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NicVAX, a nicotine vaccine (3'AmNic-rEPA), has been clinically evaluated to determine whether higher antibody (Ab) concentrations are associated with higher smoking abstinence rates and whether dosages and frequency of administration are associated with increased Ab response. This randomized, double-blinded, placebo-controlled multicenter clinical trial (N = 301 smokers) tested the results of 200- and 400-µg doses administered four or five times over a period of 6 months, as compared with placebo. 3'AmNic-rEPA recipients with the highest serum antinicotine Ab response (top 30% by area under the curve (AUC)) were significantly more likely than the placebo recipients (24.6% vs. 12.0%, P = 0.024, odds ratio (OR) = 2.69, 95% confidence interval (CI), 1.14-6.37) to attain 8 weeks of continuous abstinence from weeks 19 through 26. The five-injection, 400-µg dose regimen elicited the greatest Ab response and resulted in significantly higher abstinence rates than placebo. This study demonstrates, as proof of concept, that 3'AmNic-rEPA elicits Abs to nicotine and is associated with higher continuous abstinence rates (CAR). Its further development as a treatment for nicotine dependence is therefore justified.

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., Competing Interests: At the time the research was conducted: i) TT, ZB, and MK are/were employees and shareholders of Antidote Therapeutics, Inc. (ATI), ii) S. Fuller was a consultant to ATI and shareholder, iii) NB and S. Fallot were employees and shareholders of Blink Biomedical, SAS, iv) DS was an employee of BioFactura, Inc. and ATI shareholder, v) SH was an employee of Noble Life Sciences, Inc. MK, TT, NB, S. Fallot, ZB, S. Fuller, MLS, and PP are coinventors on the following patent application: “Novel Nicotine-binding Antibodies” US201816639050 20180814. MR, NB, S. Fallot, MLS, AS, PP, S. Fuller, SH, DS, and BZ and their respective institutions received research funding from Antidote for salaries, experiments, and manufacturing as subawardees1,5,6 or subcontractors3,4 to Antidote under R01 DA038877 from the National Institute on Drug Abuse (NIDA) at the National Institutes of Health.

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.

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.

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.

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).

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.

Protein-phospholipid as well as protein-protein interactions may be critical for tight binding of the serine protease factor VIIa (VIIa) to its receptor cofactor tissue factor (TF). To elucidate the role of protein-protein interactions, we analyzed the interaction of VII/VIIa with TF in the absence of phospholipid. Binding of VII occurred with similar affinity to solubilized and phospholipid-reconstituted TF. Lack of the gamma-carboxyglutamic acid (Gla)-domain (des-(1-38)-VIIa) resulted in a 10- to 30-fold increase of the Kd for the interaction, as did blocking the Gla-domain by Fab fragments of a specific monoclonal antibody. These results suggest that the VII Gla-domain can participate in protein-protein interaction with the TF molecule per se rather than only in interactions with the charged phospholipid surface. Gla-domain-independent, low affinity binding of VII to TF required micromolar Ca2+, indicating involvement of high affinity calcium ion binding sites suggested to be localized in VII rather than TF. Interference with Gla-domain-dependent interactions with TF did not alter the TF. VIIa-dependent cleavage of a small peptidyl substrate, whereas the proteolytic activation of the protein substrate factor X was markedly decreased, suggesting that the VIIa Gla-domain not only participates in the formation of a more stable TF. VIIa complex but contributes to extended substrate recognition.

Structural features at extra thymidine bulge sites in DNA duplexes have been elucidated from a two-dimensional NMR analysis of through-bond and through-space connectivities in the otherwise self-complementary d(C-C-G-T-G-A-A-T-T-C-C-G-G) (GTG 13-mer) and d(C-C-G-G-A-A-T-T-C-T-C-G-G) (CTC 13-mer) duplexes in aqueous solution. These studies establish that the extra thymidine flanked by guanosines in the GTG 13-mer duplex is in a conformational equilibrium between looped out and stacked states. The looped-out state is favored at low temperature (0 degrees C), whereas the equilibrium shifts in favor of the stacked state at elevated temperatures (35 degrees C) prior to the onset of the duplex-strand transition. By contrast, the extra thymidine flanked by cytidines in the CTC 13-mer duplex is looped out independent of temperature in the duplex state. Our results demonstrate that temperature and flanking sequence modulate the equilibrium between looped-out and stacked conformations of single base thymidine bulges in DNA oligomer duplexes.

The pairing of O6etG with C located four base pairs in from either end of the self-complementary d(C1-G2-C3-O6etG4-A5-G6-C7-T8-C9-G10-C11-G12) duplex (designated O6etG.C 12-mer) has been investigated from an analysis of proton and phosphorus two-dimensional NMR experiments. The structural consequences of increasing the alkyl group size were elucidated from a comparative study of the pairing of O6meG4 with C9 in a related sequence (designated O6meG.C 12-mer). The NMR parameters for both O6alkG-containing dodecanucleotides are also compared with those of the control sequence containing G4.C9 base pairs (designated G.C 12-mer). The NOE cross-peaks detected in the two-dimensional NOESY spectra of the O6alkG.C 12-mer duplexes in H2O solution establish that the O6etG4/O6meG4 and C9 bases at the lesion site stack into the helix between the flanking C3.G10 and A5.T8 Watson-Crick base pairs. The amino protons of C9 at the O6alkG4-C9 lesion site resonate as an average resonance at 7.78 and 7.63 ppm in the O6etG.C 12-mer and O6meG.C 12-mer duplexes, respectively. The observed NOEs between the amino protons of C9 and the CH3 protons of O6alkG4 establish a syn orientation of the O6-alkyl group with respect to the N1 of alkylated guanine. A wobble alignment of the O6alkG4.C9 base pair stablized by two hydrogen bonds, one between the amino group of C9 and N1 of O6alkG and the other between the amino group of O6alkG and N3 of C9, is tentatively proposed on the basis of the NOEs between the amino protons of C9 at the lesion site and the imino protons of flanking Watson-Crick base pairs. The proton and phosphorus chemical shift differences between the O6etG.C 12-mer and O6meG.C 12-mer duplexes are small compared to the differences between these O6alkG-containing duplexes and the control G.C 12-mer duplex.

Structural features at the extra adenosine bulge sites in DNA duplexes have been elucidated from an NMR analysis of two-dimensional through space and through bond connectivities in the self-complementary d(C-C-G-G-A-A-T-T-C-A-C-G-G) (CAC 13-mer) and d(C-C-G-A-G-A-A-T-T-C-C-G-G) (GAG 13-mer) duplexes in aqueous solution. These studies establish that the extra adenosine stacks into the helix at all temperatures below the onset of the melting transition in solution, and the results are independent of whether the extra adenosine is flanked by cytidines (CAC 13-mer) or guanosines (GAG 13-mer). The NMR parameters establish that the extra adenosine can be accommodated into the helix with the flanking base pairs adopting a wedge-shaped orientation. The resulting perturbation extends out to the C10-G11 phosphodiester backbone adjacent to the bulge segment in both the CAC 13-mer and GAG 13-mer duplexes.

High-resolution homonuclear and heteronuclear two-dimensional NMR studies have been carried out on the self-complementary d(C-C-G-C-G-A-A-T-T-C-C-G-G) duplex (designated GCG 13-mer) in aqueous solution. This sequence contains an extra cytidine located between residues G3 and G4 on each strand of the duplex. The exchangeable and nonexchangeable proton resonances have been assigned from an analysis of two-dimensional nuclear Overhauser enhancement (NOESY) and correlated (COSY and relay COSY) spectra for the GCG 13-mer duplex in H2O and D2O solution. The extra cytidine at the bulge site (designated CX) results in more pronounced changes in the NOE distance connectivities for the G3-CX-G4 segment centered about the CX residue compared to the C9-C10 segment on the partner strand opposite the CX residue for the GCG 13-mer duplex at 25 degrees C. The cross-peak intensities in the short mixing time NOESY spectrum also establish that all glycosidic torsion angles including that of CX are anti in the GCG 13-mer duplex at 25 degrees C. The observed chemical shift changes for the CX base protons and the G3pCX phosphorus resonance with temperature between 0 and 40 degrees C demonstrate a temperature-dependent conformational equilibrium in the premelting transition region. The NOE and chemical shift parameters establish that the predominant conformation at low temperature (0 degree C) has the extra cytidine looped out of the helix with the flanking G3.C10 and G4.C9 base pairs stacked on each other. These results support conclusions based on earlier one-dimensional NMR studies of extra cytidine containing complementary duplexes in aqueous solution [Morden, K. M., Chu, Y. G., Martin, F. H., & Tinoco, I., Jr. (1983) Biochemistry 22, 5557-5563. Woodson, S. A., & Crothers, D. M. (1987) Biochemistry 26, 904-912]. By contrast, the chemical shift and NOE parameters demonstrate that the conformational equilibrium shifts toward a structure with a stacked extra cytidine on raising the temperature to 40 degrees C prior to the helix-coil melting transition. The most downfield shifted phosphorus resonance in the GCG 13-mer duplex has been assigned to the phosphate in the C2-G3 step, and this observation demonstrates that the perturbation in the phosphodiester backbone extends to regions removed from the (G3-CX-G4).(C9-C10) bulge site.

Proton and phosphorus NMR studies are reported for the complementary d(C-A-T-G-A-G-T-A-C).d(G-T-A-C-F-C-A-T-G) nonanucleotide duplex (designated APF 9-mer duplex) which contains a stable abasic site analogue, F, in the center of the helix. This oligodeoxynucleotide contains a modified tetrahydrofuran moiety, isosteric with 2-deoxyribofuranose, which serves as a structural analogue of a natural apurinic/apyrimidinic site [Takeshita, M., Chang, C.N., Johnson, F., Will, S., & Grollman, A.P. (1987) J. Biol. Chem. 262, 10171-10179]. Exchangeable and nonexchangeable base and sugar protons, including those located at the abasic site, have been assigned in the complementary APF 9-mer duplex by recording and analyzing two-dimensional phase-sensitive NOESY data sets in H2O and D2O solution at low temperature (0 degrees C). These studies indicate that A5 inserts into the helix opposite the abasic site F14 and stacks with flanking G4.C15 and G6.C13 Watson-Crick base pairs. Base-sugar proton NOE connectivities were measured through G4-A5-G6 on the unmodified strand and between the base protons of C15 and the sugar protons of the 5'-flanking residue F14 on the modified strand. These studies establish that all glycosidic torsion angles are anti and that the helix is right-handed at and adjacent to the abasic site in the APF 9-mer duplex. Two of the 16 phosphodiester groups exhibit phosphorus resonances outside the normal spectral dispersion indicative of altered torsion angles at two of the phosphate groups in the backbone of the APF 9-mer duplex.

Structural features of A.C mismatches and A.O4meT pairs in the interior of oligodeoxynucleotide duplexes have been investigated by high-resolution two-dimensional proton NMR spectroscopy on the self-complementary d(C-G-C-A-A-G-C-T-C-G-C-G) duplex (designated A.C 12-mer) and and the self-complementary d(C-G-C-A-A-G-C-T-O4meT-G-C-G) duplex (designated A.O4meT 12-mer) containing A.C and A.O4meT pairs at identical positions four base pairs in from either end of and A.O4meT pairs at identical positions four base pairs in from either end of the duplex. Proton NMR shows that there are similar pH-dependent changes in the structure in the A.C 12-mer and A.O4meT 12-mer duplexes. Our studies have focused on the low-pH (pH 5.5) conformation where high-quality two-dimensional NOESY data sets were collected from the exchangeable and nonexchangeable protons in these duplexes. The spectral parameters for the A.C 12-mer and the A.O4meT 12-mer duplexes were very similar, indicating that they must have similar structures at this pH in aqueous solution. Both structures are right-handed double helices with all the bases adopting the normal anti configuration about the glycosidic bond. The same atoms are involved in hydrogen-bond pairing for the A.C mismatch and the A.O4meT pair, and these pairs have a similar spatial relationship to flanking base pairs.(ABSTRACT TRUNCATED AT 250 WORDS)

High-resolution two-dimensional NMR studies have been completed on the self-complementary d(C-G-C-G-A-G-C-T-T-G-C-G) duplex (designated G.T 12-mer) and the self-complementary d(C-G-C-G-A-G-C-T-O4meT-G-C-G) duplex (designated G.O4meT 12-mer) containing G.T and G.O4meT pairs at identical positions four base pairs in from either end of the duplex. The exchangeable and nonexchangeable proton resonances have been assigned from an analysis of two-dimensional nuclear Overhauser enhancement (NOESY) spectra for the G.T 12-mer and G.O4meT 12-mer duplexes in H2O and D2O solution. The guanosine and thymidine imino protons in the G.T mismatch resonate at 10.57 and 11.98 ppm, respectively, and exhibit a strong NOE between themselves and to imino protons of flanking base pairs in the G.T 12-mer duplex. These results are consistent with wobble pairing at the G.T mismatch site involving two imino proton-carbonyl hydrogen bonds as reported previously [Hare, D. R., Shapiro, L., & Patel, D. J. (1986) Biochemistry 25, 7445-7456]. In contrast, the guanosine imino proton in the G.O4meT pair resonates at 8.67 ppm. The large upfield chemical shift of this proton relative to that of the imino proton resonance of G in the G.T mismatch or in G.C base pairs indicates that hydrogen bonding to O4meT is either very weak or absent. This guanosine imino proton has an NOE to the OCH3 group of O4meT across the pair and NOEs to the imino protons of flanking base pairs.(ABSTRACT TRUNCATED AT 250 WORDS)

Proton and phosphorus NMR studies are reported for two complementary nonanucleotide duplexes containing acyclic abasic sites. The first duplex, d(C-A-T-G-A-G-T-A-C).d(G-T-A-C-P-C-A-T-G), contains an acyclic propanyl moiety, P, located opposite a deoxyadenosine at the center of the helix (designated APP 9-mer duplex). The second duplex, d(C-A-T-G-A-G-T-A-C).d(G-T-A-C-E-C-A-T-G), contains a similarly located acyclic ethanyl moiety, E (designated APE 9-mer duplex). The ethanyl moiety is one carbon shorter than the natural carbon-phosphodiester backbone of a single nucleotide unit of DNA. The majority of the exchangeable and nonexchangeable base and sugar protons in both the APP 9-mer and APE 9-mer duplexes, including those at the abasic site, have been assigned by recording and analyzing two-dimensional phase-sensitive NOESY data sets in H2O and D2O solution between -5 and 5 degrees C. These spectroscopic observations establish that A5 inserts into the helix opposite the abasic site (P14 and E14) and stacks between the flanking G4.C15 and G6.C13 Watson-Crick base pairs in both the APP 9-mer and APE 9-mer duplexes. The helix is right-handed at and adjacent to the abasic site, and all glycosidic torsion angles are anti in both 9-mer duplexes. Proton NMR parameters for the APP 9-mer and APE 9-mer duplexes are similar to those reported previously for the APF 9-mer duplex (F = furan) in which a cyclic analogue of deoxyribose was embedded in an otherwise identical DNA sequence [Kalnik, M. W., Chang, C. N., Grollman, A. P., & Patel, D. J. (1988) Biochemistry 27, 924-931]. These proton NMR experiments demonstrate that the structures at abasic sites are very similar whether the five-membered ring is open or closed or whether the phosphodiester backbone is shortened by one carbon atom. Phosphorus spectra of the APP 9-mer and APE 9-mer duplexes (5 degrees C) indicate that the backbone conformation is similarly perturbed at three phosphodiester backbone torsion angles. These same torsion angles are also distorted in the APF 9-mer but assume a different conformation than those in the APP 9-mer and APE 9-mer duplexes.