Behavioral characterization of early nicotine withdrawal in the mouse: a potential model of acute dependence.

BACKGROUND: Clinical and preclinical research have demonstrated that short-term exposure to nicotine during the initial experimentation stage can lead to early manifestation of withdrawal-like signs, indicating the state of "acute dependence". As drug withdrawal is a major factor driving the progression toward regular drug intake, characterizing and understanding the features of early nicotine withdrawal may be important for the prevention and treatment of drug addiction. In this study, we corroborate the previous studies by showing that withdrawal-like signs can be precipitated after short-term nicotine exposure in mice, providing a potential animal model of acute dependence on nicotine. RESULTS: To model nicotine exposure from light tobacco use during the initial experimentation stage, mice were treated with 0.5 mg/kg (-)-nicotine ditartrate once daily for 3 days. On the following day, the behavioral tests were conducted after implementing spontaneous or mecamylamine-precipitated withdrawal. In the open field test, precipitated nicotine withdrawal reduced locomotor activity and time spent in the center zone. In the elevated plus maze test, the mecamylamine challenge increased the time spent in the closed arm and reduced the number of entries irrespective of nicotine experience. In the examination of the somatic aspect, precipitated nicotine withdrawal enhanced the number of somatic signs. Finally, nicotine withdrawal did not affect cognitive functioning or social behavior in the passive avoidance, spatial object recognition, or social interaction test. CONCLUSIONS: Collectively, our data demonstrate that early nicotine withdrawal-like signs could be precipitated by the nicotinic antagonist mecamylamine in mice, and that early withdrawal from nicotine primarily causes physical symptoms.

Anti-smoking drugs cytisine and varenicline reduce cardiac reperfusion injury in rat model of myocardial ischemia.

Evidence to date indicates that activation of nicotinic acetylcholine receptors (nAChRs) can reduce cardiac injury from ischemia and subsequent reperfusion. The use of nAChR agonists in various animal models leads to a reduction in reperfusion injury. Earlier this effect was shown for the agonists of α7 nAChR subtype. In this work, we demonstrated the expression of mRNA encoding α4, α6 and ß2 nAChR subunits in the left ventricle of rat heart. In a rat model of myocardial ischemia, we studied the effect of α4ß2 nAChR agonists cytisine and varenicline, medicines used for the treatment of nicotine addiction, and found them to significantly reduce myocardium ischemia-reperfusion injury, varenicline manifesting a higher protection. Dihydro-ß-erythroidine, antagonist of α4ß2 nAChR, as well as methyllycaconitine, antagonist of α7 and α6ß2-containing nAChR, prevented protective effect of varenicline. This together with the presence of α4, α6 and ß2 subunit mRNA in the left ventricule of rat heart raises the possibility that the varenicline effect is mediated by α4ß2 as well as by α7 and/or α6ß2-containing receptors. Our results point to a new way for the use of cytisine and varenicline as cardioprotective agents.

Nicotinic acetylcholine receptor subtype expression, function, and pharmacology: Therapeutic potential of α-conotoxins.

The pentameric nicotinic acetylcholine receptors (nAChRs) are typically classed as muscle- or neuronal-type, however, the latter has also been reported in non-neuronal cells. Given their broad distribution, nAChRs mediate numerous physiological and pathological processes including synaptic transmission, presynaptic modulation of transmitter release, neuropathic pain, inflammation, and cancer. There are 17 different nAChR subunits and combinations of these subunits produce subtypes with diverse pharmacological properties. The expression and role of some nAChR subtypes have been extensively deciphered with the aid of knock-out models. Many nAChR subtypes expressed in heterologous systems are selectively targeted by the disulfide-rich α-conotoxins. α-Conotoxins are small peptides isolated from the venom of cone snails, and a number of them have potential pharmaceutical value.

More than Smoke and Patches: The Quest for Pharmacotherapies to Treat Tobacco Use Disorder.

Tobacco use is a persistent public health issue. It kills up to half its users and is the cause of nearly 90% of all lung cancers. The main psychoactive component of tobacco is nicotine, primarily responsible for its abuse-related effects. Accordingly, most pharmacotherapies for smoking cessation target nicotinic acetylcholine receptors (nAChRs), nicotine's major site of action in the brain. The goal of the current review is twofold: first, to provide a brief overview of the most commonly used behavioral procedures for evaluating smoking cessation pharmacotherapies and an introduction to pharmacokinetic and pharmacodynamic properties of nicotine important for consideration in the development of new pharmacotherapies; and second, to discuss current and potential future pharmacological interventions aimed at decreasing tobacco use. Attention will focus on the potential for allosteric modulators of nAChRs to offer an improvement over currently approved pharmacotherapies. Additionally, given increasing public concern for the potential health consequences of using electronic nicotine delivery systems, which allow users to inhale aerosolized solutions as an alternative to smoking tobacco, an effort will be made throughout this review to address the implications of this relatively new form of nicotine delivery, specifically as it relates to smoking cessation. SIGNIFICANCE STATEMENT: Despite decades of research that have vastly improved our understanding of nicotine and its effects on the body, only a handful of pharmacotherapies have been successfully developed for use in smoking cessation. Thus, investigation of alternative pharmacological strategies for treating tobacco use disorder remains active; allosteric modulators of nicotinic acetylcholine receptors represent one class of compounds currently under development for this purpose.

Advances in the In vitro and In vivo pharmacology of Alpha4beta2 nicotinic receptor positive allosteric modulators.

Receptors containing α4 and ß2 subunits are a major neuronal nicotinic acetylcholine receptor (nAChR) subtype in the brain. This receptor plays a critical role in nicotine addiction, with potential smoking cessation therapeutics producing modulation of α4ß2 nAChR. In addition, compounds that act as agonists at α4ß2 nAChR may be useful for the treatment of pathological pain. Further, as the α4ß2 nAChR has been implicated in cognition, therapeutics that act as α4ß2 nAChR agonists are also being examined as treatments for cognitive disorders and neurological diseases that impact cognitive function, such as Alzheimer's disease and schizophrenia. This review will cover the molecular in vitro evidence that allosteric modulators of the α4ß2 neuronal nAChR provide several advantages over traditional α4ß2 nAChR orthosteric ligands. Specifically, we explore the concept that nAChR allosteric modulators allow for greater pharmacological selectivity, while minimizing potential deleterious off-target effects. Further, here we discuss the development and preclinical in vivo behavioral assessment of allosteric modulators at the α4ß2 neuronal nAChR as therapeutics for smoking cessation, pathological pain, as well as cognitive disorders and neurological diseases that impact cognitive function. This article is part of the special issue on 'Contemporary Advances in Nicotine Neuropharmacology'.

α-Conotoxin TxIB: A Uniquely Selective Ligand for α6/α3ß2ß3 Nicotinic Acetylcholine Receptor Attenuates Nicotine-Induced Conditioned Place Preference in Mice.

Abstract: α-Conotoxin TxIB is a specific antagonist of α6/α3ß2ß3(α6ß2*) nicotinic acetylcholine receptor (nAChR) with an IC50 of 28 nM. Previous studies have shown that α6ß2* nAChRs are abundantly expressed in midbrain dopaminergic neurons and play an important role in mediating the mechanism of nicotine and other drugs reward effect. It provided important targets for the development of anti-addiction drugs. The present study evaluated the pharmacological activity of TxIB in vivo with conditioned place preference (CPP) model, which were induced by subcutaneous injection (s.c.) of nicotine (NIC, 0.5 mg/kg). α-Conotoxin TxIB inhibited the expression and reinstatement of CPP in mice dose-dependently, but had no significant effect on locomotor activity. The concentrations of dopamine (DA), γ-aminobutyric acid (GABA) and noradrenaline (NE) in different brain regions were measured by enzyme-linked immunosorbent assay (ELISA). We found that TxIB could inhibit the concentrations of DA, GABA and NE in different brain regions (such as nucleus accumbens (NAc), hippocampus (HIP) and prefrontal cortex (PFC)) in NIC-induced mice. The concentrations of DA and NE were decreased in ventral tegmental area (VTA), while GABA had little change. The current work described the inhibition activity of TxIB in NIC-induced CPP, suggesting that α6ß2* nAChR-targeted compound may be a promising drug for nicotine addiction treatment.

Membrane protein-regulated networks across human cancers.

Alterations in membrane proteins (MPs) and their regulated pathways have been established as cancer hallmarks and extensively targeted in clinical applications. However, the analysis of MP-interacting proteins and downstream pathways across human malignancies remains challenging. Here, we present a systematically integrated method to generate a resource of cancer membrane protein-regulated networks (CaMPNets), containing 63,746 high-confidence protein-protein interactions (PPIs) for 1962 MPs, using expression profiles from 5922 tumors with overall survival outcomes across 15 human cancers. Comprehensive analysis of CaMPNets links MP partner communities and regulated pathways to provide MP-based gene sets for identifying prognostic biomarkers and druggable targets. For example, we identify CHRNA9 with 12 PPIs (e.g., ERBB2) can be a therapeutic target and find its anti-metastasis agent, bupropion, for treatment in nicotine-induced breast cancer. This resource is a study to systematically integrate MP interactions, genomics, and clinical outcomes for helping illuminate cancer-wide atlas and prognostic landscapes in tumor homo/heterogeneity.

Cervical Cancer Correlates with the Differential Expression of Nicotinic Acetylcholine Receptors and Reveals Therapeutic Targets.

Nicotinic acetylcholine receptors (nAChRs) are associated with various cancers, but the relation between nAChRs and cervical cancer remains unclear. Therefore, this study investigated the differential expression of nAChR subunits in human cervical cancer cell lines (SiHa, HeLa, and CaSki) and in normal ectocervical cell lines (Ect1/E6E7) at mRNA and protein levels. Two specific nAChR subtype blockers, αO-conotoxin GeXIVA and α-conotoxin TxID, were then selected to treat different human cervical cancer cell lines with specific nAChR subtype overexpression. The results showed that α3, α9, α10, and ß4 nAChR subunits were overexpressed in SiHa cells compared with that in normal cells. α9 and α10 nAChR subunits were overexpressed in CaSki cells. α*-conotoxins that targeted either α9α10 or α3ß4 nAChR were able to significantly inhibit cervical cancer cell proliferation. These findings may provide a basis for new targets for cervical cancer targeted therapy.

Tobacco Smoking: Risk to Develop Addiction, Chronic Obstructive Pulmonary Disease, and Lung Cancer.

BACKGROUND: The morbidity and mortality associated with tobacco smoking is well established. Nicotine is the addictive component of tobacco. Nicotine, through the non-neuronal α7nicotinic receptor, induces cell proliferation, neo-angiogenesis, epithelial to mesenchymal transition, and inhibits drug-induced apoptosis. OBJECTIVE: To understand the genetic, molecular and cellular biology of addiction, chronic obstructive pulmonary disease and lung cancer. METHODS: The search for papers to be included in the review was performed during the months of July- September 2018 in the following databases: PubMed (http://www.ncbi.nlm.nih.gov), Scopus (http://www.scopus.com), EMBASE (http://www.elsevier.com/online-tools/embase), and ISI Web of Knowledge (http://apps.webofknowledge.com/). The following searching terms: "nicotine", "nicotinic receptor", and "addiction" or "COPD" or "lung cancer" were used. Patents were retrieved in clinicaltrials.gov (https://clinicaltrials.gov/). All papers written in English were evaluated. The reference list of retrieved articles was also reviewed to identify other eligible studies that were not indexed by the above-mentioned databases. New experimental data on the ability of nicotine to promote transformation of human bronchial epithelial cells, exposed for one hour to Benzo[a]pyrene-7,8-diol-9-10-epoxide, are reported. RESULTS: Nicotinic receptors variants and nicotinic receptors upregulation are involved in addiction, chronic obstructive pulmonary disease and/or lung cancer. Nicotine through α7nicotinic receptor upregulation induces complete bronchial epithelial cells transformation. CONCLUSION: Genetic studies highlight the involvement of nicotinic receptors variants in addiction, chronic obstructive pulmonary disease and/or lung cancer. A future important step will be to translate these genetic findings to clinical practice. Interventions able to help smoking cessation in nicotine dependence subjects, under patent, are reported.

Green tobacco sickness: mecamylamine, varenicline, and nicotine vaccine as clinical research tools and potential therapeutics.

INTRODUCTION: Green tobacco sickness occurs from transdermal absorption of chemicals from freshly harvested, green tobacco leaves. Signs and symptoms include nausea, vomiting, headache, and abdominal cramps. Prevalence has shifted from the United States and Europe to China, India, and Brazil. Worldwide 8 million individuals are afflicted, including women and children. Areas covered: Mecamylamine (Inversine®, Vecamyl®), a nicotinic acetylcholine receptor (nAChR) antagonist, should be tested as a remedy for green tobacco sickness. Mecamylamine is approved as an oral tablet for the treatment of hypertension, is safe, and is off-patent. Mecamylamine attenuates many of the effects of nicotine and tobacco including seizures, thereby supporting its use as an effective pharmacotherapy for tobacco dependence. Varenicline (Chantix®) and cytisine (Tabex®) are low efficacy (i.e. intrinsic activity) nAChR agonists, are used as smoking cessation aids, and are viable options to test as remedies against green tobacco sickness. Nicotine immunization strategies may provide further options for future testing. Expert commentary: Efforts to demonstrate reversal and/or prevention of green tobacco sickness by mecamylamine will underscore the importance of nicotine in this illness and highlight a new medication for effective treatment of tobacco poisoning.

Protective effect of α-asarone against nicotine-induced seizures in mice, but not by its interaction with nicotinic acetylcholine receptors.

Alpha-asarone is one of the bioactive phytochemicals present in the rhizomes of Acorus species and demonstrated its anticonvulsant activity in rodents. Alpha-asarone protected mice from the gamma-aminobutyric acid (GABA) type A receptor antagonist or N-methyl-d-aspartate (NMDA) receptor agonist-induced seizures. In our recent study, α-asarone attenuated the nicotine withdrawal-induced depression-like behavior in mice. The seizures induced by nicotine is mediated through the activation of nicotinic acetylcholine receptors (nAChRs) and stimulation of NMDA receptors. Therefore, we hypothesized that α-asarone might be effective against nicotine-induced seizures. Also, the interaction of α-asarone with nAChRs is unknown. In this study, we investigated the effect of α-asarone on the locomotor activity and body temperature in mice. In addition, we studied the effect of α-asarone on nicotine-induced seizures in mice. Finally, we assessed in vivo pharmacodynamic interaction of α-asarone with nAChRs using nicotine-induced hypomotility and hypothermia tests in mice. The results of this study showed that the α-asarone (50-200 mg/kg, i.p.) and diazepam (5 mg/kg, i.p.) treatment significantly decreased the locomotor activity and body temperature in mice. Furthermore, α-asarone (50-200 mg/kg, i.p.) and diazepam (5 mg/kg, i.p.) pretreatment significantly prolonged the onset time of nicotine-induced seizures in mice. However, α-asarone (30 and 50 mg/kg, i.p.) pretreatment did not inhibit the nicotine-induced hypomotility or hypothermia in mice. Conversely, mecamylamine (1 mg/kg, s.c.) pretreatment completely blocked the nicotine-induced seizures and significantly prevents the nicotine-induced hypomotility and hypothermia in mice. Overall, these results suggest that the protective effect of α-asarone against nicotine-induced seizures did not mediate through the antagonism of nAChRs. We also postulated that the GABAergic and glutamatergic activities of α-asarone could be involved in its protective effect against nicotine-induced seizures and based on this aspect further studies are required.

The contribution of agonist and antagonist activities of α4ß2* nAChR ligands to smoking cessation efficacy: a quantitative analysis of literature data.

RATIONALE AND OBJECTIVE: Two mechanisms underlie smoking cessation efficacies of α4ß2* nicotinic acetylcholine receptor (nAChR) agonists: a "nicotine-like" agonist activity reduces craving by substituting for nicotine during a quit attempt, and a "nicotine-blocking" antagonist activity attenuates reinforcement by competing with inhaled nicotine during a relapse. To evaluate the contribution of each mechanism to clinical efficacy, we estimated the degree of agonist and antagonist activities of nicotine replacement therapy (NRT), varenicline, cytisine, and the discontinued nAChR agonists dianicline, ABT-418, ABT-089, CP-601927, and CP-601932, relative to the functional effects of nicotine from smoking. METHODS: Functional activities that occur in vivo with clinical doses were predicted from literature data on binding and functional potencies at the target α4ß2 nAChR, as well as at α6ß2* nAChRs, and from estimates of free drug exposures in human brain. Agonist activity is comprised of nAChR activation and desensitization, which were expressed as percentages of desensitization and activation by nicotine from smoking. Antagonist activity was expressed as the reduction in nAChR occupancy by nicotine during smoking in the presence of an agonist. RESULTS: Comparisons with odds ratios at end of treatment suggest that extensive α4ß2 and α6ß2* nAChR desensitization combined with α6ß2* nAChR activation at similar levels as nicotine from smoking is associated with clinical efficacy (NRT, varenicline, cytisine, ABT-418). Effective competition with inhaled nicotine for α4ß2 and α6ß2* nAChRs further improves clinical efficacy (varenicline). Other discontinued nAChR agonists have lower agonist and antagonist activities at α4ß2 nAChRs and are inactive or less efficacious than NRT (dianicline, ABT-089, CP-601927, CP-601932). CONCLUSION: Three pharmacological effects appear to be key factors underlying smoking cessation efficacy: the degree of activation of α6ß2* nAChRs, desensitization of α4ß2 and α6ß2* nAChRs (agonist activity), and the reduction of nicotine occupancy at α4ß2 and α6ß2* nAChRs (antagonist activity). No single activity is dominant, and the level of smoking cessation efficacy depends on the profile of these activities achieved at clinical doses. While adequate agonist activity alone seems sufficient for a clinical effect (e.g., NRT, cytisine), clinical efficacy is improved with substantial competitive antagonism of α4ß2 nAChRs, i.e., if the drug has a dual agonist-antagonist mechanism of action (e.g., varenicline).

Structure-Activity Studies Reveal the Molecular Basis for GABAB-Receptor Mediated Inhibition of High Voltage-Activated Calcium Channels by α-Conotoxin Vc1.1.

α-Conotoxins are disulfide-bonded peptides from cone snail venoms and are characterized by their affinity for nicotinic acetylcholine receptors (nAChR). Several α-conotoxins with distinct selectivity for nAChR subtypes have been identified as potent analgesics in animal models of chronic pain. However, a number of α-conotoxins have been shown to inhibit N-type calcium channel currents in rodent dissociated dorsal root ganglion (DRG) neurons via activation of G protein-coupled GABAB receptors (GABABR). Therefore, it is unclear whether activation of GABABR or inhibition of α9α10 nAChRs is the analgesic mechanism. To investigate the mechanisms by which α-conotoxins provide analgesia, we synthesized a suite of Vc1.1 analogues where all residues, except the conserved cysteines, in Vc1.1 were individually replaced by alanine (A), lysine (K), and aspartic acid (D). Our results show that the amino acids in the first loop play an important role in binding of the peptide to the receptor, whereas those in the second loop play an important role for the selectivity of the peptide for the GABABR over α9α10 nAChRs. We designed a cVc1.1 analogue that is >8000-fold selective for GABABR-mediated inhibition of high voltage-activated (HVA) calcium channels over α9α10 nAChRs and show that it is analgesic in a mouse model of chronic visceral hypersensitivity (CVH). cVc1.1[D11A,E14A] caused dose-dependent inhibition of colonic nociceptors with greater efficacy in ex vivo CVH colonic nociceptors relative to healthy colonic nociceptors. These findings suggest that selectively targeting GABABR-mediated HVA calcium channel inhibition by α-conotoxins could be effective for the treatment of chronic visceral pain.

Differential regulation of alcohol taking and seeking by antagonism at α4ß2 and α3ß4 nAChRs.

RATIONALE: Alcoholism is a serious public health problem throughout the world. Current pharmacotherapies for the treatment of this disorder are poorly effective. Preclinical and clinical findings point to nicotinic acetylcholine receptors (nAChRs) as a promising target for the development of novel and effective medications. Assuage Pharmaceuticals, in collaboration with Torrey Pines Institute for Molecular Studies, has discovered a new class of potent and selective α4ß2 nAChR antagonists. OBJECTIVE: Here, it was hypothesized that α4ß2 nAChR antagonism is a viable approach for treatment of alcohol use disorders. RESULTS: When tested in rats, one lead compound, AP-202, attenuated both operant alcohol and nicotine self-administration in a paradigm in which the two reinforcers were concurrently available. The conotoxin TP2212-59, a selective α3ß4 nAChR antagonist, was only effective in reducing nicotine self-administration. AP-202 also reduced alcohol but not food responding when alcohol was presented as the only reinforcer, whereas the commercially available α4ß2 nAChR antagonist dihydro-ß-erythroidine failed to alter alcohol self-administration. AP-202 did not block relapse-like behavior induced by previously alcohol-associated stimuli or yohimbine stress. In a reinstatement paradigm, in which alcohol seeking was triggered by a nicotine challenge, a behavior successfully inhibited by the nonselective nAChR antagonist mecamylamine, AP-202 was not effective, while pretreatment with TP2212-59 abolished nicotine-induced reinstatement of alcohol seeking. CONCLUSIONS: These findings suggest differential roles for α4ß2 and α3ß4 nAChR on alcohol taking and seeking with selective blockade of α4ß2 nAChR being more implicated in modulating alcohol taking while selective blockade of α3ß4 nAChR is involved in nicotine-induced alcohol seeking.

Effects of the nicotinic agonist varenicline, nicotinic antagonist r-bPiDI, and DAT inhibitor (R)-modafinil on co-use of ethanol and nicotine in female P rats.

RATIONALE: Co-users of alcohol and nicotine are the largest group of polysubstance users worldwide. Commonalities in mechanisms of action for ethanol (EtOH) and nicotine proposes the possibility of developing a single pharmacotherapeutic to treat co-use. OBJECTIVES: Toward developing a preclinical model of co-use, female alcohol-preferring (P) rats were trained for voluntary EtOH drinking and i.v. nicotine self-administration in three phases: (1) EtOH alone (0 vs. 15%, two-bottle choice), (2) nicotine alone (0.03 mg/kg/infusion, active vs. inactive lever), and (3) concurrent access to both EtOH and nicotine. Using this model, we examined the effects of (1) varenicline, a nicotinic acetylcholine receptor (nAChR) partial agonist with high affinity for the α4ß2* subtype; (2) r-bPiDI, a subtype-selective antagonist at α6ß2* nAChRs; and (3) (R)-modafinil, an atypical inhibitor of the dopamine transporter (DAT). RESULTS: In phases 1 and 2, pharmacologically relevant intake of EtOH and nicotine was achieved. In the concurrent access phase (phase 3), EtOH consumption decreased while nicotine intake increased relative to phases 1 and 2. For drug pretreatments, in the EtOH access phase (phase 1), (R)-modafinil (100 mg/kg) decreased EtOH consumption, with no effect on water consumption. In the concurrent access phase, varenicline (3 mg/kg), r-bPiDI (20 mg/kg), and (R)-modafinil (100 mg/kg) decreased nicotine self-administration but did not alter EtOH consumption, water consumption, or inactive lever pressing. CONCLUSIONS: These results indicate that therapeutics which may be useful for smoking cessation via selective inhibition of α4ß2* or α6ß2* nAChRs, or DAT inhibition, may not be sufficient to treat EtOH and nicotine co-use.

Mecamylamine treatment for alcohol dependence: a randomized controlled trial.

BACKGROUND AND AIMS: The nicotinic acetylcholine receptor antagonist, mecamylamine, is a potential novel pharmacotherapy for alcohol use disorder. The aims were to compare alcohol consumption between mecamylamine and placebo and test if smoking status modified treatment effects. DESIGN: Out-patient, randomized, double-blind clinical trial for 12 weeks of treatment with mecamylamine (10 mg) (n = 65) versus placebo (n = 63). SETTING: Connecticut, USA. PARTICIPANTS: Individuals had current alcohol dependence (n = 128), had an average age of 48.5 [standard deviation (SD) = 9.4], 110 (85.9%) were men, and included 74 smokers (57.8%) and 54 non-smokers (42.2%). Participants were randomized to mecamylamine 10 mg per day or placebo. All subjects also received medical management therapy administered by trained research personnel. MEASUREMENTS: Primary outcome was percentage of heavy drinking days during the last month of treatment; other outcomes included drinking days, drinks per drinking days, alcohol craving, smoking, symptoms of nicotine withdrawal and side effects. FINDINGS: There were no significant differences in the percentage of heavy drinking days at 3 months between the mecamylamine (mean = 18.4, SD = 29.0) and placebo treatment groups (mean = 20.4, SD = 29.2) [F1, 100  = 1.3, P = 0.25; effect size d = 0.07; mean difference = 2.06, 95% confidence interval (CI) = -8.96 to 13.08]. There were no significant differences in percentage of drinking days or in drinks per drinking day at month 3 between the mecamylamine and placebo groups; there were no significant interactions. CONCLUSIONS: Mecamylamine 10 mg per day did not reduce alcohol consumption significantly in treatment-seeking smokers and non-smokers with alcohol use disorder.

The Nicotinic Cholinergic Pathway Contributes to Retinal Neovascularization in a Mouse Model of Retinopathy of Prematurity.

Purpose: To investigate the role of nicotinic acetylcholine receptors (nAChRs) in retinal vascular development and ischemia-induced retinal neovascularization (NV). Methods: The expression of nAChR subtypes and VEGF signaling pathway components was assessed in mice with and without oxygen-induced ischemic retinopathy by comparing expression levels at postnatal day (P) 14 and P17 in mice exposed to 75% oxygen from P7 to P12 and returned to room air versus mice pups that were exposed to ambient oxygen levels during the same period. The effect of topical or intraocular injection of mecamylamine, a nonspecific nAChR antagonist, or targeted deletion of α7- or α9-nAChRs on ischemia-induced retinal NV was determined by comparing the amount of retinal NV at P17 in these mice versus appropriate controls. Results: The expression of nAChR subunits and components of the VEGF signaling pathways was increased in ischemic retina. Topical application or intraocular injection of mecamylamine decreased retinal NV in this model. Mecamylamine had no effect on normal retinal vascular development or on revascularization of the central retinal area of nonperfusion in mice with ischemic retinopathy. Targeted deletion of α9, but not α7, nAChR receptor subunits reduced retinal NV in mice with ischemic retinopathy. Conclusion: These data suggest that nAChR signaling, primarily through the α9 nAChR subunit, contributes to ischemia-induced retinal NV, but not retinal vascular development. Mecamylamine or a specific α9 nAChR antagonist could be considered for treatment of retinopathy of prematurity and other ischemic retinopathies.

Pharmacokinetics and pharmacodynamics of oral mecamylamine - development of a nicotinic acetylcholine receptor antagonist cognitive challenge test using modelling and simulation.

A pharmacologic challenge model with a nicotinic antagonist could be an important tool not only to understand the complex role of the nicotinic cholinergic system in cognition, but also to develop novel compounds acting on the nicotinic acetylcholine receptor. The objective was to develop a pharmacokinetic-pharmacodynamic (PKPD) model using nonlinear mixed effects (NLME) methods to quantitate the pharmacokinetics of three oral mecamylamine doses (10, 20 and 30 mg) and correlate the plasma concentrations to the pharmacodynamic effects on a cognitive and neurophysiologic battery of tests in healthy subjects. A one-compartment linear kinetic model best described the plasma concentrations of mecamylamine. Mecamylamine's estimated clearance was 0.28 ± 0.015 L min-1. The peripheral volume of distribution (291 ± 5.15 L) was directly related to total body weight. Mecamylamine impaired the accuracy and increased the reaction time in tests evaluating short term working memory with a steep increase in the concentration-effect relationship at plasma concentrations below 100 µg L-1. On the other hand, mecamylamine induced a decrease in performance of tests evaluating visual and fine motor coordination at higher plasma concentrations (EC50 97 µg L-1). Systolic and diastolic blood pressure decreased exponentially after a plasma mecamylamine concentration of 80 µg L-1, a known effect previously poorly studied in healthy subjects. The developed mecamylamine PKPD model was used to quantify the effects of nicotinic blockade in a set of neurophysiological tests in humans with the goal to provide insight into the physiology and pharmacology of the nicotinic system in humans and the possibility to optimize future trials that use mecamylamine as a pharmacological challenge.

Diurnal Variation of the Peripheral Cholinergic Antiinflammatory Function in Mice.

AIMS: Cholinergic antiinflammatory (CAI) pathway functions importantly in inflammation via α7 nicotinic acetylcholine receptors (α7nAChR). The present work tested circadian rhythm in peripheral CAI activity and validities of CAI activity and glucocorticoids in chronotherapy for lipopolysaccharide (LPS)-induced shock. METHODS: Vesicular acetylcholine transporter (VAChT) expressed in liver and kidney was examined every 3 h in C57BL/6 mice. Proinflammatory cytokines in serum and survival time in shock were monitored after LPS injection every 3 h. Mifepristone, antagonist of glucocorticoid receptors, and methyllycaconitine (MLA), antagonist of α7nAChR, were administrated before LPS to block antiinflammatory function of endogenous glucocorticoids and acetylcholine. RESULTS: Both levels of tumor necrosis factor α, interleukin 1ß, and interleukin 6 and mortality exhibited diurnal variations with prominent peaks when LPS was given at 15:00, and the minimum mortality occurred at 00:00. Expression of VAChT increased during resting period. MLA increased serum proinflammatory cytokines slightly, but not affected survival rate. Both differences in cytokines and in survival times between LPS injection at 15:00 and 00:00 were eliminated by mifepristone, but not by MLA. CONCLUSION: Peripheral CAI pathway exerts more powerful antiinflammatory effect during resting period. Glucocorticoids appear to be efficient in chronotherapy for septic shock.

The α9α10 nicotinic receptor antagonist α-conotoxin RgIA prevents neuropathic pain induced by oxaliplatin treatment.

Oxaliplatin, a third-generation diaminocyclohexane platinum drug, is widely used alone or in combination with 5-fluorouracil and leucovorin to treat metastatic colorectal, ovarian, and pancreatic cancers. Oxaliplatin long-term treatment is associated with the development of a dose-limiting painful neuropathy that dramatically impairs the patient's quality of life and therapy possibility. To study novel strategies to treat oxaliplatin-induced neuropathy, we evaluated α-conotoxin RgIA, a peptide that potently blocks the α9α10 nicotinic acetylcholine receptor (nAChR) subtype in a rat model of oxaliplatin-dependent neurotoxicity (2.4mgkg(-1) oxaliplatin intraperitoneally daily for 21days). The administration of RgIA (2 and 10nmol injected intramuscularly once a day concomitantly with oxaliplatin treatment), reduced the oxaliplatin-dependent hypersensitivity to mechanical and thermal noxious and non-noxious stimuli. Moreover, morphological modifications of L4-L5 dorsal root ganglia were significantly prevented. In the spinal cord the numerical increase of astrocyte cell density present in oxaliplatin-treated rats is partially prevented by RgIA treatment. Nevertheless, the administration of the α-conotoxin is able per se to elicit a numerical increase and a morphological activation of microglia and astrocytes in specific brain areas.