Nicotine degradation in smokers: will a new and potent enzymatic approach work where nicotine vaccines have failed?

Abstract

Smoking and tobacco use continue to be the largest preventable causes of death. Although there are current pharmaceutical and behavioural therapies, the one-year sustained quit rate of these therapies is only 20-25% at best. Recently, an alternative biotherapeutic strategy has been proposed: enzymatic degradation of nicotine in the bloodstream preventing accumulation in the brain. The bacterial enzyme NicA2 oxidizes nicotine into pseudo-oxynicotine, a non-addictive compound already found in smokers. Proof-of-concept animal studies have shown that NicA2 can rapidly reduce the levels of nicotine accumulating in the brain after intravenous nicotine dosing, and NicA2 has shown to have efficacy in a continuous nicotine access self-administration rat model. Enzymatic elimination of nicotine upon smoke inhalation to combat tobacco addiction is an innovative therapeutic concept. However, it is in line with recent clinical studies demonstrating that reduction in nicotine content in cigarettes (to 2.5% of normal levels) lead to significant reduction in the number of cigarettes smoked and higher smoking cessation rates compared to a control group smoking normal nicotine level cigarettes. Enzymatic degradation of nicotine appears to be more potent than nicotine-specific antibodies or vaccines for reducing nicotine distribution to brain, and if this proves to be the case in humans, it could also be more effective for enhancing smoking cessation rates and succeed where nicotine vaccines have failed thus far. The work reviewed in this article constitutes promising initial steps towards an urgently needed new effective treatment strategy in smoking cessation therapy. *Correspondence to: van Schayk Onno, Maastricht University, Faculty of Health Medicine & Lifesciences, Maastricht, Netherlands, E-mail: onno.vanschayck@ maastrichtuniversity.nl Received: November 22, 2018; Accepted: December 14, 2018; Published: December 19, 2018 Introduction Smoking and tobacco use continue to be the largest preventable causes of death [1]. In 2015, approximately 6.4 million deaths were attributed to smoking worldwide. Although most smokers are aware of the health risks, smoking cessation is usually difficult to maintain. Current pharmacological therapies for smoking cessation combined with counselling have significant clinical effects compared to counselling alone [2]. However, only 20-25% of smokers remain abstinent for at least 1 year after treatment [3]. This fact means that new, more efficacious drugs need to be developed. Multiple meta-analyses have been conducted to investigate the pharmaceutical interventions for smoking cessation, and guidelines have been published by many organizations [2,4]. The first-line pharmacological therapy for smoking cessation are nicotine replacement products (patches, gums, inhalers, nasal sprays, tablets, and oral sprays). It evokes its effects by stimulating the nicotinic receptors in the ventral tegmental area of the brain releasing dopamine in the nucleus accumbens [5]. NRT can lead to a reduction in withdrawal symptoms in smokers who would like to quit. Varenicline works as a partial agonist of the nACh receptor also releasing dopamine [6]. Furthermore, Bupropion, a tricyclic antidepressant, can be used in smoking cessation therapy. It inhibits reuptake of dopamine, noradrenaline, and serotonin in the central nervous system, and is a non-competitive nicotine receptor antagonist. The inhibition of the levels of dopamine and noradrenalin are thought to be important for Bupropion to have its antismoking actions [7]. Varenicline and bupropion are usually prescribed and when used for 2-3 months achieve a doubling of the quit rate compared to placebo [8]. Furthermore, counselling should be given to help in smoking cessation. Brief advice alone given by a general practitioner result in a 2-3% increase in quit rates [9]. To stop smoking is to break a complex habit and addiction and, to achieve reasonable quit rates, it is necessary to provide psychological support combined with pharmacological drugs. However, even with optimal pharmacological therapies only 20–25% of smokers remain abstinent for at least 1 year after treatment. This means that new therapies need to be developed. As an alternative to small-molecule-based therapies, immunotherapeutic approaches to smoking cessation and vaccination against nicotine were investigated in the last three decades [10]. Researchers showed that it is possible to link or conjugate psychoactive drugs (such as cocaine, heroin or nicotine) to carrier proteins, thus making these small molecules antigenic. This work led to the hypothesis that it may be possible to develop vaccines which can prevent or treat addiction to these drugs. The proposed mechanism of action is that vaccine-elicited antibodies target and capture the drug in the periphery, reducing the concentrations reaching the brain and reducing its reinforcing effects. Nicotine conjugate vaccines showed early promise preclinically but failed to demonstrate broad clinical van Schayck OCP (2018) Nicotine degradation in smokers: will a new and potent enzymatic approach work where nicotine vaccines have failed? Volume 1(4): 2-4 Prev Med Commun Health , 2018 doi: 10.15761/PMCH.1000120 efficacy in large clinical randomised controlled trials [10,11]. Although increased efficacy was observed in those individuals who attained the highest anti-nicotine antibody titres [10-12], indicating that an antibody-mediated strategy in smoking cessation could work, the levels of antibodies generally were too low and too variable to have a clinically relevant outcome [13]. Essentially, the challenge has been a lack of potency to alter the pharmacokinetics of nicotine sufficiently in order to eliminate its reinforcing effects across a broad population of smokers. Recently, an alternative biotherapeutic strategy has been proposed: nicotine degradation via an enzymatic approach, eliminating its exposure to the brain [14]. Pseudomonas putida S16 is an example of a nicotine-degrading bacterial strain that can use nicotine as its nitrogen and carbon source. It was originally isolated from a field underneath continuous tobacco cropping in China and is able to metabolise nicotine to fumaric acid [15]. The enzyme found in the first committed step of S16’s nicotine degradation is NicA2, an amine oxidase. NicA2 oxidises nicotine to N-methylmyosmine, which undergoes rapid, spontaneous hydrolysis to pseudooxynicotine, a non-addictive compound already found in smokers. Xue and colleagues studied the features of NicA2 in vitro to evaluate its potential as a starting point for the development of a nicotinedegrading drug for use in smoking cessation therapies [14]. They demonstrated that NicA2 has favourable characteristics such as high stability in buffer and mouse serum, as well as high catalytic activity at nicotine concentrations typically found in smokers’ blood [14]. NicA2 was subsequently evaluated in vivo through single-dose nicotine pharmacokinetic (PK) studies in rats pre-treated with a range of NicA2 doses [16,17]. Reduction in nicotine blood and brain levels was measured 1, 3 and 5 minutes after an intravenous bolus dose of 0.03 mg/kg nicotine. This nicotine dose is equivalent to 2 cigarettes with regard to milligrams of nicotine per kilogram of body weight. Short intervals were used, as the enzyme’s effectiveness is expected to be dependent on the rapid elimination of nicotine. While smokers achieve maximum levels of brain nicotine in 3 to 5 minutes, nicotine is initially detected in the brain 7 seconds after the first inhalation [18]. NicA2’s effects on nicotine distribution to the blood and brain were dependent on dose and time, as shown in the Figure 1 below [17,19]. When dosed at 5 mg/kg, blood levels of nicotine dropped to below the limit of quantitation of the assay (2 ng/ml), virtually eliminating nicotine from the bloodstream within 1 minute as compared to the control group. The levels of nicotine in the brain were also assessed, with a 10-mg/kg NicA2 dose lowering brain nicotine levels by 95% at 3 and 5 minutes after nicotine dosing as compared to the control group, while a higher dose of 20 mg/kg was needed for reducing brain nicotine levels to the same extent within one minute. As one minute is a practical time limit to euthanise the rats and to collect blood and brain samples, the onset of enzyme activity was evaluated in blood samples in vitro, where typical maximum blood levels of nicotine were degraded to below the level of detection within 10 seconds [17]. In repeated nicotine dose experiments that simulated very heavy smoking, 5 doses of 0.03 mg/kg nicotine spaced 10 minutes apart (equivalent to 10 cigarettes over 40 minutes) were given intravenously to rats pre-treated with 10 mg/kg NicA2. Brain nicotine levels were lowered by the same degree after the 5th dose as after the 1st dose of nicotine, a potency never observed for immunotherapeutic approaches [17,19,20]. In order to enable longer-term in vivo testing, two different constructs fusing NicA2 to an albumin-binding domain (NicA2ABD) [21] have been independently reported [17,22]. Circulating half-life was extended from a few hours to 2.5 days in rats, similar to that of endogenous serum albumin, without affecting its catalytic activity [16,17]. Consistent with the effects of NicA2 on reduced nicotine distribution in the brain, when such an enzyme fusion was administered to rats during a 7-day nicotine infusion, it reduced signs of withdrawal following termination of the nicotine infusion compared to the control group. A significant impact was observed on nicotine’s behavioral effects by preventing the development of irritability-like behavior, hyperalgesia and somatic signs of withdrawal in animals exposed to chronic nicotine, strongly supporting the theory that NicA2 may prevent the development of addiction-like behavior. Moreover, there was no nicotine detected in the