modulation. Among the diverse nAChRs, the alpha7 subtype has been considered as a potential therapeutic target for treating cognitive deficits associated with neuropsychiatric and neurodegenerative diseases. Although a number of mechanisms including neurotransmitter and biochemical effects linking alpha7 nAChR activation and cognitive function are beginning to be described, the underlying molecular processes especially following repeated administration remain unclear. To address this, we have performed gene expression analysis in rats treated with nicotine and a selective alpha7 nAChR agonist, PNU-282987. Our results showed significant overlap in gene expression changes induced by PNU-282987 and nicotine, suggesting convergent pathways triggered by these compounds. Treatment with nicotine also resulted in regulation of a number of genes that were not regulated by PNU-282987, consistent with the interaction of nicotine with other nAChRs beyond the alpha7 subtype. Interestingly, these gene expression changes were observed 24 h post-dose, suggesting that both nicotine and PNU-282987 cause protracted changes in gene expression. Overall, our results identify gene expression changes that may contribute to further defining the roles of nAChR activation in cognitive function.

The potential of neuronal nicotinic acetylcholine receptor agonists for treating CNS conditions.

BACKGROUND: Nicotine has a variety of effects in humans and in experimental animals that are suggestive of therapeutic potential. However, nicotine has a variety of adverse effects that preclude its development as a therapeutic agent, with the exception of its short-term use as a smoking cessation aide. OBJECTIVE: In this paper, we describe the clinical evidence supporting the potential of new nicotinic acetylcholine receptor (nAChR) agonists for the treatment of CNS conditions and review discovery strategies for targeting neuronal nAChRs as well as some of the opportunities and challenges still ahead. METHOD: The scientific literature and company websites are used for this literature review. CONCLUSION: Advances in the understanding of nAChRs during the past two decades have revealed evidence of considerable subtype diversity and hence the possibility of targeted nAChR ligands as improved therapeutics. Several new nAChR agonists have advanced to Phase II clinical trials and have produced signals of efficacy in Alzheimer's disease, attention deficit hyperactivity disorder, cognitive deficits of schizophrenia and pain. In addition, the neuronal nAChR partial agonist varenicline recently received regulatory approval for use in smoking cessation.

[3H]A-585539 [(1S,4S)-2,2-dimethyl-5-(6-phenylpyridazin-3-yl)-5-aza-2-azoniabicyclo[2.2.1]heptane], a novel high-affinity alpha7 neuronal nicotinic receptor agonist: radioligand binding characterization to rat and human brain.

Receptor binding was characterized for [(3)H](1S,4S)-2,2-dimethyl-5-(6-phenylpyridazin-3-yl)-5-aza-2-azoniabicyclo[2.2.1]heptane ([(3)H]A-585539), a selective high-affinity alpha7 nicotinic acetylcholine receptor (nAChR) agonist with rapid kinetics, low nonspecific binding, and high specific activity. At 4 degrees C, the association was monophasic and rapid (t((1/2)) = 8.0 min); dissociation was slower (t((1/2)) = 64.2 min). The K(d) in rat brain at 4 degrees C was 0.063 nM, whereas at 22 and 37 degrees C, the K(d) values were 0.188 and 0.95 nM, respectively. In contrast, the B(max) (34 fmol/mg protein) was unaffected by temperature. In human cortex, [(3)H]A-585539 bound with a K(d) of 0.066 nM and a B(max) of 5.8 fmol/mg protein at 4 degrees C, whereas under similar conditions, specific [(3)H]methyllycaconitine ([(3)H]MLA) binding was not measurable. A number of agonist and antagonist nAChR ligands displaced binding to rat brain membranes with rank order of affinity similar to that for [(3)H]MLA, and in general, a 5 to 10-fold higher affinity was observed for [(3)H]A-585539 binding. There was also a good correlation of K(i) values between [(3)H]A-585539 binding to rat brain and human cortex. The use of a alpha7/5-hydroxytryptamine type-3 chimera revealed that the N-terminal domain of alpha7 nAChR was sufficient to faithfully reproduce the pharmacology of [(3)H]A-585539 binding. Autoradiographic studies comparing [(3)H]A-585539 and [(125)I]alpha-bungarotoxin revealed a similar pattern of labeling in the rat. In summary, [(3)H]A-585539 was shown to have excellent binding characteristics in rat and human brain and represents the first high-affinity alpha7 agonist radioligand with utility in the characterization of this important nAChR subtype that is targeted toward ameliorating cognitive deficits underlying neuropsychiatric and neurodegenerative disorders.

An allosteric modulator of the alpha7 nicotinic acetylcholine receptor possessing cognition-enhancing properties in vivo.

Augmentation of nicotinic alpha7 receptor function is considered to be a potential therapeutic strategy aimed at ameliorating cognitive and mnemonic dysfunction in relation to debilitating pathological conditions, such as Alzheimer's disease and schizophrenia. In the present report, a novel positive allosteric modulator of the alpha7 nicotinic acetylcholine receptor (nAChR), 1-(5-chloro-2-hydroxy-phenyl)-3-(2-chloro-5-trifluoromethyl-phenyl)-urea (NS1738), is described. NS1738 was unable to displace or affect radioligand binding to the agonist binding site of nicotinic receptors, and it was devoid of effect when applied alone in electrophysiological paradigms. However, when applied in the presence of acetylcholine (ACh), NS1738 produced a marked increase in the current flowing through alpha7 nAChRs, as determined in both oocyte electrophysiology and patch-clamp recordings from mammalian cells. NS1738 acted by increasing the peak amplitude of ACh-evoked currents at all concentrations; thus, it increased the maximal efficacy of ACh. Oocyte experiments indicated an increase in ACh potency as well. NS1738 had only marginal effects on the desensitization kinetics of alpha7 nAChRs, as determined from patch-clamp studies of both transfected cells and cultured hippocampal neurons. NS1738 was modestly brain-penetrant, and it was demonstrated to counteract a (-)-scopolamine-induced deficit in acquisition of a water-maze learning task in rats. Moreover, NS1738 improved performance in the rat social recognition test to the same extent as (-)-nicotine, demonstrating that NS1738 is capable of producing cognitive enhancement in vivo. These data support the notion that alpha7 nAChR allosteric modulation may constitute a novel pharmacological principle for the treatment of cognitive dysfunction.

Distinct profiles of alpha7 nAChR positive allosteric modulation revealed by structurally diverse chemotypes.

Selective modulation of alpha7 nicotinic acetylcholine receptors (nAChRs) is thought to regulate processes impaired in schizophrenia, Alzheimer's disease, and other dementias. One approach to target alpha7 nAChRs is by positive allosteric modulation. Structurally diverse compounds, including PNU-120596, 4-naphthalene-1-yl-3a,4,5,9b-tetrahydro-3-H-cyclopenta[c]quinoline-8-sulfonic acid amide (TQS), and 5-hydroxyindole (5-HI) have been identified as positive allosteric modulators (PAMs), but their receptor interactions and pharmacological profiles remain to be fully elucidated. In this study, we investigated interactions of these compounds at human alpha7 nAChRs, expressed in Xenopus laevis oocytes, along with genistein, a tyrosine kinase inhibitor. Genistein was found to function as a PAM. Two types of PAM profiles were observed. 5-HI and genistein predominantly affected the apparent peak current (type I) whereas PNU-120596 and TQS increased the apparent peak current and evoked a distinct weakly decaying current (type II). Concentration-responses to agonists [ACh, 3-[(3E)-3-[(2,4-dimethoxyphenyl)methylidene]-5,6-dihydro-4H-pyridin-2-yl]pyridine dihydrochloride (GTS-21), and N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide hydrochloride (PNU-282987)] were potentiated by both types, although type II PAMs had greater effects. When applied after alpha7 nAChRs were desensitized, type II, but not type I, PAMs could reactivate alpha7 currents. Both types of PAMs also increased the ACh-evoked alpha7 window currents, with type II PAMs generally showing larger potentiation. None of the PAMs tested increased nicotine-evoked Ca(2+) transients in human embryonic kidney 293 cells expressing human alpha4beta2 or alpha3beta4 nAChRs, although some inhibition was noted for 5-HI, genistein, and TQS. In summary, our studies reveal two distinct alpha7 PAM profiles, which could offer unique opportunities for modulating alpha7 nAChRs in vivo and in the development of novel therapeutics for central nervous system indications.

Effects of substitution on 9-(3-bromo-4-fluorophenyl)-5,9-dihydro-3H,4H-2,6-dioxa-4- azacyclopenta[b]naphthalene-1,8-dione, a dihydropyridine ATP-sensitive potassium channel opener.

Structure-activity relationships were investigated on the tricyclic dihydropyridine (DHP) KATP openers 9-(3-bromo-4-fluorophenyl)-5,9-dihydro-3H,4H-2,6-dioxa-4-azacyclopenta[b]naphthalene-1,8-dione (6) and 10-(3-bromo-4-fluorophenyl)-9,10-dihydro-1H,8H-2,7-dioxa-9-azaanthracene-4,5-dione (65). Substitution off the core of the DHP, absolute stereochemistry, and aromatic substitution were evaluated for KATP channel activity using Ltk- cells stably transfected with the Kir6.2/SUR2B exon 17- splice variant and in an electrically stimulated pig bladder strip assay. A select group of compounds was evaluated for in vitro inhibition of spontaneous bladder contractions. Several compounds were found to have the unique characteristic of partial efficacy in both the cell-based and electrically stimulated bladder strip assays but full efficacy in inhibiting spontaneous bladder strip contractions. For compound 23b, this profile was mirrored in vivo where it was fully efficacious in inhibiting spontaneous myogenic bladder contractions but only partially able to reduce neurogenically mediated reflex bladder contractions.

Alpha4beta2 nicotinic receptor stimulation contributes to the effects of nicotine in the DBA/2 mouse model of sensory gating.

RATIONALE: Nicotine improves the deficiencies of sensory gating function in schizophrenic patients and in dilute brown non-Agouti (DBA/2) mice. This effect of nicotine has been attributed to activation of the alpha7 nicotinic acetylcholine receptor (nAChR) subtype. OBJECTIVE: The aim of this study was to determine whether the activation of another nAChR subtype, the central nervous system (CNS) prominent alpha4beta2 receptor, also contributes to the effects of nicotine on sensory gating in DBA/2 mice. METHODS: Unanesthetized DBA/2 mice were treated either with nicotine, the alpha4beta2 antagonist dihydro-beta-erythroidine, the noncompetitive nAChR antagonist mecamylamine, or a combination of an antagonist and nicotine. Thereafter, gating was assessed by recording hippocampal evoked potentials (EP), which were elicited by pairs of auditory clicks. The EP response to the second click, or test amplitude (TAMP), was divided by the EP response to the first click, or condition amplitude (CAMP), to derive gating T:C ratios. RESULTS: Nicotine significantly (p<0.05) lowered T:C ratios by 42%, while significantly increasing CAMP by 55%. After a pretreatment with dihydro-beta-erythroidine, nicotine still significantly lowered T:C ratios by 28%; however, the nicotine-induced increase of CAMP was blocked. Mecamylamine blocked the effect of nicotine on both T:C ratios and CAMP. CONCLUSIONS: Activation of alpha4beta2 receptors by nicotine increases CAMP. However, under conditions where alpha4beta2 receptors are blocked, nicotine still lowers T:C ratios and may improve sensory gating, possibly through the activation of other nAChR subtypes such as alpha7. These effects of nicotine on auditory EPs may be indicative of a profile that would improve information processing in schizophrenia and other CNS diseases.