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

ABSTRACT: 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.

SUBMITTER: Pentel PR

PROVIDER: S-EPMC6056991 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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

Pentel Paul R PR Raleigh Michael D MD LeSage Mark G MG Thisted Thomas T Horrigan Stephen S Biesova Zuzana Z Kalnik Matthew W MW

BMC biotechnology 20180724 1

<h4>Background</h4>The bacterial nicotine-degrading enzyme NicA2 isolated from P. putida was studied to assess its potential use in the treatment of tobacco dependence.<h4>Results</h4>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 bloo ...[more]

PMID: 30041697

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Project description: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??M 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.

Project description:INTRODUCTION:The rate of nicotine metabolism, estimated by the nicotine metabolite ratio (NMR), is an important determinant of tobacco dependence. This study investigated the effect of NMR on smoking behavior due to nicotine reinforcement during ad libitum smoking. AIMS AND METHODS:As part of a larger study, participants were stratified based on saliva NMR as fast and slow metabolizers. After smoking a cigarette and measuring nicotine blood concentrations, participants smoked as desired over a 90-minute period. Analysis included time to first cigarette, total number of cigarettes, total number of puffs, and weight of tobacco consumed. RESULTS:Sixty-one (48%) participants were fast metabolizers and 66 (52%) slow metabolizers by NMR. No significant differences were found regarding the smoking topography variables by NMR. Normal metabolizers by genotype (n = 79) had a shorter time to first cigarette than reduced metabolizers (n = 39; p = .032). Blacks smoked fewer cigarettes (p = .008) and took fewer total puffs (p = .002) compared with Whites. Among Whites, fast metabolizers by NMR had a shorter time to first cigarette compared with slow metabolizers (p = .014). Among fast metabolizers, Whites had, compared with Blacks, shorter latency to first cigarette (p = .003) and higher number of total puffs (p = .014) and cigarettes smoked (p = .014). Baseline cigarettes per day and nicotine elimination half-life significantly predicted topography outcomes. CONCLUSIONS:Saliva NMR did not predict cigarette reinforcement during a relatively brief period of ad libitum smoking. Differences were seen by race, with White fast metabolizers by NMR having shorter time to first cigarettes compared with slow metabolizers. IMPLICATIONS:After a 90-minute period of nicotine abstinence, NMR was not significantly associated with smoking reinforcement. Slow and fast metabolizers had similar time to first cigarette, number of cigarettes smoked, total number of puffs, and tobacco consumed; however, within-race differences show that within Whites, fast metabolizers had a faster time to first cigarette than slow metabolizers.

Project description:Drug-induced alterations in transcriptional regulation play a central role in establishing the persistent neuroplasticities that occur during drug addiction. Additionally, changes in gene expression associated with drug administration provide valuable insight into the molecular basis of drug abuse. The molecular mechanisms that underlie susceptibility to psychostimulant addiction remain unknown. Identifying the common gene transcriptional responses to psychostimulants can provide a mechanistic insight to elucidate the molecular nature of drug dependence. Male Wistar rats (4 weeks old) were acclimatized for a week to their housing conditions prior to experiments. Thereafter, they were divided into two groups: (cohort 1) 4 groups of rats (n=12 rats per group) given saline only (i.p., "drug-naive" rats); and (cohort 2): 3 groups of rats given either methamphetamine, amphetamine or methylphenidate (5 mg/kg,i.p., M-CM-"M-bM-^BM-,M-EM-^Sdrug-pretreated") for 7 days (2x daily) prior to behavioral assays. Conditioned place preference (CPP) tests were conducted a day after the final drug administration. Rats which showed CPP were evaluated for self-administration of the psychostimulants. [Cohort 1] A day after the final self-administration test, brains of 2-3 rats from each subgroups (i.e. those which showed the most robust self-administration), as well as 5 rats from the control group were removed for microarray analysis. The striatum (rostral part of the caudate putamen and the nucleus accumbens [NAc]) and the prefrontal cortex were dissected and immediately frozen at -70M-CM-^BM-BM-0C. Total RNA was isolated and gene expression profiling was conducted on independent pools of total RNA from 2-5 animals per group. [Cohort 2] A day after the final self-administration test, brains of 3 rats from each subgroups (i.e. those which showed the most robust self-administration), as well as 3 rats from the control group were removed for microarray analysis. The striatum (rostral part of the caudate putamen and the nucleus accumbens [NAc]) and the prefrontal cortex were dissected and immediately frozen at -70M-BM-0C. Total RNA was isolated and gene expression profiling was conducted on independent pools of total RNA from three animals per group.

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

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

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