Background:Smoking and tobacco use continue to be the largest preventable causes of death globally. A novel therapeutic approach has recently been proposed: administration of an enzyme that degrades nicotine, the main addictive component of tobacco, minimizing brain exposure and reducing its reinforcing effects. Pre-clinical proof of concept has been previously established through dosing the amine oxidase NicA2 from Pseudomonas putida in rat nicotine self-administration models of addiction. Results: This paper describes efforts towards optimizing NicA2 for potential therapeutic use: enhancing potency, improving its pharmacokinetic profile, and attenuating immunogenicity. Libraries randomizing residues located in all 22 active site positions of NicA2 were screened. 58 single mutations with 2-to 19-fold enhanced catalytic activity compared to wt at 10 μM nicotine were identified. A novel nicotine biosensor assay allowed efficient screening of the many primary hits for activity at nicotine concentrations typically found in smokers. 10 mutants with improved activity in rat serum at or below 250 nM were identified. These catalytic improvements translated to increased potency in vivo in the form of further lowering of nicotine blood levels and nicotine accumulation in the brains of Sprague-Dawley rats. Examination of the X-ray crystal structure suggests that these mutants may accelerate the rate limiting re-oxidation of the flavin adenine dinucleotide cofactor by enhancing molecular oxygen's access. PEGylation of NicA2 led to prolonged serum half-life and lowered immunogenicity observed in a human HLA DR4 transgenic mouse model, without impacting nicotine degrading activity.Conclusions: Systematic mutational analysis of the active site of the nicotine-degrading enzyme NicA2 has yielded 10 variants that increase the catalytic activity and its effects on nicotine distribution in vivo at nicotine plasma concentrations found in smokers. In addition, PEGylation substantially increases circulating half-life and reduces the enzyme's immunogenic potential. Taken together, these results provide a viable path towards generation of a drug candidate suitable for human therapeutic use in treating nicotine addiction.

Although tobacco smoking is the world’s most important preventable cause of many chronic diseases (including COPD and asthma) and premature death, many physicians do not routinely apply smoking cessation in the daily health care of their patients. Two widely felt important concerns of physicians are that smoking cessation as part of a treatment is time-consuming and may jeopardize their relationship with patients. Very Brief Advice (VBA) is a non-confrontational method, which could assist general practitioners (GPs) as a simple, quick first step in getting patients to stop smoking. In this study, we investigated the opinions and experiences of GPs with VBA in their routine care in two rounds of telephone interviews with 19 GPs. The interviews were recorded and transcribed and subsequently analysed with NVivo12. We observed that the GPs had a very positive experience with using VBA. They found the method to be efficient as to the time involved, patient-friendly and easy to implement.

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.

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