An enzymatic approach reverses nicotine dependence, decreases compulsive-like intake, and prevents relapse

Abstract

Tobacco use disorder is the leading cause of disease and preventable death worldwide, but current medications that are based on pharmacodynamics have low efficacy. Novel pharmacokinetic approaches to prevent nicotine from reaching the brain have been tested using vaccines, but these efforts have failed because antibody affinity and concentration are not sufficient to completely prevent nicotine from reaching the brain. We provide preclinical evidence of the efficacy of an enzymatic approach to reverse nicotine dependence, reduce compulsive-like nicotine intake, and prevent relapse in rats with a history of nicotine dependence. Chronic administration of NicA2-J1, an engineered nicotine-degrading enzyme that was originally isolated from Pseudomonas putida S16, completely prevented nicotine from reaching the brain and reversed somatic signs of withdrawal, hyperalgesia, and irritability-like behavior in nicotine-dependent rats with a history of escalation of nicotine self-administration. NicA2-J1 also decreased compulsive-like nicotine intake, reflected by responding despite the adverse consequences of contingent footshocks, and prevented nicotine- and stress (yohimbine)-induced relapse. These results demonstrate the efficacy of enzymatic therapy in treating nicotine addiction in advanced animal models and provide a strong foundation for the development of biological therapies for smoking cessation in humans.

Fig. 1. Blood nicotine levels after escalation of nicotine intake.

(A) Detailed timeline of the experiments. (B) After the last session of escalation of nicotine intake, blood nicotine levels were detected in rats that were pretreated with NicA2-J1 (2 mg/kg) (red circles) and rats that were pretreated with PBS (blue circles). Student’s t test showed no difference in blood nicotine levels between groups (t = 1.15, df = 14, P > 0.05). (C) At the termination of escalation of nicotine intake, rats that were pretreated with NicA2-J1 (10 mg/kg) exhibited nearly undetectable levels of nicotine in blood compared with the PBS-pretreated group (Student’s t test; t = 4.198, df = 14, P < 0.001). **P < 0.01.

Fig. 2. NicA2-J1 prevents nicotine addiction–like behavior during withdrawal.

(A) Detailed timeline of the experiments. (B) NicA2-J1 suppressed hyperalgesia during nicotine withdrawal. The two-way mixed-factorial analysis of variance (ANOVA), with group (PBS versus NicA2-J1) as the between-subjects factor and time (baseline, pre-escalation hyperalgesia, and post-escalation hyperalgesia) as the within-subjects factor, revealed significant effects of group (F_1,14 = 4.91, P = 0.04) and time (F_2,14 = 10.89, P = 0.0003) and a significant group × time interaction (F_2,28 = 5.11, P = 0.012). The Newman-Keuls post hoc analysis revealed that mechanical sensitivity significantly decreased during withdrawal (pre-escalation) in PBS-treated rats compared with their mechanical sensitivity before nicotine exposure (P = 0.017) and compared with the NicA2-J1 group (P = 0.007). Mechanical sensitivity after pretreatment with NicA2-J1 was comparable to baseline sensitivity before nicotine exposure (P > 0.05), suggesting that NicA2-J1 treatment completely reversed withdrawal-induced hyperalgesia (pre-escalation versus post-escalation, P = 0.043), whereas this effect was not detected in the PBS group, which exhibited more severe hyperalgesia during withdrawal (post-escalation versus baseline, P = 0.0018). Moreover, the Newman-Keuls post hoc analysis showed that hyperalgesia during post-escalation was completely reversed in the NicA2-J1 group (post-escalation in the PBS group versus post-escalation in the NicA2-J1 group, P = 0.041). (C) Effect of NicA2-J1 on irritability-like behavior, reflected by defensive and aggressive responses. The baseline of defensive and aggressive responses was measured during 48 hours of spontaneous nicotine withdrawal before treatment with NicA2-J1 or PBS. All the other measures were performed during 48 hours of spontaneous nicotine withdrawal after the last escalation phase after the completion of NicA2-J1 treatment (10 mg/kg). A significant decrease in defensive responses (n = 8; t = 4.5, df = 7, P < 0.01) and aggressive responses (n = 8; t = 5.22, df = 7, P < 0.01) was observed in NicA2-J1–pretreated rats. No changes from baseline were observed in the PBS group. Student’s paired t test revealed a significant reduction of aggressive responses (t = 2.27, df = 14, P < 0.05) but not defensive responses (t = 1.85, df = 14, P > 0.05) in NicA2-J1–pretreated rats compared with PBS-pretreated rats. *P < 0.05, **P < 0.01, versus baseline; ^#P < 0.05, versus pre-escalation; ^&P < 0.05, post-escalation comparisons between the PBS and NicA2-J1 groups.

Fig. 3. Acute administration of NicA2-J1 decreased withdrawal-induced hyperalgesia.

(A) Detailed timeline of the experiments. (B) Mechanical nociceptive thresholds immediately after nicotine escalation and 48 hours into withdrawal. During withdrawal, a significant decrease in hyperalgesia thresholds was observed compared with baseline (BSL) thresholds (n = 11; t = 11.9, df = 10, P < 0.01). (C) In PBS-treated animals, a significant decrease in hyperalgesia thresholds was observed compared with baseline thresholds at 24 hours into withdrawal (n = 11; t = 13.89, df = 10, P < 0.01) and 48 hours into withdrawal (n = 11; t = 9.96, df = 10, P < 0.01) but not during nicotine self-administration. (D) In NicA2-J1–treated rats, a decrease in hyperalgesia thresholds was observed at 24 hours into withdrawal (n = 11; t = 13.89, df = 10, P < 0.05). *P < 0.05, **P < 0.01.

Fig. 4. Effect of NicA2-J1 on the escalation of nicotine intake (1 and 21 hours).

(A) Detailed timeline of the experiments. (B) Nicotine self-administration and escalation of nicotine intake during the first hour of nicotine exposure. Two separate one-way ANOVAs showed that the animals significantly escalated their nicotine intake in the first hour of the session during escalation in the PBS group (F_15,105 = 9.92, P = 0.0001) and NicA2-J1 group (F_15,105 = 51.691, P = 0.0001). The Newman-Keuls post hoc analysis revealed significant escalation on the last three intermittent-access days compared with the last three continuous-access days (P = 0.001) in both groups. (C) Inactive lever responses did not change over time in either the PBS group (F_15,105 = 0.62, P = 0.54) or NicA2-J1 group (F_15,105 = 0.79, P = 0.32). (D) The two-way mixed-factorial ANOVA, with treatment (PBS and NicA2-J1) as the between-subjects factor and time as the within-subjects factor, did not show a significant treatment × time interaction (F_10,140 = 0.991, P = 0.45). (E) Inactive lever responding was unaffected by NicA2-J1 treatment (F_10,140 = 0.782, P = 0.55). (F) Nicotine self-administration and escalation of nicotine intake during the 21 hours of nicotine exposure. Two separate one-way ANOVAs showed that the animals significantly escalated their nicotine intake in the PBS group (F_15,105 = 5.53, P = 0.0001) and NicA2-J1 group (F_15,105 = 4.186, P = 0.001). The Newman-Keuls post hoc analysis indicated significant escalation on the last three intermittent-access days compared with the last three continuous-access days (P = 0.01) in both groups. (G) Inactive lever responses did not change over time in either the PBS group (F_15,105 = 0.32, P = 0.23) or NicA2-J1 group (F_15,105 = 0.49, P = 0.41). (H) The two-way mixed-factorial ANOVA, with treatment (PBS and NicA2-J1) as the between-subjects factor and time as the within-subjects factor, showed a significant effect of time (F_10,14 = 2.37, P = 0.013) but no treatment × time interaction (F_10,140 = 0.991, P = 0.45). (I) Inactive lever responding was unaffected by NicA2-J1 treatment (F_10,140 = 0.65, P = 0.76).

Fig. 5. NicA2-J1 reduces compulsive-like responding for nicotine in dependent rats.

(A) Detailed timeline of the experiments. (B) When footshock (0.1 mA) was introduced, the results showed that nicotine-dependent animals that exhibited escalation of nicotine intake and were pretreated with PBS continued responding for nicotine despite the adverse consequences of footshocks at a higher level compared with their intake prior to footshock exposure (n = 8; t = 3.063, df = 7, P = 0.028). No change in responding was observed in NicA2-J1 (10 mg/kg)–pretreated rats (n = 8; t = 0.39, df = 7, P = 0.7). Student’s t test revealed a significant difference between groups (t = 2.315, df = 14, P = 0.04). (C) When a higher footshock intensity (0.2 mA) was introduced, the results showed that nicotine-dependent animals that were pretreated with PBS exhibited no changes in responding for nicotine despite the adverse consequences of footshocks compared with their intake prior to footshock exposure (t = 1.88, df = 7, P = 0.118), whereas a significant reduction was observed in NicA2-J1–pretreated rats (t = 3.78, df = 7, P = 0.0091). Student’s t test revealed a significant difference between groups (t = 3.5, df = 14, P = 0.0048). *P < 0.05, **P < 0.01, versus baseline; ^#P < 0.05, ^##P < 0.01, comparison between the PBS and NicA2-J1 groups.

Fig. 6. NicA2-J1 prevents nicotine- and stress-induced reinstatement after extinguished nicotine intake.

(A) Detailed timeline of the experiments. (B) Extinction phase. (C) Pretreatment with NicA2-J1 (10 mg/kg) prevented nicotine prime-induced reinstatement of nicotine-seeking behavior. The two-way mixed-factorial ANOVA, with group (PBS versus NicA2-J1) as the between-subjects factor and treatment (extinction, vehicle, and 0.03 mg/kg per injection of nicotine) as the within-subjects factor, showed a significant effect of treatment (F_2,14 = 9.14, P = 0.0008) and a significant group × treatment interaction (F_2,28 = 3.48, P = 0.044). The Newman-Keuls post hoc analysis revealed that one injection of nicotine (0.03 mg/kg per injection) significantly increased the number of lever presses compared with the extinction phase in PBS-treated rats (P < 0.0008). Nicotine priming did not induce the reinstatement of nicotine-seeking behavior in rats that were pretreated with NicA2-J1 during escalation (nicotine priming versus extinction, P = 0.61). The post hoc analysis showed that nicotine-induced priming was abolished in the NicA2-J1 group compared with the PBS group (nicotine priming in the PBS group versus nicotine priming in the NicA2-J1 group, P = 0.047). (D) Inactive lever responses were unaffected by nicotine infusions in either group (F_2,28 = 0.64, P = 0.53). (E) NicA2-J1 (10 mg/kg) prevented the stress (yohimbine)–induced reinstatement of nicotine-seeking behavior in the PBS-pretreated group. The two-way mixed-factorial ANOVA, with group (PBS versus NicA2-J1) as the between-subjects factor and treatment (extinction, vehicle, and yohimbine) as the within-subjects factor, showed a significant effect of treatment (F_2,14 = 3.31, P = 0.05) and a significant group × treatment interaction (F_2,28 = 5.12, P = 0.0127). The Newman-Keuls post hoc analysis revealed that yohimbine significantly increased the number of lever presses in the PBS-pretreated group compared with the extinction phase (P = 0.011). No effect of yohimbine-induced reinstatement was observed in the NicA2-J1–pretreated group. (F) Inactive lever responses were unaffected by yohimbine in either group (F_2,28 = 0.15, P = 0.85). *P < 0.05, **P < 0.01, versus extinction; ^&P < 0.05, nicotine priming–induced reinstatement between the PBS and NicA2-J1 groups.