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.

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

“…Recently, a biotherapeutic strategy has been proposed: degradation of nicotine in blood via an enzymatic approach in order to reduce nicotine delivery to the brain [6]. Pre-clinical proof of concept has been obtained dosing the amine oxidase NicA2 from Pseudomonas putida in well-accepted rat behavioral models of nicotine addiction [7, 8].…”

“…NicA2 has been evaluated in vivo in single-dose nicotine pharmacokinetic (PK) studies in rats pre-treated with a range of NicA2 doses [7, 11]. Reduction in nicotine blood and brain levels were measured 1, 3, and 5 minutes (min) after an i.v.…”

“…A NicA2-albumin binding domain (ABD) fusion with extended circulating half-life was shown to attenuate nicotine discrimination and to decrease nicotine reinforcement in a continuous nicotine access self-administration model, which closely resembles human nicotine exposure [7]. After a high dose of NicA2-ABD (70 mg/kg), nicotine seeking behaviour was extinguished over 6 days (d) of testing with rats having continuous access to nicotine [7]. Furthermore, chronic administration of NicA2 reversed signs of withdrawal, hyperalgesia, and irritability-like behavior in nicotine-dependent rats, as well as prevented nicotine- and stress-induced relapse [8].…”

“…This putative anti-smoking medication may play an important role in preventing relapse within the quit process and in maintening abstinence. These initial studies provided proof of concept for the use of NicA2 as a basis for development of a smoking cessation drug [7, 8].…”

“…Kinetic characterization of NicA2 indicated a low K m (92 nM at 37 °C which compares favorably to the serum nicotine concentration in a typical smoker of 250 nM), but a relatively slow catalytic turnover rate [6]. This could be part of the reason for the relatively high doses required for efficacy in the rat nicotine-self-administration studies [7]. While mg/kg dose levels in this animal model do not necessarily reflect human dose levels (e.g.…”