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Objective: The attenuation of renal ischemia reperfusion (I/R) injury by stimulating cholinergic antiinflamatory pathway has been demonstrated in the literature. The purpose of this study is to investigate the protective effects of epibatidine, a non selective nicotinic acethycholine receptor agonist, administered before ischemia on renal ischemia-reperfusion injury. Methods: After Ethics Committee approval, 24 Wistar Albino rats were randomly allocated into 4 groups. Group S: Sham (n=6), Group I/R: Ischemia/reperfusion (n=6), Group I/R-E: Ischemia/reperfusion-Epibatidine (n=6), Group E: Epibatidine (n=6). All groups underwent laparotomy and bilateral renal artery and vein dissection under anesthesia. No further action was taken in Group S. In Group I/R and Group I/R-E, renal ischemia was applied for 25 minutes and then reperfusion for 24 hours. A single dose of 5 μg kg-1 epibatidine was administered subcutaneously before the laparotomy in Group I/R-E and Group E. At the end of the reperfusion period, blood and urine samples were taken for plasma BUN, creatine, plasma and urinary neutrophil gelatinase associated lipocalin (NGAL) and cystatin C, plasma IL-6 and TNF-α assessment. Results: Plasma BUN, creatine, NGAL, cystatin C, IL-6 and TNF-α levels were significantly higher in Group I/R when compared to Group S (p<0.05). All the levels of the above biomarkers were comparable in Group S and Group I/R-E. Conclusion: These results indicate that epibatidine administration before ischemia has a protective effect on kidney in renal I/R injury induced rats. [ABSTRACT FROM AUTHOR]

Abstract Background Some dendrobatid poison frogs sequester the toxin epibatidine as a defense against predators. We previously identified an amino acid substitution (S108C) at a highly conserved site in a nicotinic acetylcholine receptor β2 subunit of dendrobatid frogs that decreases sensitivity to epibatidine in the brain-expressing α4β2 receptor. Introduction of S108C to the orthologous high-sensitivity human receptor similarly decreased sensitivity to epibatidine but also decreased sensitivity to acetylcholine, a potential cost if this were to occur in dendrobatids. This decrease in the acetylcholine sensitivity manifested as a biphasic acetylcholine concentration–response curve consistent with the addition of low-sensitivity receptors. Surprisingly, the addition of the β2 S108C into the α4β2 receptor of the dendrobatid Epipedobates anthonyi did not change acetylcholine sensitivity, appearing cost-free. We proposed that toxin-bearing dendrobatids may have additional amino acid substitutions protecting their receptors from alterations in acetylcholine sensitivity. To test this, in the current study, we compared the dendrobatid receptor to its homologs from two non-dendrobatid frogs. Results The introduction of S108C into the α4β2 receptors of two non-dendrobatid frogs also does not affect acetylcholine sensitivity suggesting no additional dendrobatid-specific substitutions. However, S108C decreased the magnitude of neurotransmitter-induced currents in Epipedobates and the non-dendrobatid frogs. We confirmed that decreased current resulted from fewer receptors in the plasma membrane in Epipedobates using radiolabeled antibodies against the receptors. To test whether S108C alteration of acetylcholine sensitivity in the human receptor was due to (1) adding low-sensitivity binding sites by changing stoichiometry or (2) converting existing high- to low-sensitivity binding sites with no stoichiometric alteration, we made concatenated α4β2 receptors in stoichiometry with only high-sensitivity sites. S108C substitutions decreased maximal current and number of immunolabeled receptors but no longer altered acetylcholine sensitivity. Conclusions The most parsimonious explanation of our current and previous work is that the S108C substitution renders the β2 subunit less efficient in assembling/trafficking, thereby decreasing the number of receptors in the plasma membrane. Thus, while β2 S108C protects dendrobatids against sequestered epibatidine, it incurs a potential physiological cost of disrupted α4β2 receptor function.

Background: Some dendrobatid poison frogs sequester the toxin epibatidine as a defense against predators. We previously identified an amino acid substitution (S108C) at a highly conserved site in a nicotinic acetylcholine receptor β2 subunit of dendrobatid frogs that decreases sensitivity to epibatidine in the brain-expressing α4β2 receptor. Introduction of S108C to the orthologous high-sensitivity human receptor similarly decreased sensitivity to epibatidine but also decreased sensitivity to acetylcholine, a potential cost if this were to occur in dendrobatids. This decrease in the acetylcholine sensitivity manifested as a biphasic acetylcholine concentration–response curve consistent with the addition of low-sensitivity receptors. Surprisingly, the addition of the β2 S108C into the α4β2 receptor of the dendrobatid Epipedobates anthonyi did not change acetylcholine sensitivity, appearing cost-free. We proposed that toxin-bearing dendrobatids may have additional amino acid substitutions protecting their receptors from alterations in acetylcholine sensitivity. To test this, in the current study, we compared the dendrobatid receptor to its homologs from two non-dendrobatid frogs. Results: The introduction of S108C into the α4β2 receptors of two non-dendrobatid frogs also does not affect acetylcholine sensitivity suggesting no additional dendrobatid-specific substitutions. However, S108C decreased the magnitude of neurotransmitter-induced currents in Epipedobates and the non-dendrobatid frogs. We confirmed that decreased current resulted from fewer receptors in the plasma membrane in Epipedobates using radiolabeled antibodies against the receptors. To test whether S108C alteration of acetylcholine sensitivity in the human receptor was due to (1) adding low-sensitivity binding sites by changing stoichiometry or (2) converting existing high- to low-sensitivity binding sites with no stoichiometric alteration, we made concatenated α4β2 receptors in stoichiometry with only high-sensitivity sites. S108C substitutions decreased maximal current and number of immunolabeled receptors but no longer altered acetylcholine sensitivity. Conclusions: The most parsimonious explanation of our current and previous work is that the S108C substitution renders the β2 subunit less efficient in assembling/trafficking, thereby decreasing the number of receptors in the plasma membrane. Thus, while β2 S108C protects dendrobatids against sequestered epibatidine, it incurs a potential physiological cost of disrupted α4β2 receptor function. [ABSTRACT FROM AUTHOR]

This investigation defines the roles of various amino acids, neighboring key conserved amino acids in loops C and D of the nicotinic acetylcholine (ACh) receptor (nAChR), in the selective molecular recognition of nicotinic ligands with diverse pharmacophores using Aplysia californica ACh binding protein Y55W ( Ac -AChBP) mutants (+Q57R; + Q57R+S189 V; + Q57R+S189E; + Q57T; + Q57T+S189 V; + Q57T+S189E) and Lymnaea stagnalis AChBP ( Ls- AChBP) mutants (Q55T; Q55T+S186E; Q55R) as insect and mammalian nAChR structural surrogates, respectively. N -nitro/cyanoimine insecticides show high affinity to four Ac -AChBPs containing Arg57 or Thr57 and Ser189 or Val189, except for those with Glu189. Pyrazinoyl compound selectively interacts with the three Ac -AChBPs containing Arg57 and Ser189, Val189, or Glu189. Cationic ligands prefer three Ac -AChBPs with Thr57 and Ser189, Val189, or Glu189 and two Ls -AChBPs providing Thr55 ± Glu186 over the four Ac - and Ls -AChBPs with Arg57/55. Accordingly, loop C contributes to N -nitro/cyanoimine insecticide action, and loop D controls the affinity of the pyrazinoyl or cationic ligand.

Rationale: Prolinol aryl ethers and their rigidified analogues pyrrolidinyl benzodioxanes have a high affinity for mammalian α4β2 nicotinic acetylcholine receptors (nAChRs). Electrophysiological studies have shown that the former are full agonists and the latter partial agonists or antagonists of human α4β2 receptors, but their in vivo effects are unknown. Objectives and methods: As α4β2 nAChRs play an important role in the cognition and the rewarding effects of nicotine, we tested the effects of two full agonists and one antagonist on spatial learning, memory and attention in zebrafish using a T-maze task and virtual object recognition test (VORT). The effect of a partial agonist in reducing nicotine-induced conditioned place preference (CPP) was also investigated. Results: In comparison with the vehicle alone, the full agonists MCL-11 and MCL-28 induced a significant cognitive enhancement as measured by the reduced running time in the T-maze and increased attention as measured by the increased discrimination index in the VORT. MCL-11 was 882 times more potent than nicotine. The two compounds were characterised by an inverted U-shaped dose-response curve, and their effects were blocked by the co-administration of the antagonist MCL-117, which alone had no effect. The partial agonist MCL-54 induced CPP and had an inverted U-shaped dose-response curve similar to that of nicotine but blocked the reinforcing effect of co-administered nicotine. Binding studies showed that all of the compounds have a higher affinity for heteromeric [3H]-epibatidine receptors than [125I]-αBungarotoxin receptors. MCL-11 was the most selective of heteromeric receptors. Conclusions: These behavioural studies indicate that full agonist prolinol aryl ethers are very active in increasing spatial learning, memory and attention in zebrafish. The benzodioxane partial agonist MCL-54 reduced nicotine-induced CPP, and the benzodioxane antagonist MCL-117 blocked all agonist-induced activities. [ABSTRACT FROM AUTHOR]

7-Azabicyclo[2.2.1]heptane (7-azanorbornane) is a bridged heterocyclic nucleus found in epibatidine, the alkaloid isolated from the skin of the tropical poison frog Epipedobates tricolor. Since epibatidine is known as one of the most potent acetylcholine nicotinic receptor agonists, a plethora of literature has been devoted to this alkaloid. However, there are no structural data on the unsubstituted 7-azanorbornane, the parent bicyclic ring of epibatidine and its derivatives. We herein present the structural characterization of the 7-azabicyclo[2.2.1]heptane parent ring as its hydrochloride salt, namely 7-azabicyclo[2.2.1]heptan-7-ium chloride, C6H12N+·Cl−. The compete cation is generated by a crystallographic mirror plane with the N atom lying on the mirror, as does the chloride anion. In the crystal, the cations are linked to the anions by N—H...Cl hydrogen bonds, which generate [001] chains.

Epibatidine is a natural alkaloid that acts at nicotinic acetylcholine receptors (nAChRs). The present review aims to carefully discuss the affinity of epibatidine and its synthetic derivatives, analogues to nAChRs for α4β2 subtype, pharmacokinetic parameters, and its role in health. Published literature shows a low affinity and lack of binding of epibatidine and its synthetic analogues to plasma proteins, indicating their availability for metabolism. Because of its high toxicity, the therapeutic use of epibatidine is hampered. However, new synthetic analogs endowed from this molecule have been developed, with a better therapeutic window and improved selectivity. All these aspects are also discussed here. On the other hand, many reports are devoted to structure–activity relationships to obtain optically active epibatidine and its analogues, and to access its pharmacological effects. Although pharmacological results are obtained from experimental studies and only a few clinical trials, new perspectives are open for the discovery of new drug therapies.

The determination of the conformational states corresponding to diverse functional roles of ligand gated ion channels is subject of intense investigation with various techniques, from X-rays structure determination to electrophysiology and computational modeling. Even with a certain number of structures becoming recently available, only few major structural features distinguishing conductive open channel from the non conductive resting protein have been highlighted, while high-resolution details are still missing. The characterization of the desensitized conformation(s) is even more complex, and only few specific characteristics have been identified. Furthermore, experimental data provide conflicting information for different ion channels, adding further complexity to the topic. Desensitization is defined as the transition of the agonist-bound open channel into an ion channel configuration inactive even in the presence of agonists. In this work, we analyze a conformation corresponding to a non conductive state obtained via molecular dynamics simulations of a homology model of the human α 7 nicotinic receptor complexed with agonists. We highlight some characteristics that could associate it to a desensitized state. The obtained structure is assessed against experimental data for other ligand gated ion channels that have been putatively associated to active, inactive and desensitized conditions. [ABSTRACT FROM AUTHOR]

7-Azabicyclo[2.2.1]heptane (7-azanorbornane) is a bridged heterocyclic nucleus found in epibatidine, the alkaloid isolated from the skin of the tropical poison frog Epipedobates tricolor. Since epibatidine is known as one of the most potent acetylcholine nicotinic receptor agonists, a plethora of literature has been devoted to this alkaloid. However, there are no structural data on the unsubstituted 7-azanorbornane, the parent bicyclic ring of epibatidine and its derivatives. We herein present the structural characterization of the 7- azabicyclo[2.2.1]heptane parent ring as its hydrochloride salt, namely 7- azabicyclo[2.2.1]heptan-7-ium chloride, C6H12N+ºCl-. The compete cation is generated by a crystallographic mirror plane with the N atom lying on the mirror, as does the chloride anion. In the crystal, the cations are linked to the anions by N--H ... Cl hydrogen bonds, which generate [001] chain [ABSTRACT FROM AUTHOR]

Epibatidine is an alkaloid toxin that binds with high affinity to nicotinic and muscarinic acetylcholine receptors, and has been extensively used as a research tool. To examine binding interactions at the nicotinic receptor, it has been co-crystallised with the structural homologue acetylcholine binding protein (AChBP; PDB ID 2BYQ ), and with an AChBP chimaera (3SQ6) that shares 64% sequence identity with the α7 nACh receptor. However, the binding orientations revealed by AChBP co-crystal structures may not precisely represent their receptor homologues and experimental evidence is needed to verify the ligand poses. Here we identify potential binding site interactions between epibatidine and AChBP residues, and substitute equivalent positions in the α7 nACh receptor. The effects of these are probed by [ 3 H]epibatidine binding following the expression α7 nACh receptor cysteine mutants in HEK 293 cells. Of the sixteen mutants created, the affinity of epibatidine was unaffected by the substitutions Q55C, L106C, L116C, T146C, D160C and S162C, reduced by C186A and C187A, increased by Q114C and S144C, and abolished by W53C, Y91C, N104C, W145C, Y184C and Y191C. These results are consistent with the predicted orientations in AChBP and suggest that epibatidine is likely to occupy a similar location at α7 nACh receptors. We speculate that steric constraints placed upon the C-5 position of the pyridine ring in 3SQ6 may account for the relatively poor affinities of epibatidine derivatives that are substituted at this position. [ABSTRACT FROM AUTHOR]

An ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the simultaneous quantification of batrachotoxin and epibatidine in plasma. Plasma samples were pretreated by liquid-liquid extraction with acetonitrile and methanol. The toxins were separated on a reversed phase C18-column (2.1 mm × 50 mm, 1.7 μm) using a formic acid/acetonitrile gradient elution. Quantification was carried out by mass chromatography with each product ion referenced against midazolam-d 4 as an internal standard (IS). The two toxins and the IS were separated within 2 min. The calibration curves for the two toxins spiked into human plasma showed good linearities in the range from 2.5 to 250 ng/mL. The detection limits were estimated to be 0.5 ng/mL for batrachotoxin and 1 ng/mL for epibatidine with a signal-to-noise ratio of 3:1. Overall recoveries ranged from 69.6% to 98.2%, and no significant matrix effects were observed. The intra- and interday accuracies were 94.7–102.3%, and the precisions were 1.0–10.3%. This method was successfully applied for the quantification of batrachotoxin and epibatidine in rat plasma samples taken after intraperitoneal administration of the toxins. This is the first report to use UPLC-MS/MS to simultaneously quantify batrachotoxin and epibatidine in plasma samples. [ABSTRACT FROM AUTHOR]

Autoradiography was used to investigate nicotinic acetylcholine receptor (nAChR) binding in the brains of two groups of macaque monkeys with parkinsonism produced by different types of MPTP exposure: animals with cognitive deficits but no motor symptoms (motor-asymptomatic) and animals with typical motor symptoms of parkinsonism (motor-symptomatic). Motor-asymptomatic animals had no significant changes in [125I]epibatidine binding to β2⁎–β4⁎ nAChRs and [125I]A85380 binding to β2⁎ nAChRs in cognition-related cortical regions such as Broadman’s area 46, orbitofrontal cortex, the anterior cingulate sulcus and the hippocampus, but binding of both radioligands was decreased 70–80% in the caudate and putamen. Motor-symptomatic animals had decreases in β2⁎ and β4⁎ nAChR in the principal sulcus (40–60%), anterior cingulate sulcus (30–55%), and orbitofrontal cortex (30–41%), but not in the hippocampus, plus significant decreases in binding (70–80%) in the caudate and putamen. These results suggest that while nAChR expression is similarly decreased in the striatum of motor-asymptomatic and motor-symptomatic MPTP-treated monkeys, there are differences in β2⁎ and β4⁎ nAChR expression in cortical regions in these two conditions. Therefore, our data suggest that a therapeutic strategy based on nAChR agonist administration that might improve cognition in early PD patients may, due to a changing nAChR profile, have little or no effect on the same symptoms in more advanced patients.

Palladium-catalyzed hydroarylations and additional domino reactions of aza- bicyclic and tricyclic norbornene derivatives were investigated and a series of new epibatidine analogues were synthesized.

Both enantiomers of [18F]flubatine are promising radioligands for neuroimaging of α4ß2 nicotinic acetylcholine receptors (nAChRs) by positron emission tomography (PET). To support clinical studies in patients with early Alzheimer's disease, a detailed examination of the metabolism in vitro and in vivo has been performed. (+)- and (--)-flubatine, respectively, were incubated with liver microsomes from mouse and human in the presence of NADPH (α-nicotinamide adenine dinucleotide 20-phosphate reduced tetrasodium salt). Phase I in vitro metabolites were detected and their structures elucidated by LC-MS/MS (liquid chromatography-tandem mass spectrometry). Selected metabolite candidates were synthesized and investigated for structural confirmation. Besides a high level of in vitro stability, the microsomal incubations revealed some species differences as well as enantiomer discrimination with regard to the formation of monohydroxylated products, which was identified as the main metabolic pathway in this assay. Furthermore, after injection of 250 MBq (+)-[18F]flubatine (specific activity > 350 GBq/κmol) into mouse, samples were prepared from brain, liver, plasma, and urine after 30 min and investigated by radio-HPLC (high performance liquid chromatography with radioactivity detection). For structure elucidation of the radiometabolites of (+)-[18F]flubatine formed in vivo, identical chromatographic conditions were applied to LC-MS/MS and radio-HPLC to compare samples obtained in vitro and in vivo. By this correlation approach, we assigned three of four main in vivo radiometabolites to products that are exclusively C- or N-hydroxylated at the azabicyclic ring system of the parent molecule. [ABSTRACT FROM AUTHOR]

Teratogenic alkaloids can cause developmental defects due to the inhibition of fetal movement that results from desensitization of fetal muscle-type nicotinic acetylcholine receptors (nAChRs). We investigated the ability of two known teratogens, the piperidinyl-pyridine anabasine and its 1,2-dehydropiperidinyl analog anabaseine, to activate and desensitize peripheral nAChRs expressed in TE-671 and SH-SY5Y cells. Activation-concentration response curves for each alkaloid were obtained in the same multi-well plate. To measure rapid desensitization, cells were first exposed to five potentially-desensitizing concentrations of each alkaloid in log10 molar increments from 10 nM to 100 μM and then to a fixed concentration of acetylcholine (ACh), which alone produces near-maximal activation. The fifty percent desensitization concentration (DC50) was calculated from the alkaloid concentration-ACh response curve. Agonist fast desensitization potency was predicted by the agonist potency measured in the initial response. Anabaseine was a more potent desensitizer than anabasine. Relative to anabaseine, nicotine was more potent to autonomic nAChRs, but less potent to the fetal neuromuscular nAChRs. Our experiments have demonstrated that anabaseine is more effective at desensitizing fetal muscle-type nAChRs than anabasine or nicotine and, thus, it is predicted to be more teratogenic. [ABSTRACT FROM AUTHOR]

Rationale: Recent studies in rodents suggest that non-nicotine constituents of tobacco smoke (e.g., minor tobacco alkaloids) may promote tobacco consumption-either through their own pharmacological effects or by augmenting the effects of nicotine. However, there is scant information on the behavioral pharmacology of minor tobacco alkaloids in primate species. Objective: The present studies were conducted to determine whether the minor tobacco alkaloids nornicotine, anabasine, anatabine, myosmine, and cotinine exhibit nicotine-like behavioral effects in squirrel monkeys. Methods: Initial experiments were conducted to determine the effects of nicotine (0.032-1.0 mg/kg) and the minor tobacco alkaloids nornicotine (1-1.8 mg/kg), anabasine (0.1-1.0 mg/kg), anatabine (10-32 mg/kg), myosmine (0.32-1.8 mg/kg), and cotinine (10-180 mg/kg) on food-maintained performance ( n = 4). Next, the ability of tobacco alkaloids to substitute for the α4β2-selective nicotinic agonist (+)-epibatidine in drug discrimination experiments was evaluated in a separate group of monkeys ( n = 4). Results: Results show that nicotine and each minor tobacco alkaloid except cotinine (a) produced dose-related decreases in food-maintained responding; (b) substituted for (+)-epibatidine and, in additional experiments, produced additive effects when combined with nicotine; (c) induced emesis or tremor at doses that reduced food-maintained responding and had (+)-epibatidine-like discriminative-stimulus effects; and (d) based on correlation with reported receptor binding affinities, likely produced their behavioral effects through α4β2 receptor mechanisms. Conclusion: Selected minor tobacco alkaloids have nicotinic-like effects that may contribute to tobacco consumption and addiction. [ABSTRACT FROM AUTHOR]

Effects of nicotinic receptor agonists (epibatidine and nicotine) on mechano-sensitive bladder afferent nerve (MS-BAN) activity were studied in an in vitro bladder–pelvic afferent nerve preparation. MS-BAN activity was induced by isotonic distention of the bladder at pressures of 10–40 cmH 2 O. The effect of epibatidine varied according to the concentration, route of administration and the intravesical pressure stimulus. Epibatidine (300–500 nM) administered in the perfusate to the serosal surface of the bladder decreased distension evoked afferent firing by 30–50% depending on the bladder pressure. However these concentrations also produced an immediate increase in tonic afferent firing in the empty bladder. Lower concentrations (50–100 nM) elicited weaker and more variable effects. The inhibitory effects were blocked by bath application of mecamylamine (150 µM) a nicotinic receptor antagonist. Bath application of nicotine (20 µM) elicited similar effects. Intravesical administration of epibatidine (500 nM) significantly increased MS-BAN firing by 15–30%; while lower concentrations (200–300 nM) were ineffective. This facilitatory effect of epibatidine was blocked by intravesical administration of mecamylamine (250 µM). Electrical stimulation on the surface of the bladder elicited action potentials (AP) in BAN. Bath application of epibatidine (300 nM) or nicotine (20 µM) did not change either the voltage threshold or the area of evoked AP. These results indicate that nicotinic agonists: (1) enhance MS-BAN activity originating at afferent receptors near the urothelium, (2) inhibit MS-BAN activity originating at afferent receptors located at other sites in the bladder, (3) directly excite unidentified afferents, (4) do not alter afferent axonal excitability. [ABSTRACT FROM AUTHOR]

The nicotinic acetylcholine receptor (nAChR) subtypes were characterized in the superior cervical ganglion (SCG) of wild-type and dystrophin-lacking mdx mice. The binding of Epibatidine and αBungarotoxin, ligands for α3- and α7-containing receptors, respectively, revealed, for each ligand, a single class of high-affinity binding sites, with similar affinity in both wild-type and mdx mice. The Epibatidine-labeled receptors were immunoprecipitated by antibodies against the α3, β2, and β4 subunits. Immunocytochemistry showed that the percentage of α3-, β2-, and β4- but not of α7-immunopositive postsynaptic specializations was significantly lower in mdx than in wild-type mouse SCG. These observations suggest that the mouse SCG contains nAChRs, stabilized by dystrophin, in which the α3 subunit is associated with the β2 and/or β4 subunits. Conversely, dystrophin is not involved in the stabilization of the α7-containing nAChRs, as the percentage of α7-immunopositive synapses is similar in both wild-type and mdx mouse SCG.

Nicotinic acetylcholine receptors (nAChRs) play a crucial role in a number of clinically relevant mental and neurological pathways, as well as autonomic and immune functions. The development of subtype-selective ligands for nAChRs therefore is potentially useful for targeted therapeutic management of conditions where nAChRs are involved. We tested if selectivity for a particular nAChR subtype can be achieved through small structural modifications of a lead compound containing the nicotinic pharmacophore by changing the distance between the electronegative elements. For this purpose, analogs of A-84543 were designed, synthesized and characterized as potentially new nAChR subtype-selective ligands. Compounds were tested for their binding properties in rat cerebral cortical tissue homogenates, and subtype-selectivity was determined using stably transfected HEK cells expressing different nAChR subtypes. All compounds synthesized were found to competitively displace [H]-epibatidine ([H]EB) from the nAChR binding site. Of all the analogues, H-11MNH showed highest affinity for nAChRs compared to a ~fivefold to tenfold lower affinity of A-84543. All other compounds had affinities >10,000 nM. Both A-84543 and H-11MNH have highest affinity for α2β2 and α4β2 nAChRs and show moderate affinity for β4- and α7-containing receptors. H-11MNH was found to be a full agonist with high potency at α3β4, while A-84543 is a partial agonist with low potency. Based on their unique pharmacological binding properties we suggest that A-84543 and its desmethylpyrrolidine analog can be useful as pharmacological ligands for studying nAChRs if selective pharmacological and/or genetic tools are used to mask the function of other receptors subtypes. [ABSTRACT FROM AUTHOR]

The nicotine (NIC) withdrawal syndrome is considered to be a major cause of the high relapse rate among individuals undergoing smoking cessation. The aim of the present study was to evaluate a possible role of GABA B receptors in NIC withdrawal, by comparing GABA B1 knockout mice and their wild-type littermates. We analysed the time course of the global withdrawal score, the anxiety-like effects, monoamine concentrations, the brain-derived neurotrophic factor (BDNF) expression, the corticosterone plasmatic levels and [ 3 H]epibatidine binding sites during NIC withdrawal precipitated by mecamylamine, a nicotinic receptor antagonist (MEC). In NIC withdrawn wild-type mice, we observed a global withdrawal score, an anxiety-like effect in the elevated plus maze, a decrease of the striatal dopamine and 3,4-dihydroxyphenylacetic acid concentrations, an increase of corticosterone plasma levels, a reduction of BDNF expression in several brain areas and an increase of [ 3 H]epibatidine binding sites in specific brain regions. Interestingly, the effects found in NIC withdrawn wild-type mice were absent in GABA B1 knockout mice, suggesting that GABA B1 subunit of the GABA B receptor is involved in the regulation of the behavioural and biochemical alterations induced by NIC withdrawal in mice. These results reveal an interaction between the GABA B receptors and the neurochemical systems through which NIC exerts its long-term effects. [ABSTRACT FROM AUTHOR]

A method has been developed for the determination of 2- endo(6′-chloropyridin-3′-yl)-7-azabicyclo[2.2.1]heptane (epibatidine) and 2- endo(6′-chloropyridin-3′-yl)-7-azabicyclo[2.2.1]heptane-7-ol (epibatidine biomarker) in blood plasma at a level of 1 × 10-1 × 10 mg/mL. The method is based on the extraction of components from blood plasma by liquid-liquid extraction followed by gas-chromatographic determination using thermionic or mass spectrometric detectors. To reduce the limit of detection for epibatidine and its biomarker in using the mass-spectrometric detector, a derivatization stage using N, O-bis(trimethylsilyl)trifluoroacetamide or 2,3,4,5,6-pentafluorobenzyl bromide has been included into the procedure. [ABSTRACT FROM AUTHOR]

A convenient one-step method for the preparation of 8-azabicyclo[3.2.1]octan-3-one derivatives, analogs of epibatidine, was suggested, which consists in the reaction of succinic aldehyde with heterocyclic amines and acetonedicarboxylic acid in the presence of sodium hydrogen phosphate. [ABSTRACT FROM AUTHOR]

Both enantiomers of [18F]flubatine are promising radioligands for neuroimaging of α4β2 nicotinic acetylcholine receptors (nAChRs) by positron emission tomography (PET). To support clinical studies in patients with early Alzheimer’s disease, a detailed examination of the metabolism in vitro and in vivo has been performed. (+)- and (−)-flubatine, respectively, were incubated with liver microsomes from mouse and human in the presence of NADPH (β-nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt). Phase I in vitro metabolites were detected and their structures elucidated by LC-MS/MS (liquid chromatography-tandem mass spectrometry). Selected metabolite candidates were synthesized and investigated for structural confirmation. Besides a high level of in vitro stability, the microsomal incubations revealed some species differences as well as enantiomer discrimination with regard to the formation of monohydroxylated products, which was identified as the main metabolic pathway in this assay. Furthermore, after injection of 250 MBq (+)-[18F]flubatine (specific activity > 350 GBq/μmol) into mouse, samples were prepared from brain, liver, plasma, and urine after 30 min and investigated by radio-HPLC (high performance liquid chromatography with radioactivity detection). For structure elucidation of the radiometabolites of (+)-[18F]flubatine formed in vivo, identical chromatographic conditions were applied to LC-MS/MS and radio-HPLC to compare samples obtained in vitro and in vivo. By this correlation approach, we assigned three of four main in vivo radiometabolites to products that are exclusively C- or N-hydroxylated at the azabicyclic ring system of the parent molecule.

Teratogenic alkaloids can cause developmental defects due to the inhibition of fetal movement that results from desensitization of fetal muscle-type nicotinic acetylcholine receptors (nAChRs). We investigated the ability of two known teratogens, the piperidinyl-pyridine anabasine and its 1,2-dehydropiperidinyl analog anabaseine, to activate and desensitize peripheral nAChRs expressed in TE-671 and SH-SY5Y cells. Activation-concentration response curves for each alkaloid were obtained in the same multi-well plate. To measure rapid desensitization, cells were first exposed to five potentially-desensitizing concentrations of each alkaloid in log10 molar increments from 10 nM to 100 µM and then to a fixed concentration of acetylcholine (ACh), which alone produces near-maximal activation. The fifty percent desensitization concentration (DC50) was calculated from the alkaloid concentration-ACh response curve. Agonist fast desensitization potency was predicted by the agonist potency measured in the initial response. Anabaseine was a more potent desensitizer than anabasine. Relative to anabaseine, nicotine was more potent to autonomic nAChRs, but less potent to the fetal neuromuscular nAChRs. Our experiments have demonstrated that anabaseine is more effective at desensitizing fetal muscle-type nAChRs than anabasine or nicotine and, thus, it is predicted to be more teratogenic.

Abstract: Previous studies indicate that 3,4-methylenedioxy-methamphetamine (MDMA, ecstasy) can induce a heteromeric nicotinic acetylcholine receptor (nAChR, mainly of α4β2 subtype) up-regulation. In this study we treated male Sprague–Dawley rats twice-daily for 10days with either saline or MDMA (7mg/kg) and sacrificed them the day after to perform [125I]Epibatidine binding autoradiograms on serial coronal slices. MDMA induced significant increases in nAChR density in the substantia nigra, ventral tegmental area, nucleus accumbens, olfactory tubercle, anterior caudate–putamen, somatosensory, motor, auditory and retrosplenial cortex, laterodorsal thalamus nuclei, amygdala, postsubiculum and pontine nuclei. These increases ranged from 3% (retrosplenial cortex) to 30 and 34% (amygdala and substantia nigra). No increased α4 subunit immunoreactivity was found in up-regulated areas compared with saline-treated rats, suggesting a post-translational mechanism as occurs with nicotine. The heteromeric nAChR up-regulation in certain areas could account, at least in part, for the reinforcing, sensitizing and psychiatric disorders observed after long-term consumption of MDMA. [Copyright &y& Elsevier]

Anatoxin- a and epibatidine are natural toxins with a high affinity to nicotinic acetylcholine receptors (nAChR). Nicotinic ligands have the potential to become novel therapeutic agents for various cognitive disorders such as Alzheimer's and Parkinson's diseases. The determination of the physicochemical and biological properties of anatoxin- a and epibatidine derivatives is important because these might lead to the development of new cholinergic therapeutic agents. To study these features, the toxins and a set of their derivatives were subjected to a molecular modelling study and QSAR analysis. The structural analyses indicated that the geometric and steric features are important determinants of the compound's activities. The descriptors selected for the QSAR model also highlighted the roles of the geometric and steric features, together with the importance of electronic features. [ABSTRACT FROM AUTHOR]

A preparative-scale method was developed for the production of the analgesic epibatidine, yielding its endo and exo isomers with yields of 9.9% and 8.8% respectively. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate layer and species variations in detrusor muscle strip responses to myogenic, neurogenic, and nicotinic, and muscarinic receptor stimulations. Strips from bladders of 9 dogs and 6 human organ transplant donors were dissected from inner and outer longitudinal muscle layers, at least 1 cm above urethral orifices. Strips were mounted in muscle baths and maximal responses to neurogenic stimulation using electrical field stimulation (EFS) and myogenic stimulation using potassium chloride (KCl, 120 mM) determined. After washing and re-equilibration was completed, responses to nicotinic receptor agonist epibatidine (10 μM) were determined followed by responses to EFS and muscarinic receptor agonist bethanechol (30 μM) in continued presence of epibatidine. Thereafter, strips and full-thickness bladder sections from four additional dogs and three human donors were examined for axonal density and intramural ganglia. In dog bladders, contractions to KCl, epibatidine, and bethanechol were 1.5- to 2-fold higher in the inner longitudinal muscle layer, whereas contractions to EFS were 1.5-fold higher in the outer (both pre- and post-epibatidine). Human bladders showed 1.2-fold greater contractions to epibatidine in the inner layer and to EFS in the outer, yet no layer differences to KCl or bethanechol were noted. In both species, axonal density was 2- to 2.5-fold greater in the outer layer. Dogs had more intramural ganglia in the adventitia/serosa layer, compared with more internal layers and to humans. These findings indicate several layer-dependent differences in receptor expression or distribution, and neurogenic responses in dog and human detrusor muscles, and myogenic/muscarinic differences between dog versus humans.