Attenuating nicotine's effects with high affinity human anti-nicotine monoclonal antibodies.

Use of nicotine-specific monoclonal antibodies (mAbs) to sequester and reduce nicotine distribution to brain has been proposed as a therapeutic approach to treat nicotine addiction (the basis of tobacco use disorder). A series of monoclonal antibodies with high affinity for nicotine (nic•mAbs) was isolated from B-cells of vaccinated smokers. Genes encoding 32 unique nicotine binding antibodies were cloned, and the mAbs expressed and tested by surface plasmon resonance to determine their affinity for S-(-)-nicotine. The highest affinity nic•mAbs had binding affinity constants (KD) between 5 and 67 nM. The 4 highest affinity nic•mAbs were selected to undergo additional secondary screening for antigen-specificity, protein properties (including aggregation and stability), and functional in vivo studies to evaluate their capacity for reducing nicotine distribution to brain in rats. The 2 most potent nic•mAbs in single-dose nicotine pharmacokinetic experiments were further tested in a dose-response in vivo study. The most potent lead, ATI-1013, was selected as the lead candidate based on the results of these studies. Pretreatment with 40 and 80 mg/kg ATI-1013 reduced brain nicotine levels by 56 and 95%, respectively, in a repeated nicotine dosing experiment simulating very heavy smoking. Nicotine self-administration was also significantly reduced in rats treated with ATI-1013. A pilot rat 30-day repeat-dose toxicology study (4x200mg/kg ATI-1013) in the presence of nicotine indicated no drug-related safety concerns. These data provide evidence that ATI-1013 could be a potential therapy for the treatment of nicotine addiction.

Optimization of a nicotine degrading enzyme for potential use in treatment of nicotine addiction.

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.

The nicotine-degrading enzyme NicA2 reduces nicotine levels in blood, nicotine distribution to brain, and nicotine discrimination and reinforcement in rats.

BACKGROUND: The bacterial nicotine-degrading enzyme NicA2 isolated from P. putida was studied to assess its potential use in the treatment of tobacco dependence. RESULTS: Rats were pretreated with varying i.v. doses of NicA2, followed by i.v. administration of nicotine at 0.03 mg/kg. NicA2 had a rapid onset of action reducing blood and brain nicotine concentrations in a dose-related manner, with a rapid onset of action. A 5 mg/kg NicA2 dose reduced the nicotine concentration in blood by > 90% at 1 min after the nicotine dose, compared to controls. Brain nicotine concentrations were reduced by 55% at 1 min and 92% at 5 min post nicotine dose. To evaluate enzyme effects at a nicotine dosing rate equivalent to heavy smoking, rats pretreated with NicA2 at 10 mg/kg were administered 5 doses of nicotine 0.03 mg/kg i.v. over 40 min. Nicotine levels in blood were below the assay detection limit 3 min after either the first or fifth nicotine dose, and nicotine levels in brain were reduced by 82 and 84%, respectively, compared to controls. A 20 mg/kg NicA2 dose attenuated nicotine discrimination and produced extinction of nicotine self-administration (NSA) in most rats, or a compensatory increase in other rats, when administered prior to each daily NSA session. In rats showing compensation, increasing the NicA2 dose to 70 mg/kg resulted in extinction of NSA. An enzyme construct with a longer duration of action, via fusion with an albumin-binding domain, similarly reduced NSA in a 23 h nicotine access model at a dose of 70 mg/kg. CONCLUSIONS: These data extend knowledge of NicA2's effects on nicotine distribution to brain and its ability to attenuate addiction-relevant behaviors in rats and support its further investigation as a treatment for tobacco use disorder.

Therapeutic vaccines against tobacco addiction.

Most smokers are aware of the dangers of smoking and want to quit, yet few are successful owing to the highly addictive properties of nicotine. Available smoking cessation tools include pharmacotherapies that act in the CNS and show modest long-term efficacy. Additionally, there are emerging concerns that they may cause adverse neuropsychiatric events. Antinicotine vaccines have been used experimentally as aids to smoking cessation. It is hypothesized that antibody capture of nicotine in the bloodstream would prevent it from crossing the blood-brain barrier and reaching the nicotinic receptors. The advantage of the approach includes the relatively gradual rise of antibody levels, which may reduce nicotine withdrawal symptoms, and the possible persistence of the antibodies potentially provides long-term protection, possibly preventing relapse. Proof-of-concept studies of at least two vaccine candidates have shown correlations between antinicotine antibody exposure and smoking abstinence. Unfortunately, the only vaccine tested in two large, randomized Phase III trials, 3'-amino-methyl-nicotine r-exoprotein A conjugate vaccine (NicVAX(®), Nabi Biopharmaceuticals, MD, USA), did not demonstrate efficacy. However, despite the lack of efficacy, there is good reason for continued optimism. This review will summarize the current status of the development of nicotine vaccines, discuss possible causes for the mixed results and review future scientific directions.

The efficacy and safety of a nicotine conjugate vaccine (NicVAX®) or placebo co-administered with varenicline (Champix®) for smoking cessation: study protocol of a phase IIb, double blind, randomized, placebo controlled trial.

BACKGROUND: A potential new treatment in smoking cessation and relapse prevention is nicotine vaccination which is based on active immunization against the nicotine molecule. This immunization will elicit the immune system to produce nicotine-specific antibodies that sequester nicotine in the blood stream, after inhaling tobacco products. The resulting antibody-antigen is too large to cross the blood-brain barrier and is therefore postulated to attenuate the rewarding effect of nicotine by preventing the latter from reaching its receptors in the brain and causing the release of dopamine. The aim of this paper is to describe the design of a phase IIb, multi-center, double blind, randomized, placebo controlled trial to assess the efficacy of the nicotine vaccine NicVAX® co-administered with varenicline (Champix®) and intensive counseling as an aid in smoking cessation and relapse prevention. METHODS/DESIGN: Two centers will include a total of 600 smokers who are motivated to quit smoking. At week -2 these smokers will be randomized, in a 1:1 ratio, to either 6 injections of NicVAX® or placebo, both co-administered with 12-weeks of varenicline treatment, starting at week 0. The target quit day will be set after 7 days of varenicline treatment at week 1. Smokers will be followed up for 54 weeks. The primary outcome is defined as biochemically validated prolonged smoking abstinence from week 9 to 52. Secondary outcomes include safety, immunogenicity, smoking abstinence from week 37 to 52, abstinence from week 9 to 24, abstinence in the subset of subjects with the highest antibody response, and lapse/relapse rate. DISCUSSION: This is the first study to assess the efficacy of a nicotine conjugate vaccine in combination with an evidence-based smoking cessation pharmacotherapy (varenicline) to quit smoking. Although NicVAX® is primarily designed as an aid to smoking cessation, our study is designed to explore its potential to maintain abstinence and prevent relapse. The results of this trial will give a unique insight in the potential of nicotine vaccination for relapse prevention. TRIAL REGISTRATION: ClinicalTrials.gov: (NCT00995033).

Nicotine vaccines.

Smoking is a global healthcare problem. Current smoking cessation rates using behavioral counseling and pharmacotherapeutic interventions have had modest success, with ∼1:5 smokers remaining abstinent long-term. Nicotine vaccines are a new class of immunotherapeutics under development. It is believed that anti-nicotine antibodies arising from vaccination capture nicotine and prevent or reduce its entry into the brain, as the antibody-bound nicotine is too large to cross the blood-brain barrier. This in turn decreases the pleasurable effects of smoking, reducing or eliminating positive reinforcement, thereby making it easier for a smoker to quit smoking. Four vaccine candidates have advanced into clinical testing with mixed success. Proof-of-concept has been established in that individuals with higher levels of anti-nicotine antibodies were observed to have higher smoking cessation and abstinence rates. Recently, the most advanced candidate vaccine, NicVAX, failed to meet the primary endpoint in two large phase III studies, although the correlation of higher abstinence rates in subjects with higher immunity to nicotine was observed. Although the field has had setbacks, the magnitude of the tobacco epidemic and the positive pre-clinical research and observed clinical trends indicate continued research is warranted. Several avenues are being actively pursued: a) improving vaccine potency by introducing novel carriers and/or adjuvants to stimulate higher immune response b) targeting subjects who have a robust response (e.g. personalized medicine) c) combining vaccines with pharmacotherapy for maintenance of abstinence/relapse prevention.