RUOLO DEI RECETTORI NICOTINICI CENTRALI NELL'ADDICTION' E NELLA 'DEPENDENCE' DA NICOTINA: POTENZIALE STRATEGIA FARMACOLOGICA DI NUOVI AGONISTI NICOTINICI PARZIALI

Rationale 1: Cigarette smoking is one of the most serious health problems worldwide and people trying to stop smoking have high rates of relapse. Nicotine is the main psychoactive ingredient in cigarettes and its binding to neuronal nicotinic acetylcholine receptors (nAChRs) located in the mesolimbic areas induces reward (Picciotto and Kenney, 2013). Current first-line pharmacotherapies aiming at encouraging people to stop smoking include nicotine replacement therapy, bupropion hydrochloride and varenicline tartrate. The atypical antidepressant bupropion reduces the severity of nicotine craving during withdrawal, but its long-term beneficial effects on relapse are not clear (Hughes et al., 2007). Cytisine-related varenicline tartrate is a partial agonist of α4β2 and α6β2 nAChR subtypes and a full agonist of α7 and α3β4 nAChRs. Clinical trials indicate that varenicline is effective in decreasing relapse to smoking in humans (Cahill et al., 2011; Gonzales et al., 2006; Joreby et al., 2006; Tonstad et al., 2006; Zierler-Brown and Kyle, 2007) but adverse cardiovascular effects and/or neuropsychiatric events have recently been reported including depression, suicidal ideation, suicide attempts and completed suicide (Freedman, 2007; Moore et al., 2011; Singh et al., 2011). Aim 1: In search for new drugs that act on nicotine-induced dopamine release, firstly we tested the ability of new nicotinic partial agonists derivated from cytisine, 1,2-bis(cytisin-12-ul)ethane (CC4) and 1,4-bis(cytisin-12-yl)-2-butyne (CC26), compared to nicotine, cytisine and varenicline, to reduce nicotine-induced Conditioned Place Preference (CPP) in zebrafish, a promising animal model for rapidly screening new compounds to induce smoking cessation. Results 1: Our results demonstrated that CC4, CC26, cytisine and varenicline induced per se CPP with an inverted U-shaped dose-response curve similar to that of nicotine. However, when co-administered with the maximally effective dose of nicotine, they blocked its reinforcing effect. Since very little is known about the pharmacology of the native nicotinic subtypes expressed in zebrafish, we used binding and pharmacological experiments to identify the native nicotinic receptors in adult zebrafish brain by means of subtypes-selective antagonists. The results demonstrated that zebrafish brain expresses two distinct classes of nicotinic receptors: one containing the α7 subunit that binds [125I]-αbungarotoxin with high affinity and another that binds [3H]-epibatidine. We used also three antagonists α-conotoxin MII, methyllycaconitine (MLA) and dihydro-β-erythroidine (DhβE), selective respectively for α6β2, α7 and β2. Mecamilamine, a non selective antagonist, DhβE and MLA blocked the nicotine rewarding effect, demonstrating that nicotine exerted its addictive properties acting on α7 and β2 nAChRs. Since the results obtained in zebrafish looked promising, CC4 was also tested on rats, to verify if in this animal model it was effective in reducing nicotine rewarding effect. Our results demonstrated that CC4, like cytisine and nicotine, induced CPP and is ICV self-administered with an inverted-U dose response curve. Both models (CPP and ICV self-administration) showed that CC4 is per se slighty reinforcing, although to a lesser extent than nicotine. Moreover, in line with the fact that CC4 is a partial agonist, pre-treatment with non-reinforcing doses of CC4 significantly antagonized the rewarding effects induced by nicotine, both in CPP and in self administration task, without affecting motor functions. These findings indicate that this compound has a selective effect in reducing nicotine addiction-associated behaviours. Conclusions 1: Our results, obtained in zebrafish and in rats, demonstrated the possible development of CC4 or its derivatives as a new medication specific to tobacco smoking cessation with fewer side effects due to its lack of action on β4 nAChR subtypes. Rationale 2: In humans, however, the addiction is not just due to nicotine, but at least also to a complex of 4000 substances that constitute cigarette smoke. Recently electronic-cigarette (e-cigarette) has been introduced into the market with the purpose of mimic nicotine inhalation through traditional cigarette, whithout the negative effects of tobacco combustion. However, under now there are no long-term studies about the use of e-cigarette effects. In addition, the way in which nicotine is administered to experimental animals is very different from that used by humans: they do not mimic the route of administration used in the latter case, they are invasive and do not allow to study the effects of cigarette smoke compared to e-cigarette vapour (Cohen and George 2013). Aim 2: Thus, another aim of our study was to validate a rodent smoking model (tobacco smoke or e-cigarette vapour) and investigate the behavioural and biochemical changes after 7 weeks exposure. Male Balb/C mice were exposed to a mechanical ventilator delivering the smoke of 7 traditional cigarettes (cig) or e-cigarette vapour (e-cig) containing 5.6 mg of nicotine for three 30-minutes session/day for seven weeks. One hour after the last session, a group of animals was sacrified for biochemical assays and another group underwent mecamylamine precipitated or spontaneous withdrawal for behavioural analysis. Results 2: Mice submitted to cig or e-cig showed an increase in α4β2 nicotinic receptor subtypes in cerebral cortex, nucleus accumbens and hippocampus. To quantify the amount of nicotine intake and its metabolism, after 1, 4 and 7 weeks of exposure the urinary cotinine levels (the main nicotine metabolite) were quantified: Chronic intermittent exposure to cig or e-cig induces a similar increase of urinary cotinine concentrations (in a range of 700 ng/ml). During cig smoke or e-cig vapour exposure we monitored body weight and food intake: mice exposed to cig showed a significant progressive reduction across the weeks accompanied by a decrease in food intake while only a mild decrease in these parameters was shown in e-cig group. At the end of the exposure, we measured nicotine and cotinine levels in the brain of exposure animals: mice exposed to cig showed an increase in cotinine level, in contrast, mice exposed to e-cig showed an increase in cerebral nicotine level. To verify that our exposure method produces nicotine dependence we precipitated withdrawal syndrome with mecamylamine (1 mg/kg, s.c.), a non selective antagonist: mice submitted to cig or e-cig showed a significant increase in withdrawal-precipitated symptoms. However e-cig group showed a less severe withdrawal syndrome. There was also a reduction of motor function only in cig exposed mice probably due to the presence of severe signs. Finally we monitored spontaneous withdrawal syndrome from 24 hours to 90 days after the end of exposure. In comparison with the control group, both groups exposed to cig or e-cig showed impaired spatial memory (evaluated using the spatial object recognition task) and a progressive increase in anxiety-like behavior (evaluated using elevated plus maze test and marble burying test) starting from 24 hour to at least 90 days after spontaneous withdrawal. Finally, after 60 days e-cig animals and after 90 days cig group, showed a depressive-like behaviour, measured with Tail Suspension test and Sucrose Preference test. Conclusions 2: In conclusion, we propose an innovative procedure for investigating the short- and long- term alterations induced by exposure to tobacco smoke and, for the first time, we describe the short- and long- term effects of e-cig. Finally, this rodent model is also most suitable for testing new compounds for smoking cessation, such as CC4, whose effectiveness has already been tested on zebrafish and rats. Future: We propose to investigate the effect of CC4 on nicotine dependence in both cig and e-cig exposed animals. 1. Picciotto MR, Kenny PJ; “Molecular mechanisms underlying behaviors related to nicotine addiction” Cold Spring Harbor perspectives in medicine 3: a012112, 2013 2. Hughes JR, Stead LF, Lancaster T; “Antidepressants for smoking cessation” The Cochrane database of systematic reviews CD000031, 2007 3. Cahill K, Stead LF, Lancaster T; “Nicotine receptor partial agonists for smoking cessation” The Cochrane database of systematic reviews CD006103, 2011 4. Gonzales D, Rennard SI, Nides M, Oncken C, Azoulay S, Billing CB, Watsky EJ, Gong J, Williams KE, Reeves KR; “Varenicline, an alpha4beta2 nicotinic acetylcholine receptor partial agonist, vs sustained-release bupropion and placebo for smoking cessation: a randomized controlled trial” JAMA: the journal of the American Medical Association 296: 47-55, 2006 5. Joreby DE, Hays JT, Rigotti NA, Azoulay S, Watsky EJ, Williams KE, Billing CB, Gong J, Reeves KR; “Efficacy of varenicline, an alpha4beta2 nicotinic acetylcholine receptor partial agonist, vs placebo or sustained-release bupropion for smoking cessation: a randomized controlled trial” JAMA: the journal of the American Medical Association 296: 56-63, 2006 6. Tonstad S, Tonnesen P, Hajek P, Williams KE, Billing CB, Reeves KR; “Effects of maintenance therapy with varenicline on smoking cessation: a randomized controlled trial” JAMA: the journal of the American Medical Association 296: 64-71, 2006 7. Zierler-Brown SL, Kyle JA; “Oral varenicline for smoking cessation” The Annals of pharmacotherapy 41: 95-99, 2007 8. Freedman R; “Exarbation of schizophrenia by varenicline” The American journal of psychiatry 164: 1269, 2007 9. Moore TJ, Furberg CD, Glenmullen J, Maltsberger JT, Singh S; “Suicidal behavior and depression in smoking cessation treatments” PloS one 6: e27016, 2011 10. Singh S, Loke YK, Spangler JG, Furberg CD; “Risk of serious adverse cardiovascular events associated with varenicline: a systematic review and meta-analysis” CMAJ: Canadian Medical Association journal = journal de l’Association medicale canadienne 183: 1359-1366, 2011 11. Cohen A, George O; “Animal models of nicotine exposure: relevance to second-hand smoking, electronic cigarette use and compulsive smoking” Font Psychiatry doi: 10.3389/fpsyt.2013.00041, 2013