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1.  Chronic Sazetidine-A Maintains Anxiolytic Effects and Slower Weight Gain Following Chronic Nicotine Without Maintaining Increased Density of Nicotinic Receptors in Rodent Brain 
Journal of neurochemistry  2014;129(4):721-731.
Chronic nicotine administration increases the density of brain α4β2* nicotinic acetylcholine receptors (nAChRs), which may contribute to withdrawal symptoms associated with smoking cessation. Varenicline, a smoking cessation drug, also increases these receptors in rodent brain. The maintenance of this increase by varenicline as well as nicotine replacement may contribute to the high rate of relapse during the first year after smoking cessation. Recently we found that sazetidine-A, a potent partial agonist that desensitizes α4β2* nAChRs, does not increase the density of these receptors in brain at doses that decrease nicotine self-administration, increase attention in rats, and produce anxiolytic effects in mice. Here we investigated whether chronic sazetidine-A and varenicline maintain the density of nAChRs after their up-regulation by nicotine. In addition, we examined the effects of these drugs on a measure of anxiety in mice and weight gain in rats. After increasing nAChRs in the rodent brain with chronic nicotine, replacing nicotine with chronic varenicline maintained the increased nAChR binding, as well as the subunit proteins measured by western blots. In contrast, replacing nicotine treatments with chronic sazetidine-A resulted in the return of the density of nAChRs to the levels seen in saline controls. Nicotine, sazetidine-A and varenicline each demonstrated anxiolytic effects in mice, but only sazetidine-A and nicotine attenuated the gain of weight over a 6-week period in rats. These findings suggest that apart from its modest anxiolytic and weight control effects, sazetidine-A, or drugs like it, may be useful in achieving long-term abstinence from smoking.
PMCID: PMC3999245  PMID: 24422997
nicotinic receptor; nicotine dependence; receptor up-regulation; sazetidine-A; varenicline; nicotine
2.  Effects of chronic nicotine on heteromeric neuronal nicotinic receptors expressed in rat primary cultured neurons 
Journal of neurochemistry  2011;119(1):153-164.
Nicotine increases the number of neuronal nicotinic receptors (nAChRs) in brain. This study investigated the effects of chronic nicotine treatment on nAChRs expressed in primary cultured neurons. In particular, we studied the chronic effects of nicotine exposure on the total density, surface expression and turnover rate of heteromeric nAChRs. The receptor density was measured by [125I]epibatidine ([125I]EB) binding. Untreated and nicotine-treated neurons were compared from several regions of embryonic (E19) rat brain. Twelve days of treatment with 10 µM nicotine produced a two-fold up-regulation of nAChRs. Biotinylation and whole-cell binding studies indicated that up-regulation resulted from an increase in the number of cell surface receptors as well as intracellular receptors. nAChR subunit composition in cortical and hippocampal neurons was assessed by immunoprecipitation with subunit-selective antibodies. These neurons contain predominantly α4, β2 and α5 subunits, but α2, α3, α6 and β4 subunits were also detected. Chronic nicotine exposure yielded a two-fold increase in the β2-containing receptors and a smaller up-regulation in the α4-containing nAChRs. To explore the mechanisms of up-regulation we investigated the effects of nicotine on the receptor turnover rate. We found that the turnover rate of surface receptors was > two weeks and chronic nicotine exposure had no effect on this rate.
PMCID: PMC3171599  PMID: 21806615
Nicotinic Receptors; Primary neurons; Up-regulation; Cell Surface; Receptor degradation rate
3.  Quantitative analysis of the heteromeric neuronal nicotinic receptors in the rat hippocampus 
Journal of neurochemistry  2010;115(3):625-634.
The objective of this study was to identify and quantify the heteromeric neuronal nicotinic receptors (nAChRs) in the rat hippocampus. The density of nAChR subtypes was assessed by labeling them with [3H]epibatidine followed by immunoprecipitation with subunit-selective antibodies. Sequential immunoprecipitation assays were used to establish associations between two different subunits, which then allowed the full subunit composition of the receptors to be deduced. Our results show that most of the hippocampal heteromeric nAChRs contain α4 and β2 subunits. In fact, we identified two populations containing these two predominant subunits, the α4β2 and α4β2α5 subtypes which account for ~40% and ~35%, respectively, of the total [3H]epibatidine-labeled receptors. An additional heteromeric subtype with the subunit composition of α4β2α3 represented ~10% of the total nAChRs, and another 10% of the immunoprecipitated receptors contained α4 and β4 subunits, with or without the α3 subunit. To determine if α4β2 and α4β2α5 nAChR subtypes differ in their ligand binding affinities, the α3- and β4-containing receptors were first removed by immunoprecipitation and then, competition studies with acetylcholine, nicotine, cytisine and sazetidine-A against [3H]epibatidine were carried out on the remaining α4β2 and α4β2α5 subtypes. Results suggested these subtypes have comparable binding affinities for the nicotinic ligands used here.
PMCID: PMC2976603  PMID: 20796176
Neuronal nicotinic receptor; heteromeric subtypes; α5 subunit; hippocampus; immunoprecipitation; binding affinity
4.  Effects of the sazetidine-a family of compounds on the body temperature in wildtype, nicotinic receptor β2−/− and α7−/− mice 
European journal of pharmacology  2013;718(0):10.1016/j.ejphar.2013.08.037.
Nicotine elicits hypothermic responses in rodents. This effect appears to be related to nicotinic receptor desensitization because sazetidine-A, an α4β2 nicotinic receptor desensitizing agent, produces marked hypothermia and potentiates nicotine-induced hypothermia in mice. To determine the specificity of sazetidine-A induced hypothermia to β2 subunit-containing nicotinic receptors, we tested its efficacy in β2 knockout (β2−/−) mice. These effects were compared with wildtype (WT) and α7 knockout (α7−/−) mice. Confirming our earlier results, sazetidine-A elicited a pronounced and long-lasting hypothermia in WT mice. In comparison, sazetidine-A induced a much attenuated and shorter hypothermic response in β2−/− mice. This indicates that the greater proportion of sazetidine-A induced hypothermia is mediated via actions on β2-containing nicotinic receptors, while a smaller component of hypothermia induced by sazetidine-A is mediated by non-β2 nicotinic receptors. Similar to WT mice, α7−/− mice showed the full extent of the sazetidine-A effect, suggesting that the hypothermia produced by sazetidine-A did not depend on actions on α7 nicotinic receptor subtype. Three other novel nicotinic receptor desensitizing agents derived from sazetidine-A, triazetidine-O, VMY-2-95 and YL-1-127 also produced hypothermia in WT and α7−/− mice. Furthermore, unlike sazetidine-A, triazetidine-O and YL-1-127 did not show any hint of a hypothermic effect in β2−/− mice. VMY-2-95 like sazetidine-A did show a residual hypothermic effect in the β2−/− mice. These studies show that the hypothermic effects of sazetidine-A and the related compound VMY-2-95 are mainly mediated by nicotinic receptors containing β2 subunit, but that a small component of the effect is apparently mediated by non-β2 containing receptors.
PMCID: PMC3844546  PMID: 24036108
Nicotinic; Temperature Regulation; sazetidine-A; Desensitization; Knockout
5.  Derivatives of dibenzothiophene for PET imaging of α7-Nicotinic Acetylcholine Receptors 
Journal of medicinal chemistry  2013;56(19):10.1021/jm401184f.
A new series of derivatives of 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)dibenzo[b,d]thiophene 5,5-dioxide with high binding affinities and selectivity for α7-nicotinic acetylcholine receptors (α7-nAChRs) (Ki = 0.4 – 20 nM) has been synthesized for PET imaging of α7-nAChRs. Two radiolabeled members of the series [18F]7a (Ki = 0.4 nM) and [18F]7c (Ki = 1.3 nM) were synthesized. [18F]7a and [18F]7c readily entered the mouse brain and specifically labeled α7-nAChRs. The α7-nAChR selective ligand 1 (SSR180711) blocked the binding of [18F]7a in the mouse brain in a dose-dependent manner. The mouse blocking studies with non-α7-nAChR CNS drugs demonstrated that [18F]7a is highly α7-nAChR selective. In agreement with its binding affinity the binding potential of [18F]7a (BPND = 5.3 – 8.0) in control mice is superior to previous α7-nAChR PET radioligands. Thus, [18F]7a displays excellent imaging properties in mice and has been chosen for further evaluation as a potential PET radioligand for imaging of α7-nAChR in non-human primates.
PMCID: PMC3866913  PMID: 24050653
6.  [18F]ASEM, a radiolabeled antagonist for imaging the α7-nicotinic acetylcholine receptor (α7-nAChR) with positron emission tomography (PET) 
The α7-nicotinic cholinergic receptor (α7-nAChR) is a key mediator of brain communication and has been implicated in a wide variety of central nervous system disorders. None of the currently available PET radioligands for α7-nAChR are suitable for quantitative PET imaging, mostly due to insufficient specific binding. The goal of this study was to evaluate the potential of [18F]ASEM ([18F]JHU82132) as an α7-nAChR radioligand for PET.
Inhibition binding assay and receptor functional properties of ASEM were assessed in vitro. The brain regional distribution of [18F]ASEM in baseline and blockade were evaluated in DISC1 mice (dissection) and baboons (PET).
ASEM is an antagonist for the α7-nAChR with high binding affinity (Ki = 0.3 nM). [18F]ASEM readily entered the baboon brain and specifically labeled α7-nAChR. The in vivo specific binding of [18F]ASEM in the brain regions enriched with α7-nAChRs was 80–90%. SSR180711, an α7-nAChR selective partial agonist, blocked [18F]ASEM binding in the baboon brain in a dose-dependent manner, suggesting that the binding of [18F]ASEM was mediated by α7-nAChRs and the radioligand was suitable for drug evaluation studies. In the baboon baseline studies, the brain regional volume of distribution (VT) values for [18F]ASEM were 23 (thalamus), 22 (insula), 18 (hippocampus) and 14 (cerebellum), whereas in the binding selectivity (blockade) scan, all regional VT values were reduced to less than 4. The range of regional binding potential (BPND) values in the baboon brain was from 3.9 to 6.6. In vivo cerebral binding of [18F]ASEM and α7-nAChR expression in mutant DISC1 mice, a rodent model of schizophrenia, was significantly lower than in control animals, which is in agreement with previous post-mortem human data.
[18F]ASEM holds promise as a radiotracer with suitable imaging properties for quantification of α7-nAChR in the human brain.
PMCID: PMC4112566  PMID: 24556591
α7-nAChR; nicotine; PET; baboon; [18F]ASEM
7.  Effects of sazetidine-A, a selective α4β2* nicotinic receptor desensitizing agent, on body temperature regulation in mice and rats 
European journal of pharmacology  2012;682(0):110-117.
Nicotine-induced hypothermia is well established, but the nicotinic receptor actions underlying this effect are not clear. Nicotine causes activation and desensitization at a variety of nicotinic receptor subtypes. Sazetidine-A [6-(5(((S)-azetidine-2-yl)methoxy)pyridine-3-yl)hex-5-yn-1-ol] is a novel compound that potently and selectively desensitizes α4β2* nicotinic receptors. The main goal of this study was to investigate the effects of sazetidine-A, on core body temperature (Tc) in mice and rats. Sazetidine-A effects on Tc and the interactions of sazetidine-A with nicotine and selective nicotinic antagonists were investigated to determine the receptor actions underlying nicotine-induced hypothermia. Adult male mice were injected with different dose of nicotine (0.2, 0.4 and 0.8 mg/kg), sazetidine-A (0.3, 1, and 3 mg/kg), a mixture of nicotine (0.4 or 0.8 mg/kg) and sazetidine-A (0.3 or 0.6 mg/kg) or saline and Tc was monitored telemetrically. In another set of experiments, the interaction between sazetidine-A and dihydro-β-erythroidine (DHβE), an α4β2* nicotinic receptors antagonist, and methyllycaconitine (MLA), an α 7 antagonist, was investigated. Tc of mice was monitored following DHβE (1, 3 and 6 mg/kg), a combination of DHβE (3 mg/kg) and sazetidine-A (0.6 mg/kg), MLA (1.5, 3 or 6 mg/kg) or combination of MLA (6 mg/kg) and sazetidine (0.6 mg/kg) or saline. The acute effect of sazetidine-A (1, 3, and 6 mg/kg) on rats Tc was also studied. Acute sazetidine-A caused a pronounced and long-lasting hypothermia in mice; Tc decreased to about 28 °C at 100 min and recovered within 230 min. The hypothermic effect of sazetidine in rats was much less in magnitude (about 3°C) and shorter in duration compared with that in mice. Nicotine co-administration with low doses of sazetidine potentiated the magnitude and duration of hypothermia in mice. The α4β2* nicotinic receptors antagonist DHβE significantly prolonged sazetidine-A-induced hypothermia but did not increase its depth. The α7 antagonist MLA caused a modest degree of hypothermia with relatively short duration in mice. MLA failed to counteract the sazetidine-A-induced hypothermia. Overall, our results show that pharmacological modulation of α4β2* nicotinic receptors elicits changes in body temperature that may involve desensitization of these receptors.
PMCID: PMC4065797  PMID: 22387853
Hypothermia; Thermoregulation; Nicotine; Telemetry; DHβE; MLA; Body temperature
8.  Chemistry and Pharmacological Studies of 3-Alkoxy-2,5-Disubstituted-Pyridinyl Compounds as Novel Selective α4β2 Nicotinic Acetylcholine Receptor Ligands That Reduces Alcohol Intake in Rats 
Journal of medicinal chemistry  2013;56(7):10.1021/jm4000374.
Neuronal acetylcholine receptors mediate the addictive effects of nicotine and may also be involved in alcohol addiction. Varenicline, an approved smoking cessation medication, showed clear efficacy in reducing alcohol consumption in heavy-drinking smokers. More recently, sazetidine-A, which selectively desensitizes α4β2 nicotinic receptors, was shown to significantly reduce alcohol intake in a rat model. To develop novel therapeutics for treating alcohol use disorder, we designed and synthesized novel sazetidine-A analogs containing a methyl group at the 2-position of the pyridine ring. In vitro pharmacological studies revealed that some of the novel compounds showed similar overall pharmacological property profiles with that of sazetidine-A, but exhibited reduced agonist activity across all nicotinic receptor subtypes tested. In animal studies, compound (S)-9 significantly reduced alcohol uptake in rats. More importantly, preliminary results from studies in a ferret model indicate that these novel nAChR ligands have an improved adverse side-effect profile in comparison with that of varenicline.
PMCID: PMC3809750  PMID: 23540678
nicotinic acetylcholine receptors; sazetidine-A; varenicline; desensitization; addiction; alcohol use disorders
Psychopharmacology  2012;222(2):269-276.
Sazetidine-A is a selective α4β2 nicotinic receptor desensitizing agent and partial agonist. It has been shown in previous studies to significantly reduce nicotine self-administration in rats after acute or repeated injections. However, the effects of continuous chronic infusions of sazetidine-A on maintenance of nicotine self-administration and relapse after abstinence have yet to be examined.
This study evaluated the efficacy of continuous sazetidine-A infusions (sc) over a period of four weeks to reduce nicotine self-administration in male and female Sprague-Dawley rats.
Sazetidine-A was administered via Alzet osmotic minipumps to young adult female and male rats at doses of 0, 2 or 6 mg/kg/day for four weeks. The effects of sazetidine-A on IV nicotine self-administration were examined in repeated 3-hour sessions over the first two weeks of infusion followed by one week of forced abstinence from nicotine and one week of resumed nicotine access.
The 6 mg/kg/day sazetidine-A dose significantly reduced overall nicotine self-administration compared with vehicle control across the sessions for both male (p<0.001) and female (p<0.05) rats. The lower 2 mg/kg/day sazetidine-A infusion dose was effective in reducing nicotine self-administration for male (p<0.001), but not female rats. No attenuation in sazetidine-A effectiveness was seen over the course of the four-week treatment. In the vehicle control group, male rats self-administered significantly (p<0.001) more nicotine than females.
The continuing effectiveness of sazetidine-A in reducing nicotine self-administration in both male and female rats supports its promise as a new treatment to help people successfully quit smoking.
PMCID: PMC3426624  PMID: 22297831
Nicotine; Sazetidine-A; chronic; Self-administration; Sex differences
10.  Effects of sazetidine-A, a selective α4β2 nicotinic acetylcholine receptor desensitizing agent on alcohol and nicotine self-administration in selectively bred alcohol-preferring (P) rats 
Psychopharmacology  2010;211(2):161-174.
Manipulations of nicotinic cholinergic receptors have been shown to influence both alcohol and nicotine intake. Sazetidine-A [6-(5(((S)-azetidine-2-yl)methoxy)pyridine-3-yl)hex-5-yn-1-ol] is a novel compound that potently and selectively desensitizes α4β2 nicotinic receptors with only modest receptor activation.
The goal of the present study was to examine the effects of sazetidine-A on alcohol and nicotine self-administration in alcohol-preferring (P) rats.
P rats were given the choice of water or alcohol. Once stable baselines were established, the acute (0, 0.1, 0.3, 1, and 3 mg/kg, s.c.) and chronic (3 mg/kg for 10 days) effects of sazetidine-A on alcohol intake were assessed. Naltrexone (2.5 mg/kg) served as a positive control. The effect of sazetidine-A (3 mg/kg) and naltrexone (4 mg/kg) on saccharin (0.2%) preference was also assessed. In addition, the acute effects of sazetidine-A (3 mg/kg) and naltrexone (4 mg/kg) on alcohol intake after alcohol deprivation were evaluated. In another experiment, the effects of sazetidine-A (0, 1, or 3 mg/kg) on IV nicotine self-administration in P and NP rats were assessed.
Sazetidine-A caused a dose-dependent reduction in alcohol intake. Chronic sazetidine-A also effectively reduced alcohol intake until the seventh day of treatment, when partial tolerance appeared to develop. In the post-deprivation study, sazetidine-A significantly reduced alcohol intake and preference. Sazetidine-A at 3 mg/kg significantly reduced nicotine self-administration in both lines.
Sazetidine-A significantly reduced alcohol and nicotine intake in P rats that self-administer higher levels of both drugs. Sazetidine-A may hold promise for the treatment of alcohol and nicotine addiction.
PMCID: PMC3695635  PMID: 20535453
Alcoholism; P rats; Nicotinic agonists; Alcohol drinking; Naltrexone; Treatment; Animal model; Saccharin; Nicotine addiction
11.  The Biology of Tobacco and Nicotine: Bench to Bedside 
Strong epidemiologic evidence links smoking and cancer. An increased understanding of the molecular biology of tobacco-related cancers could advance progress toward improving smoking cessation and patient management. Knowledge gaps between tobacco addiction, tumorigenesis, and cancer brought an interdisciplinary group of investigators together to discuss “The Biology of Nicotine and Tobacco: Bench to Bedside.” Presentations on the signaling pathways and pathogenesis in tobacco-related cancers, mouse models of addiction, imaging and regulation of nicotinic receptors, the genetic basis for tobacco carcinogenesis and development of lung cancer, and molecular mechanisms of carcinogenesis were heard. Importantly, new opportunities to use molecular biology to identify and abrogate tobacco-mediated carcinogenesis and to identify high-risk individuals were recognized.
PMCID: PMC3459058  PMID: 15824140
12.  Toxicological Analysis of Low-Nicotine and Nicotine-Free Cigarettes 
Toxicology  2008;249(2-3):194-203.
Low-nicotine and nicotine-free cigarettes are commercially available under the brand-name Quest®. Some consumers may believe that these are safer cigarettes, and they may smoke more cigarettes or inhale more smoke to compensate for low nicotine yields. Thus, we have studied the toxicological effects of these two cigarettes and compared them with the Kentucky reference cigarette 2R4F. Also, the availability of nicotine-free cigarettes allows for the assessing the role of nicotine in cigarette smoke. In addition to nicotine, some tobacco-specific nitrosamines, aldehydes, and volatile organic compounds were also reduced in the Quest® cigarettes compared to the 2R4F. However, aromatic amines were higher in the nicotine-free compared with low nicotine cigarettes. The Ames test revealed that cigarette smoke condensates from the nicotinefree (CSC-F), low nicotine (CSC-L) and 2R4F (CSC-R) cigarettes had a similar mutagenic potency. Exposure to any CSC caused a similar dose-dependent LDH leakage from normal human bronchial epithelial cells. However, CSC-F had more inhibitory effects on the cell growth than CSC-L and CSC-R. Adding nicotine to the CSC-F attenuated this inhibition. Both Quest® CSCs decreased gap junction intercellular communication and caused cell cycle arrest. CSC exposure increased cytoplasmic nucleosomes, sub-G1/G0 population and apoptotic comet tails. Proapoptotic protein Bax increased independent of p53 induction after exposure to CSC-F. In conclusion, these studies are not consistent with a perception that low-nicotine or nicotine-free cigarettes may have less toxicity in human cells. Nicotine, as it exists in CSC, attenuates cytotoxicity possibly in part through inhibition of apoptotic pathways.
PMCID: PMC2573966  PMID: 18599178
cigarette smoke; nicotine; mutagenicity; cytotoxicity; apoptosis
13.  Rat neuronal nicotinic acetylcholine receptors containing α7 subunit: pharmacological properties of ligand binding and function 
Acta Pharmacologica Sinica  2009;30(6):842-850.
To compare pharmacological properties of heterologously expressed homomeric α7 nicotinic acetylcholine receptors (α7 nAChRs) with those of native nAChRs containing α7 subunit (α7* nAChRs) in rat hippocampus and cerebral cortex.
We established a stably transfected HEK-293 cell line that expresses homomeric rat α7 nAChRs. We studies ligand binding profiles and functional properties of nAChRs expressed in this cell line and native rat α7* nAChRs in rat hippocampus and cerebral cortex. We used [125I]-α-bungarotoxin to compare ligand binding profiles in these cells with those in rat hippocampus and cerebral cortex. The functional properties of the α7 nAChRs expressed in this cell line were studied using whole-cell current recording.
The newly established cell line, KXα7R1, expresses homomeric α7 nAChRs that bind [125I]-α-bungarotoxin with a Kd value of 0.38±0.06 nmol/L, similar to Kd values of native rat α7* nAChRs from hippocampus (Kd=0.28±0.03 nmol/L) and cerebral cortex (Kd=0.33±0.05 nmol/L). Using whole-cell current recording, the homomeric α7 nAChRs expressed in the cells were activated by acetylcholine and (−)-nicotine with EC50 values of 280±19 μmol/L and 180±40 μmol/L, respectively. The acetylcholine activated currents were potently blocked by two selective antagonists of α7 nAChRs, α-bungarotoxin (IC50=19±2 nmol/L) and methyllycaconitine (IC50=100±10 pmol/L). A comparative study of ligand binding profiles, using 13 nicotinic ligands, showed many similarities between the homomeric α7 nAChRs and native α7* receptors in rat brain, but it also revealed several notable differences.
This newly established stable cell line should be very useful for studying the properties of homomeric α7 nAChRs and comparing these properties to native α7* nAChRs.
PMCID: PMC4002377  PMID: 19448648
neurotransmitter release; memory; Alzheimer's disease
14.  Synthesis and Pharmacological Evaluation of Novel 9- and 10- Substituted Cytisine Derivatives - Nicotinic Ligands of Enhanced Subtype Selectivity 
Journal of medicinal chemistry  2006;49(9):2673-2676.
We report the synthesis and pharmacological properties of several cytisine derivatives. Among them, two 10-substituted derivatives showed much higher selectivities for the α4β2 nAChR subtype in binding assays than cytisine. The 9-vinyl derivative was found to have a very similar agonist activity profile to that of cytisine.
PMCID: PMC2504867  PMID: 16640326

Results 1-14 (14)