NicA2 preparation and in vitro characterization

NicA2 for in vitro studies was generated as described in [8]. For in vivo experiments, a similar expression construct was generated by cloning a synthetic gene encoding the same wildtype NicA2 amino acid sequence (GenBank: AEJ14620.1) [14] with an added C-terminal His 6 -tag (optimized for E. coli expression; GeneArt/Invitrogen) into pET22b(+), and was transformed into the E. coli expression strain BL21(DE3) (Agilent).

Purification of enzyme for in vivo testing added steps for endotoxin removal including 0.1% of the non-ionic surfactant octylphenol ethoxylate (Triton X-114; Sigma-Aldrich, St. Louis, MO) in the wash buffer during cobalt immobilized metal affinity chromatography purification (using Talon resin; Clontech), followed by tangential flow filtration buffer exchange and an additional polishing step in the form of anion exchange chromatography using a Q Sepharose FF column (GE Life Sciences). Fractions containing NicA2 (by SDS-PAGE/Coomassie stain) were pooled, dialyzed into PBS pH 7.4 and concentrated. Concentration was determined by UV absorbance at 280 nm using the theoretically determined extinction coefficient A 280 at 1 g/L = 1.313 [37]. Endotoxin levels were determined using an Endosafe® PTS™ instrument (Charles River). Final purity was > 95% (visual estimate based on SDS-PAGE), with an endotoxin level of 0.12 EU/mg.

Activity of purified protein was measured in vitro using the Amplex Red assay kit (Thermo). Based on NicA2’s proposed mechanism, oxidation of nicotine results in the generation of H 2 O 2 which is coupled to the conversion of the colorless Amplex Red reagent into its red-fluorescent product, resorufin by horseradish peroxidase [38]. Assays were performed as per the manufacturers protocol, including S-(−)-nicotine (Sigma) at a final assay concentration of 10 μM in 96-well black half-area flat bottom plates (Corning). Fluorescence was detected in a SpectraMax M2 plate reader using excitation at 555 nm, detection at 590 nm, and employing a “Plate Blank” well to subtract the value derived from the no enzyme control for each point in the SoftMax® Pro data evaluation software package (Molecular Devices). Activities were expressed as the relative slopes of increase in fluorescence as a function of time. Development of fluorescence was dependent on the presence of nicotine, and the rate of fluorescence-development was proportional to the concentration of NicA2 in the range used.

Substrate specificity of NicA2

Substrate specificity of NicA2 was analyzed by the Amplex Red assay described above, using 10 μM of test compound and 160 nM NicA2 enzyme. Compounds tested were: (2’S)-nicotine-1’-N-oxide (Toronto Research Chemicals; TRC), (±)-nornicotine, nicotinamide, β-nicotinamide adenine dinucleotide (NAD), acetylcholine, choline, (−)-cotinine, varenicline, bupropion, (−)-cytisine, mecamylamine (TRC), dopamine, serotonin, (±)-norepinephrine, L-glutamate, γ-amino-N-butyric acid (GABA), (R,S)-anatabine (TRC), (R,S)-anabasine (TRC), and myosmine (compounds obtained from Sigma-Aldrich unless otherwise stated). Activities were expressed relative to the activity found for S-(−)-nicotine run in parallel.

Preparation of NicA2-albumin-binding domain fusion and in vitro characterization

A gene fusion was prepared consisting of the NicA2 amino acid sequence mentioned above fused at its C-terminus to a 5 kDa albumin binding domain (ABD035, which binds albumin with high affinity across rodents, non-human primates and humans [39]) via a flexible Gly 4 Ser linker followed by a C-terminal His 6 -tag (gene optimized for E. coli expression; GeneArt/Invitrogen). This construct was cloned into pET22b(+) and transformed into the E. coli expression strain BL21(DE3) (Agilent). Expression and purification was carried out as described above.

Nicotine assay and quenching of NicA2 activity

Nicotine concentrations in blood or brain were measured using gas chromatography with nitrogen phosphorus detection [40, 41]. Concentrations that were below the limit of quantitation for the assay were considered to be at the limit of 2 ng/ml for purposes of analysis. For this assay blood undergoes solvent extraction, while brain is first digested in NaOH before extraction. Because residual NicA2 in samples could continue to degrade nicotine ex vivo, blood samples were quenched immediately upon collection by drawing blood into a tube and transferring 0.5 ml into 4 volumes of methanol and immediately vortexing.

Completeness of quenching of NicA2 activity in blood by MeOH was assessed in vitro by comparing samples prepared by adding the following to 0.5 ml blood, in this order: a) BSA 100 μg/ml → nicotine 40 ng/ml → 4 volumes of methanol; b) NicA2 100 μg/ml (the approximate blood concentration of NicA2 following a 10 mg/kg i.v. dose) → nicotine 40 ng/ml → 4 volumes of methanol; c) NicA2 100 μg/ml → 4 volumes of methanol → nicotine 40 ng/ml. Tubes were vortexed for 5 s at each step. Samples were stored at − 20 °C until assayed for nicotine levels. A similar protocol was performed using brain homogenate containing 40 ng/ml nicotine in place of blood, to determine completeness of quenching of brain samples by MeOH in vitro. Nicotine concentrations were compared using two-tailed unpaired t tests with Welch’s correction and adjusted for multiple comparisons (α = 0.025).

For in vivo studies blood was obtained either through an indwelling venous catheter or as trunk blood after decapitation, and NicA2 activity was quenched as above. Brain was rapidly removed after decapitation, rinsed in methanol, and immediately homogenized with 4 volumes of methanol. At this point brain was stored at − 20 °C with no loss of nicotine concentration. Alternatively, to facilitate storage and shipping of samples, brain could be rinsed, flash frozen in liquid nitrogen and stored at − 80 °C until assayed. When ready for assay, the frozen sample was placed in 4 volumes of methanol and processed as above. The adequacy of flash-freezing brain prior to addition of methanol was evaluated by pretreating 5 rats with 1.25 mg/kg NicA2 and administering nicotine 0.03 mg/kg 5 min later (equivalent to two cigarettes in a human). Brains were collected 3 min after nicotine administration. Brains were rinsed in methanol and one hemisphere of each brain was immediately homogenized in 4 volumes of methanol while the other half was flash frozen and stored at − 80 °C until assayed. Groups were compared with a two-tailed paired t test.

Because it was expected that very little NicA2 would cross the blood-brain barrier owing to its molecular weight of 52.5 kDa, it was initially unclear whether brain, after rinsing in methanol, required further quenching by methanol. This question was addressed by dividing samples obtained as part of the repeated nicotine dose pharmacokinetic experiment described below. In this experiment brain was obtained from rats that had been pretreated with NicA2 and then received either 1 or 5 doses of nicotine. Brains were first rinsed in MeOH and then split so that one hemisphere was processed only by rinsing the whole brain in methanol and the other hemisphere was processed by immediately placing it in 4 volumes of methanol and homogenizing. Groups were compared using two-tailed paired t tests.

Estimation of NicA2 and NicA2-ABD pharmacokinetic parameters

Female Sprague Dawley rats weighing 225–250 g were obtained with a jugular venous catheter in place (Charles River). The choice of male or female rats in this and subsequent experiments was depending upon their availability and desired weight range at the time of each experiment. An additional goal was to test efficacy of NicA2 in both male and female rats. Three rats received 5 mg/kg His-tagged NicA2 via the tail vein. Blood (0.2 ml) was collected into serum separator tubes via the jugular catheter at pre-dose and over a 5 min-24 h period for NicA2 or 5 min-10 days for NicA2-ABD, and serum was isolated and stored at − 20 °C until analysis. Assay of NicA2 or NicA2-ABD concentrations in serum samples took advantage of the C-terminal His-tag. MaxiSorp ELISA plates (Nunc) were coated overnight with anti-His tag antibody (R&D Systems). Plates were blocked with 1% non-fat dry milk (NFDM) in PBS for approximately 1 h. Dilutions of NicA2 or NicA2-ABD standards (for the latter a pre-incubation step in rat serum was conducted so the standard curve would accurately represent the NicA2-ABD:albumin complex detection in the actual samples) and serum samples in 1% NFDM in PBS + 0.1% Tween-20 were added to the plates and incubated for 2 h at room temperature. After washing away unbound substances (all wash steps performed in PBS + 0.1% Tween-20), rabbit anti-NicA2 polyclonal primary detection antibody (custom reagent generated by Noble Life Sciences) was added to the wells for a 1 h incubation. A wash step was followed by addition of horseradish peroxidase-conjugated goat anti-rabbit IgG (Fc) (KPL International). Plates were washed, and the remaining binding complex was detected with TMB substrate (3,3′,5,5′-tetramethylbenzidine; KPL International). Once stopped with acid, plates were read on a spectrophotometer at 450 nm and data analyzed in SoftMax® Pro, version 5.4 (Molecular Devices). Estimates of pharmacokinetic parameters (volume of distribution, clearance, terminal half-life) were obtained from serum concentrations using noncompartmental methods [42].

NicA2 effects on blood and brain nicotine levels: Single nicotine doses

Female Sprague Dawley rats weighing 225–250 g were purchased with jugular venous catheters in-place (Charles River Labs). Fifteen groups of 8 rats were pretreated with NicA2 through the catheter with 3 groups at each of the following NicA2 doses: 0.3125, 0.625, 1.25, 5.0, 10.0 mg/kg. Three control groups of 8 rats each received bovine serum albumin 4 mg/kg rather than NicA2. Five min later each group received 0.03 mg/kg nicotine i.v. Six groups of rats (one group at each NicA2 dose and one control group) were then sacrificed at 1, 3 or 5 min following the nicotine dose. Blood and brain samples were obtained by decapitation and quenched with methanol as described above. Two additional groups were pretreated with NicA2 20 mg/kg and studied as above but blood and brain nicotine levels measured only at 1 and 3 min based on pilot data showing blood and brain levels were undetectable at 5 min. Blood or brain nicotine concentrations were compared by Bonferroni-corrected Welch’s t-tests to accommodate heterogeneity of variance between doses or time points. Each time point or dose was considered a separate family of comparisons, such that the significance level was set at p = 0.0083 for comparing each NicA2 dose to BSA for each time point. Serum nicotine levels were not normally distributed in two groups and brain nicotine levels were non-normal in one group. This was due to the presence of one outlier in each group, confirmed by Iterative Grubb’s analysis (p < 0.01). These outliers were removed for the statistical analyses but were included in the figures.

NicA2 effects on blood and brain nicotine levels: Multiple nicotine doses

Male Holtzman Sprague Dawley rats weighing 340–470 g were anesthetized with 0.1 mg/kg fentanyl and 0.05 mg/kg dexmedetomidine i.m. and 100 mg/kg propofol i.p. and a jugular venous catheter was placed. Two groups of 10 rats received NicA2 10 mg/kg i.v. via the jugular catheter and two groups received 10 mg/kg BSA as controls. Five min later one NicA2 and one BSA group received nicotine 0.03 mg/kg i.v. These groups were sacrificed 3 min after the nicotine dose, and blood and brain obtained by decapitation. The two remaining groups received nicotine 0.03 mg/kg i.v. every 10 min × 5 and were sacrificed 3 min after the fifth nicotine dose. Blood and brain nicotine concentrations were compared between NicA2 and BSA groups using two-tailed unpaired t tests with Welch’s correction for unequal variances.

Effect of NicA2 on nicotine discrimination

Procedures were similar to those previously used in our laboratory [43, 44]. Four male Sprague Dawley rats weighing 550–600 g had been trained to discriminate nicotine alone (0.4 mg/kg s.c. from saline using a 2-lever discrimination procedure). Lever pressing was reinforced under a terminal variable-interval 15 s schedule using 45 mg food pellets. Discrimination was assessed twice weekly (Tues and Fri) during 2 min extinction test sessions. Discrimination was considered stable when a) > 80% responding occurred on the injection-appropriate lever during two consecutive saline and nicotine test sessions, b) > 90% injection-appropriate responding occurred on six consecutive training sessions, and c) response rates (total responses/session) were stable (no trend across these four test sessions and six training sessions). When performance was stable, rats were habituated to being placed in restraint tubes for tail vein injection of NicA2 by injecting saline via tail vein 10 min prior to two or more test sessions until restraint had no effect on discrimination performance. At this point, the effect of 10 mg/kg NicA2 i.v. on the ability of 0.1 mg/kg nicotine s.c. to substitute for the 0.4 mg/kg training dose was determined. During these test sessions, PBS or NicA2 was administered i.v. 10 min prior to 0.1 mg/kg nicotine s.c.. The 0.1 mg/kg nicotine s.c. dose was used because it produces serum nicotine concentrations similar to those produced by the 0.03 mg/kg i.v. dose in the pharmacokinetic studies. This dose normally produces partial substitution for a 0.4 mg/kg training dose, i.e. about 50–60% nicotine lever responding [43, 44]. The percentage of responding on the nicotine-appropriate lever (%NLR) and overall response rate (responses/second) during the 2-min extinction test sessions served as the primary dependent measures. These measures were compared between vehicle and NicA2 administration prior to the 0.1 mg/kg nicotine substitution test sessions via paired t-test.

Effect of NicA2 on nicotine or food self-administration

Procedures were similar to those previously used in our laboratory [32]. A total of 8 male Holtzman Sprague Dawley rats weighing 310–460 g were used. Four were experimentally naïve. One was previously trained to self-administer a unit nicotine dose of 0.06 mg/kg. Three had failed to self-administer anabasine in an unrelated pilot study. Naïve rats were implanted with jugular cannulas 1 week after arrival. One week later the rats were placed in operant conditioning chambers and allowed to acquire nicotine self-administration (NSA) using 2 h sessions at a 0.03 mg/kg unit nicotine dose and gradually escalating the fixed-ratio (FR) schedule to FR 3 over several weeks. Non-naïve rats were similarly trained. On average, the rats self-administered 0.51 mg/kg/day (16.9 ± 4.1 SD infusions) of nicotine over the 2 h sessions. Rats were considered to have acquired NSA when they earned at least 8 infusions per session and responding on the active lever compared to the inactive was > 2:1. After at least 1 week at FR 3, if NSA was stable (< 15% variation and no trend), rats received an i.v. infusion of PBS vehicle 10 min before one session (Mon), followed by i.v. infusion of 20 mg/kg NicA2 10 min before each of four consecutive sessions (Tues-Fri). Rats were then allowed to reacquire NSA until stable. To confirm that these rats were sensitive to changes in nicotine exposure per se, NicA2 effects were compared to extinction of NSA by substituting saline for nicotine for 4 consecutive sessions (Tues-Fri). The order of the extinction and NicA2 treatment phases was counterbalanced across subjects. Another group of 5 rats was trained to respond for sucrose pellets to examine whether NicA2 effects were selective for NSA or produced nonspecific side effects. These rats were trained under the same FR 3 schedule as the NSA group. Sessions ended after 2 h or when 50 pellets were earned. After responding was stable (no trend in response rate over 5 consecutive sessions), rats received an i.v. infusion of PBS vehicle 10 min before one session (Monday), followed by i.v. infusion of 20 mg/kg NicA2 10 min before each of the following four consecutive sessions (Tues-Fri). In two rats, a higher NicA2 dose (70 mg/kg) was then tested 6–12 weeks later in the same manner after recovery of baseline NSA performance and allowing elimination of NicA2. Reinforcement rate (reinforcers/min) was calculated for NSA (infusions delivered) and sucrose self-administration (pellets delivered). This measure for vehicle and NicA2 test sessions was transformed to a percentage of baseline (mean of the week before NicA2 testing), which served as the primary dependent measure. Paired t-tests with a Bonferroni correction for multiple comparisons were used to compare this measure during each NicA2 session to vehicle (p < 0.012 for four comparisons) for NSA or sucrose self-administration separately. Effects of the higher NicA2 dose were considered significant if the number of infusions during NicA2 treatment was below the range of infusions during baseline.

A separate group of four male Holtzman Sprague Dawley rats weighing 325–360 g was used in a pilot NSA study to test the longer-acting variant of the enzyme (NicA2-ABD) in rats with 23 h/day access to nicotine. The longer-acting enzyme allowed use of the 23 h nicotine access model which more closely resembles human nicotine exposure than does a 2 h session. Rats were trained to self-administer a unit nicotine dose of 0.03 mg/kg using the same procedures described above, except that sessions were 23 h in duration. Cage maintenance was done during the 1 h between sessions. After NSA was stable (same criteria), saline was substituted for nicotine to extinguish NSA. When the number of infusions decreased by at least 50% and there was no trend across three consecutive session, rats were allowed to reacquire stable NSA. Rats were then given a PBS vehicle infusion 10 min before one session, followed by infusion of 70 mg/kg NicA2-ABD 10 min prior to each of six consecutive sessions (the average number of session required for saline extinction). This dose of NicA2-ABD was used because of the variability in effect observed for the 20 mg/kg NicA2 dose during 2 h sessions and the higher nicotine intake occurring in the 23 h sessions (1 mg/kg/day, 33.3 ± 10.5 SD infusions v. 0.51 mg/kg/day for the 2 h sessions). Data were analyzed in the same way as the 2 h data (p < 0.0083 for six comparisons).