Investigations of Enantiopure Nicotine Haptens Using an Adjuvanting

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Investigations of Enantiopure Nicotine Haptens using an Adjuvanting Carrier in Anti-Nicotine Vaccine Development Nicholas T. Jacob, Jonathan W. Lockner, Joel E Schlosburg, Beverly A Ellis, Lisa M. Eubanks, and Kim D. Janda J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01676 • Publication Date (Web): 26 Feb 2016 Downloaded from http://pubs.acs.org on March 6, 2016

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Journal of Medicinal Chemistry

Investigations of Enantiopure Nicotine Haptens using an Adjuvanting Carrier in Anti-Nicotine Vaccine Development Nicholas T. Jacob1, Jonathan W. Lockner1, Joel E. Schlosburg3, Beverly A. Ellis1, Lisa M. Eubanks1, Kim D. Janda*, 1

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Abstract.

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Despite efforts to produce suitable smoking cessation aids, addiction to nicotine continues to carry a

substantive risk of recidivism. An attractive alternative to current therapies is the pharmacokinetic strategy of anti-nicotine vaccination. A major hurdle in the development of the strategy has been to elicit a sufficiently high antibody concentration to curb nicotine distribution to the brain. Herein, we detail investigations into a new hapten design, which was able to elicit an antibody response of significantly higher specificity for nicotine. We also explore the use of a mutant flagellin carrier protein with adjuvanting properties. These studies underlie the feasibility of improvement in anti-nicotine vaccine formulations to move towards clinical efficacy.


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Introduction. Despite half a century of anti-smoking campaigns and legislation, smoking remains the leading preventable cause of death in the United States.1 Tobacco is not only responsible for nearly six million deaths a year globally, but it is estimated that disease caused by smoking costs nearly $300 billion a year in medical expenses and lost productivity.2 In 2011, it was estimated that over 65% of current smokers desired to quit.2 Unfortunately, there is only a 13.4% chance that an attempt to quit will be successful,3,4 and most attempts to quit without cessation aids fail within the first month.5 Current cessation aids rely on nicotine replacement therapies (NRT’s) or partial agonists of the nicotinic acetylcholine receptors (nAChRs).6 NRT includes transdermal patches, gum, lozenge, or inhalation devices, but it remains unclear as to the proper use of these therapies or if they are truly effective at abating an addiction to nicotine.7-9 Partial agonists of the nAChRs, such as varenicline (Chantix), dianicline, and cytisine, often combined with NRT’s have been shown to increase the 6 month quit rate to over 50%.10 However, these therapies carry black box warnings due to their potential severe side-effects, which include hallucinations, depression, and severe mood swings.9,10 These side effects encumber the effectiveness of the therapy. Vaccines present an attractive alternative cessation therapy, as the underlying mechanism for preventing the nicotine reward is targeting the nicotine molecule, not the nAChRs.11 This strategy intends to avoid the serious side effects associated with targeting the nAChRs directly, as with currently available therapeutics. The most extensively studied antinicotine vaccines in the clinic are NicVAX (Nabi Pharm.) and NicQβ (Cytos Biotech.), which were both halted in clinical trials for failure to meet endpoint goals in the intent to treat population.12 However, these trials provided an interesting insight into the effectiveness of the vaccine on certain subgroups. It was revealed that individuals achieving the highest tertile of anti-nicotine antibody production were significantly more likely to abstain from smoking for over 6 months.13 These observations highlight the mechanistic validity of vaccination as a method for smoking cessation. Genetic analysis of smokers has also lead to the interesting observation that individual smokers who metabolize nicotine more slowly are significantly more successful at quitting smoking than individuals with faster nicotine metabolism.6,14-16 This raises the interesting prospect of targeting the timing of nicotine delivery as a method for cessation; it may simply be enough to blunt the delivery of nicotine to the central nervous system, providing a more gradual nicotine delivery to aid in cessation. These observations imply that previous anti-nicotine vaccine attempts were simply not efficacious enough, and can be improved. The goal, then, of anti-nicotine vaccine research lies in improving the specificity and concentration of antinicotine antibodies raised in response to a particular vaccine. This is achieved through optimization of the vaccine ACS Paragon Plus Environment


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formulation, including the hapten and carrier protein elements. The hapten used in NicVAX, called 3’AmNic, 1 (Figure 1), was conjugated as a racemic mixture to the 1’ position of the nicotine scaffold. Recently, we found that by making the 3’-AmNic hapten enantiopure, we could greatly increase the specificity of the antiserum for (S)-nicotine, the active enantiomer of the drug.17 Having the stereochemistry of the hapten mimic that of (S)-nicotine greatly decreased the average dissociation constant of the antibodies for nicotine. We have also recently explored the bacterial protein flagellin for its ability to act as a carrier protein in hapten vaccination.18,19 Flagellin has been shown to activate Toll-like receptor 5 (TLR5) and thus potentiate a Th2-type immune response when acting as an adjuvant.20-23 While we found that flagellin is sufficient as both a carrier and adjuvant to produce an anti-cocaine antibody response, we also found that the efficacy of flagellin as a carrier protein and adjuvant was increased when formulated with alum.18 A combination of flagellin and alum outperformed formulations containing flagellin or alum alone.18 This observation of adjuvant synergy also encouraged the use of CpG ODN 1826 as it has been shown that agonism of extracellular and intracellular TLRs has a synergistic effect in the early immune response.24 We endeavored to investigate flagellin as a carrier of our nicotine hapten in our vaccine formulation. An essential part of an effective vaccine is antigen presentation on molecules of a major histocompatibility complex (MHC) of the antigen-presenting cell (APC). In the case of a small-molecule hapten, the orientation of the covalent modification of the small-molecule will affect the epitope presented in an immunological synapse.25-27 While the most successful nicotine hapten (NicVAX) was modified through the 3’-position of the pyrrolidine, it has been demonstrated that covalent linker attachment through the pyridine moiety can bestow a greater affinity to elicited antinicotine antibodies.28 We endeavored to explore a new nicotine hapten modified at the 4-position of the pyridine ring. The present study aims to investigate the use of flagellin as a carrier protein, continue to explore the effects of enantiopurity of the hapten, as well as covalent attachment though the pyridine moiety of the nicotine scaffold. Results and discussion. The most well studied nicotine hapten modified at the pyridine ring contained covalent attachment through a pyridyl ether.28 We hypothesized the mesomeric and inductive effects on the pyridine imparted by this functional group affect the specificity of the cognate anti-serum raised by the vaccine. We reasoned that such differences in the electronics of the hapten would negatively impact the specificity of the raised antibodies for nicotine.29,30 Thus, a hapten was prepared with an aminopropyl linker attached to the 4-position of the nicotine scaffold (N4N, 2, Figure 1). The synthesis of the N4N hapten (2) was achieved in four steps from nicotine (Scheme 1). This synthesis utilizes (–)-(S)-nicotine as the starting material, allowing for retention of stereochemistry in the final hapten. ACS Paragon Plus Environment

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This is of critical importance, as our previous studies have determined that the use of racemic nicotine-based haptens negatively affects the specificity of the antiserum for nicotine.17 The N4N hapten was conjugated to either flagellin (FliC) or tetanus toxoid (TT) via a succinyl linker17, designated N4N-SucFliC and N4N-SucTT, respectively. The (S)-3’AmNic hapten (NicVAX) was also indpendently coupled to either carrier in the same manner. The 3’AmNic hapten was also made enantiopure to eliminate the effects of the racemate we previously observed.17 BALB/c mice received a subcutaneous injection of 50 µg of conjugate formulated in a 100 µL bolus of alum and CpG. The vaccine schedule is shown in Figure 2. During the course of study, no animals exhibited signs of ill health or significant weight loss, including in response to FliC.18 Mice were bled at 4, 7, and 9 weeks and the antibody titer was determined by enzyme-linked immunosorbent assay (ELISA) against the N4N and 3’AmNic hapten conjugated to bovine serum albumin (BSA) via the succinyl linker (Figure 3). The highest titer of ~22,000 was achieved by the 3’AmNic-SucTT formulation. Compared to their FliC counterparts, the TT formulations provided consistently higher titer. When compared with N4N, the 3’AmNic hapten produced a higher, but comparable (P > 0.05) titer between both carrier groups. Neither hapten formulation gave appreciable titer when run against the opposite haptenBSA conjugate (Supplementary Figure 1). The hallmark of a potent anti-nicotine vaccine is its ability to prevent the pharmacodynamic effects of nicotine treatment.11,31,32 In order to ascertain whether mice exposed to nicotine were experiencing agonism of the nicotinic cholinergic receptors, the phenotypic effects of antinociception and hypothermia in response to nicotine were measured. Antinociception was measured via response to a hot plate and nicotine-induced hypothermia was measured by rectal body temperature.31-33 All four cohorts were compared with a vaccine-naive group that had received saline injections during prime and boosting. This control group served to show the full effects of nicotine treatment in each experiment. The N4NSucFliC cohort exhibited the least antinociception to the hot plate at a dose of 3 mg/kg of nicotine was the (Figure 4), while the 3’AmNic-SucFliC group exhibited little to no attenuation of antinociception. No other group showed significant attenuation of antinociception. The N4N-SucFliC group was also able to significantly attenuate nicotine-induced hypothermia within 30 minutes at the same dose (Figure 5). Taken together, these data suggest the N4N-SucFliC cohort exhibited a significantly blunted response to nicotine. In both experiments, the N4N-SucFliC cohort performed comparably with the 3’AmNic-SucTT group. This is a striking observation, as the N4N-SucFliC raised a significantly lower average titer than the 3’AmNic-SucTT cohort based on the ELISA results (Figure 3).



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The efficacy of a vaccine against nicotine is not only dependent on the ability of the vaccine to raise a high concentration of anti-nicotine antibodies, it is also dependent on the affinity of the antibodies to bind nicotine. A rapid dissociation of nicotine from a neutralizing antibody will undermine the efficacy of a vaccine producing any appreciable antibody concentration. In order to determine the specificity of the antiserum for nicotine, a radioimmunoassay (RIA) was employed using tritium-labeled nicotine. Competitive binding curves (Supplementary Figure 2) run against pure nicotine provided dissociation constants and antibody concentrations summarized in Table 1. The trend in calculated abundance from each cohort resembled that seen in the titers from the ELISA (Figure 3). The antiserum of the N4N-SucFliC cohort was determined to have the lowest average dissociation constant for nicotine at 46.7 nM, however the average abundance of anti-nicotine antibodies was quite low at 2.29 µg/mL. While the FliC formualtions both produced a lower average concentration of anti-nicotine antibody, both formulations produced antibodies with lower average dissociation constants than their TT counterparts. A comparison of the haptens shows that the pyridine linked N4N haptens consistently produced antiserum of higher average affinity for nicotine than the pyrrolidine, as was observed previously28, indicating this mode of covalent modification may present a more suitable chemical epitope in the immunological synapse to produce an anti-nicotine antiserum. It should be noted that the ratio of average antibody concentration to dissociation constant, X17, for the N4N-SucFliC serum was only 0.050, whereas all the other groups were found to have X values more than twice as high(Supplementary Table 1), in opposition to the observed phenotypic effects. It is possible that despite the low antibody concentration, the high affinity of the polyclonal sera is able to compensate and compete effectively with the binding of nicotine to the nAChR (EC50 ≈ 2.0 nM for α2β4)34, to minimize the effective nicotine concentration at its site of action and attenuate the pharmacodynamics effects of nicotine treatment. This effect will be investigated in subsequent studies. Since it was consistently able to produce antibodies with a lower average dissociation constant for nicotine, the use of FliC as an effective carrier protein was further explored. A second study in outbred Swiss Webster mice was conducted to examine the effects of the FliC formulations in a system with more variability in the immune response. The N4N and 3’AmNic haptens were formulated as previously described and the same vaccine schedule was used (Figure 2). The results of ELISA measurements produced a similar trend as seen in the first study. The 3’AmNic hapten produced a higher, but comparable (P > 0.05) titer than the N4N hapten (Figure 6). Both formulations produced significantly lower titers in the Swiss Webster mice than they had in the BALB/c mice. Again, neither formulation produced significant cross-reactivity to the opposite hapten (Supplementary Figure 3). Both cohorts were examined for ACS Paragon Plus Environment

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antinociception and hypothermia in response to nicotine. Again, the N4N hapten was able to attenuate antinociception more significantly than the 3’AmNic hapten (Figure 7a). The same trend was observed in nicotine-induced hypothermia, albeit less significantly than in the BALB/c mice (Figure 7b). These results indicate that the effects of the N4N-SucFliC formulation are broadly applicable to more heterogeneous systems. To extrapolate these observations, mice were sacrificed 2 weeks after their final boost along with the saline control cohort 10 minutes after receiving a 2 mg/kg dose of nicotine. Serum and brain tissues were collected, extracted and the levels of nicotine present measured by LC/MS-TOF (Figure 8).33,35,36 The N4N cohort was found to have the highest serum nicotine concentration and lowest brain nicotine concentration when compared with the control group (Table 2), however the difference between the groups was statistically insignificant. The trend is still consistent with the observed behavior and does not undermine the evidence that the N4N-SucFliC formulation is able to attenuate the phenotypic effects of nicotine. However a future study covering a range of nicotine doses will need to be undertaken to confirm that this effect is indeed the result of sequestration of nicotine to the serum. Conclusions. Evidence suggests that there is extraordinary utility in being able to delay the onset of the reward effects of nicotine in addiction therapy.6,13-16 The results of this study suggest that formation of a nicotine hapten through covalent modification of the pyridine ring produces an antiserum of superior affinity for free nicotine. Despite being of extremely low abundance, vaccination with the N4N-SucFliC formulation was able to delay the effects of nicotine within the first 10 minutes, a time when nicotine concentrations are highest after insufflation.37 Evidence that the N4N-SucFliC formulation is able to sequester nicotine to the serum is reinforced by the observation that vaccinated mice exhibited a lower concentration of nicotine in the brain. The results of this study also reinforce our previous observations that FliC serves as a suitable carrier in conjugate vaccines. The N4N hapten appears to consistently raise antisera of higher affinity for nicotine than the 3’AmNic hapten, demonstrating that conjugation through the pyridine ring of the nicotine scaffold presents an epitope more closely related to nicotine. Future efforts will focus on producing an increased antibody concentration in response to the N4N-SucFliC vaccine formulation through experimentation with various adjuvants, as well as to examine the formulation in rats, which will provide better insight into the clinical feasibility of the formulation as therapy for nicotine relapse and addiction cessation. Experimental section. Chemistry. All reactions were carried out under inert atmosphere with anhydrous solvents unless otherwise stated. Purity of all compounds was determined by an Agilent 1260 Infinity HPLC to be 95% or greater, unless otherwise ACS Paragon Plus Environment


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specified. Carrier proteins were used as follows: Imject BSA from Thermo Fisher, tetanus toxoid from Statens Serum Institute (Denmark), flagellin

(FliC) was prepared recombinantly and purified as previously described.18 The (–)-

3’AmNic hapten was prepared as described previously.17 L-[N-methyl-3H]-nicotine of specific activity = 81.7 Ci/mmol (PerkinElmer, Boston, MA) was used in RIA analysis. (S)-(–)-Nicotine (98%) was obtained from Toronto Research Chemicals (Toronto, Canada). Synthesis of ethyl 3-(3-((S)-1-methylpyrrolidin-2-yl)-1-pivaloyl-1,4-dihydropyridin-4-yl)propanoate (4). To a dry, argon-purged roundbottom flask was added ethyl 3-iodopropinate (5.93 g, 26 mmol). This was diluted in 40 mL of dry THF. This solution was cooled to -78 ºC via dry ice/acetone bath. To this was added zinc powder (2.04 g, 31.2 mmol), followed by transmetalation with copper (I) cyanide (1.79 g, 20 mmol) with lithium chloride (1.78 g, 42 mmol). To a separate flask was added (–)-(S)-nicotine, 3 (3.24 g, 20 mmol). To this was added trimethylacetyl chloride (2.41 g, 20 mmol). This was diluted in 40 mL dry THF. This solution was cooled to -78 ºC via dry ice/acetone bath. To this was added copper, zinc activated propionate solution. Solution was allowed to warm to ambient temperature overnight while stirring under argon.38,39 Reaction was monitored by TLC. Upon reaction completion, solution was cooled to 0 ºC via ice bath. To this was added 150 mL 10 % ammonium hydroxide. Mixture was filtered and filtrate extracted with ethyl acetate. Organic layers combined and washed with 10 % ammonium hydroxide, sodium bicarbonate, and brine, dried with sodium sulfate, filtered, and concentrated to afford a crude oil. This was purified by flash chromatography (90:10:1 EtOAc/MeOH/NH4OH) to afford 2.93 g (42 %) of 4 as an brown semi-solid. 1H NMR (500 MHz, CDCl3) δ 7.30 (s, 1H), 7.20 (dd, J = 1.5, 8.2 Hz, 1H), 5.06 (dd, J = 5.0, 8.1 Hz, 1H), 4.17 (qd, J = 0.9, 7.1 Hz, 2H), 3.16 (t, J = 8.3 Hz, 1H), 3.09 (d, J = 4.7 Hz, 1H), 2.67 – 2.53 (m, 1H), 2.39 – 2.24 (m, 5H), 2.19 (d, J = 9.5 Hz, 1H), 2.10 – 1.95 (m, 2H), 1.95 – 1.85 (m, 2H), 1.85 – 1.68 (m, 2H), 1.42 (s, 9H), 1.31 (t, J = 7.1 Hz, 3H).

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C NMR (126 MHz, CDCl3) δ 174.45, 125.42,

123.44, 110.37, 77.67, 77.42, 77.16, 70.98, 60.63, 57.20, 41.05, 34.70, 32.36, 31.23, 30.46, 28.54, 23.29, 14.61. HRMS calculated for C20H32N2O3 [m/z +H+] 349.24, found 324.2485. Synthesis of ethyl (S)-3-(3-(1-methylpyrrolidin-2-yl)pyridin-4-yl)propanoate (5). To a dry, argon-purged round-bottom flask was added 2.90 g (8.32 mmol) of compound 4. This was dissolved in 12.5 mL of xylene. To this was added 350 mg (10.82 mmol) of sulfur. Solution was refluxed for 90 minutes. Reaction mixture cooled to ambient temperature and loaded onto silica gel for purification by flash chromatography (90:10:0 -> 90:10:1 EtOAc/MeOH/NH4OH) to afford 1.09 g (50 %) of 5 as a brown semi-solid. 1H NMR (400 MHz, CDCl3) δ 8.77 (s, 1H), 8.39 (d, J = 5.1 Hz, 1H), 7.05 (d, J = 5.1 Hz, 1H), 4.14 (q, J = 7.1 Hz, 2H), 3.41 (s, 1H), 3.30 (s, 1H), 3.00 (t, J = 7.9 Hz, 2H), 2.66 – 2.53 (m, 2H), 2.38 – 2.25 (m, 2H), ACS Paragon Plus Environment

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2.22 (s, 3H), 2.02 (s, 1H), 1.85 (s, 1H), 1.72 (s, 1H), 1.25 (t, J = 7.2 Hz, 3H).

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C NMR (101 MHz, CDCl3) δ 172.31,

149.42, 123.28, 77.33, 77.02, 76.70, 60.74, 56.85, 34.53, 26.64, 22.68, 14.19. HRMS calculated for C15H22N2O2 [m/z +H+] 263.17, found 263.1723. Synthesis of (S)-3-(3-(1-methylpyrrolidin-2-yl)pyridin-4-yl)propanamide (6). To a dry, argon-filled 4 mL screw-cap vial was added 131 mg 5. To this was added 1.25 mL of ammonium hydroxide. Solution was stirred vigorously at ambient temperature for 8 hours. Reaction mixture concentrated in vacuo and purified by flash chromatography (90:10:1 -> 80:20:2 EtOAc/MeOH/NH4OH) to afford 105 mg (90 %) of 6 as colorless syrup. 1H NMR (600 MHz, CDCl3) δ 8.67 (s, 2H), 8.37 (d, J = 5.1 Hz, 2H), 7.07 (dd, J = 0.7, 5.0 Hz, 2H), 6.09 (s, 1H), 5.80 – 5.76 (m, 1H), 3.37 (t, J = 8.6 Hz, 2H), 3.25 (ddd, J = 1.9, 7.8, 9.4 Hz, 2H), 3.04 (ddt, J = 7.9, 14.5, 44.6 Hz, 4H), 2.53 (t, J = 7.9 Hz, 4H), 2.36 – 2.22 (m, 4H), 2.18 (s, 6H), 2.04 – 1.93 (m, 2H), 1.85 (ddddd, J = 1.8, 4.9, 7.9, 9.7, 12.7 Hz, 2H), 1.70 (dddd, J = 5.0, 8.7, 10.9, 13.3 Hz, 2H). 13C NMR (151 MHz, CDCl3) δ 173.90, 149.62, 148.04, 147.87, 136.30, 123.44, 77.26, 77.05, 76.84, 66.05, 56.98, 56.97, 40.69, 36.01, 34.24, 26.84, 14.19. HRMS calculated for C15H22N2O2 [m/z +H+] 234.15, found 234.1564. Synthesis of N4N hapten (2). 100 mg of 6 was azeotroped with benzene and diluted in 5 mL of toluene. To this was added 5 mL THF. This solution was added dropwise to a dry, argon-purged round-bottom flask charged with 580 µL (1.93 mmol) of 65 % by weight reductive aluminum. Reaction mixture stirred for 90 minutes at ambient temperature. Reaction cooled to 0 ºC via ice bath and quenched with a mixture of Celite, DARCO G60, and water. Reaction mixture filtered and filtrate concentrated in vaccuo to afford 88 mg of pale amber syrup. This was purified by flash chromatography (4:1 CHCl3/MeOH w/ 2% NH4OH) to afford 68 mg of a pale, yellow syrup. This material was repurified by preparative TLC (4:1 CHCl3/MeOH w/ 2% NH4OH) to afford 35 mg (37%) of the N4N hapten (5). 1H NMR (600 MHz, Methanol-d4) δ 8.64 (s, 1H), 8.31 (d, J = 5.2 Hz, 1H), 7.27 (d, J = 5.2 Hz, 1H), 3.51 (t, J = 8.5 Hz, 1H), 3.33 (p, J = 1.7 Hz, 1H), 3.27 (ddd, J = 1.9, 7.9, 9.6 Hz, 1H), 2.85 – 2.73 (m, 3H), 2.43 – 2.32 (m, 2H), 2.21 (s, 3H), 2.04 – 1.95 (m, 1H), 1.91 (dtd, J = 2.3, 3.9, 4.9, 9.9 Hz, 1H), 1.80 (p, J = 7.7 Hz, 2H), 1.71 – 1.62 (m, 1H). 13C NMR (151 MHz, MeOD) δ 150.67, 147.84, 146.55, 137.00, 124.17, 64.97, 56.45, 40.73, 39.42, 34.22, 33.04, 28.72, 22.11. HRMS calculated for C13H21N3 [m/z +H+] 220.17, found 220.1812. Conjugation Protocol. To a solution of BSA (~4 mg/mL), TT (~2 mg/mL), or FliC (~2 mg/mL) in Tris buffer (pH = 8.65) was added succinic anhydride (1.0 M in DMSO, 750-3000 equiv) and solution allowed to stir at ambient temperature for 1 h. This solution was dialyzed against MES buffer (pH = 5.8). To a solution of SucBSA (~4 mg/mL), SucTT (~2 mg/mL), or SucFliC (~2 mg/mL) was added N4N or 3’AmNic hapten (0.2 M in H2O, 100-800 equiv) and ACS Paragon Plus Environment


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EDC (0.2 M in H2O, 1000 equiv) and mixture stirred at ambient temperature for 24 hours. Solution dialyzed into PBS (pH = 7.4). Hapten densities determined by MALDI-TOF analysis. Vaccine Formulation. Protein-hapten conjugates (50 µg) were formulated with 50 µg CpG ODN 182640 (Eurofins MWG Operon, Huntsville, AL) and 20 µL Alhydrogel (10 mg/mL Al, vac-alu-50, InvivoGen, San Diego, CA). Mice (n = 8) were injected subcutaneously with 100 µL total volume at 0, 3, and 6 weeks. Vertebrate animals. Male BALB/c and Swiss Webster mice were obtained from Taconic (Oxnard, CA). All animals were housed three per cage in a temperature-controlled (22 ºC) vivarium on a 12-h light cycle with ad libitum access to food and water. Nonterminal bleeds were obtained by tail tip amputation (~200 µL) at 4 and 7 weeks. Terminal bleeds were obtained by cardiac puncture (~1.25 mL) at 8 weeks. All procedures adhere to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and all protocols were approved by the Institutional Animal Care and Use Committee of The Scripps Research Institute (La Jolla, CA). Hot plate assessment. Antinociception was measured by the hot-plate test as described previously.31,32 The percent maximum possible effect (% MPE) was determined using the standard formula: (test-baseline)/(cutoff-baseline) x 100, with a cutoff time of 30 seconds. Statistical analysis was performed using Prism 4 (GraphPad) using betweensubjects analysis of variance (ANOVA). Significant results were followed by Dunnett’s or Tukey’s post-hoc test where appropriate. Hypothermia assessment. Rectal temperature was measured by inserting a thermocouple probe 2 cm into the rectum, and temperature obtained from BAT-10 telethermometer (Physitemp Instruments, Clifton, NJ). Baseline readings were taken just before injection, with measurements at 10, 30 and 60 mins after injection. The difference in rectal temperature was calculated for each mouse and significance analyzed by ANOVA with a Tukey’s post-hoc test using Prism 4 (GraphPad). ELISA. Assays were performed as previously described.17 Briefly, 96-well plates (Costar 3690) were coated with 25 µL/well BSA conjugate in PBS (pH = 7.2) at 5 µg/mL and incubated overnight at 37 ºC. Antigen was methanol fixed at ambient temperature for 5 min, blocked with 5 % milk solution at 37ºC for 30 min, then charged with 25 µL 2 % BSA in PBS (pH = 7.2). Serum samples were diluted 1:100 in 1% BSA and serially diluted across 12 wells. Plate incubated at 37 ºC for 90 min, washed extensively with H2O, then incubated with goat-antimouse Ig(H+L)-HRP (Southern Biotech) secondary antibody for 30 min at 37 ºC. Plates were washed extensively with H2O, and HRP detected with TMB substrate kit (Pierce) in the dark for 10 - 20 min. Reaction quenched with the addition of 2 M sulfuric acid. Absorbance at ACS Paragon Plus Environment

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450 nm detected by SpectraMax M2e (Molecular Devices, Sunnyvale, CA). Midpoint titers obtained using Microsoft Excel and GraphPad Prism. Statistical significance was assessed by Bonferroni post-test using GraphPad Prism. RIA protocol. Binding curves and competitive RIA was carried out in 5 kDa MWCO Equilibrium Dialyzer-96 plates (Harvard Apparatus, Holliston, MA) as described previously.17 Pooled mouse serum was diluted in 2% BSA to a concentration that bound ~50% of nicotine tracer. Sample chambers were loaded with 75 µL of diluted serum and 75 µL of radiolabeled nicotine, and buffer chambers were loaded with 150 µL of unlabeled nicotine at concentrations in 1% BSA. Dialysis plates were equilibrated at ambient temperature for 22 h, after which a 75 µL aliquot was removed from each side and diluted into 5 mL of scintillation fluid (Ecolite(+), MP Biomedicals, Santa Ana, CA). Radioactivity (dpm) was measured in a Beckman LS 6500 scintillation counter. Average dissociation constants and antibody concentrations were obtained using Microsoft Excel and GraphPad Prism. LCMS-TOF Analysis. Blood and brain samples were prepared as previously described.33 Mice were sacrificed 10 min after nicotine administration using isoflurane followed by decapitation. Trunk blood was collected and 1 µL of 1 mM (±)-nicotine-2,4,5,6-d4 (CDN Isotopes, Pointe-Claire, Quebec, CA) added to 1 mL whole blood before sample preparation. Mouse brains were dissected following decapitation, homogenized in 1 mL PBS, and 1 µL 1 mM (±)nicotine-2,4,5,6-d4 added to homogenate.



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Supporting Information. Additional assay data, and mass analysis spectra of conjugates are available in the supporting information.

Corresponding Author Information: *Phone: (858)-785-2516. Fax: (858)-784-2595. Email: [email protected]

Present/Current Author Addresses: 1Departments of Chemistry and Immunology, Worm Institute for Research and Medicine (WIRM), The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, United States. 2Department of Chemical Physiology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, United States.

Acknowledgments: This is manuscript no. 29229 from The Scripps Research Institute. Funding for this study was provided by the Skaggs Institute and the NIH (Grant #TL1TR001113 to NTJ).

Abbreviations Used: nAChR – nicotinic acetylcholine receptor; NRT – nicotine replacement therapy; TLR – Toll-like receptor; %MPE – percent maximal possible effect; MHC – major histocompatibility complex; APC – antigenpresenting cell; MES – 2-(N-morpholino)ethanesulfonic acid; BSA – bovine serum albumin.


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Figure 1: Chemical structures of the 3’AmNic hapten (1, NicVAX) and N4N hapten (2).

Scheme 1: Synthesis of the N4N hapten, 2.



Figure 2: Immunization and bleed schedule.

300000

***

**

200000

Average Titer

* Treatment Group: N4N-SucFliC N4N-SucTT 3'AmNic-SucFliC 3'AmNic-SucTT

100000

le e B d1 le e B d2 le ed 3 B le e B d1 le e B d2 le ed 3 B le e B d1 le e B d2 le ed 3 B le e B d1 le e B d2 le ed 3

0

B

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N4N-SucBSA 3'AmNic-SucBSA Coating Antigen Figure 3: Average anti-hapten titer measured by ELISA. Serum of sham-vaccinated mice gave no appreciable measurement for either coating. Average values ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. ACS Paragon Plus Environment

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% MPE (Hot Plate)

100 80

*

60 40 20

Sa lin e

ic -

(-) -

3' -A

N m 3' -A

(-) -

Su cF liC m N ic -S uc TT

T -S uc T

4N N

4N -S

uc Fl iC

0

N

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Treatment Figure 4: Antinociception effects of nicotine (3 mg/kg) measured by response to hot plate in vaccinated and unvaccinated mice. % MPE – percent maximal possible effect. Average values ± SEM. *P < 0.01 compared to saline. ACS Paragon Plus Environment


Time Post-Nicotine (min) 30

2 10

60

***

1

**

0

∆Temp (°C)

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-1 -2 -3

***

*** *** **

*

*

**

-4

N4N-SucFliC N4N-SucTT (-)-3'-AmNic-SucFliC (-)-3'-AmNic-SucTT Saline

-5 Figure 5: Time-course of difference in body temperature measured in response to challenge with 2 mg/kg nicotine in vaccinated and unvaccinated mice. Average values ± SEM. *P < .05, **P < .005, ***P < .001 compared to Saline.

Table 1: Summary of RIA Results [Ab] (µg/mL) Hapten Copy #a Formulation Kd (nM) N4N-SucFliC 46.66 2.29 35 N4N-SucTT 344.18 49.83 36 3’AmNic-SucFliC 131.92 14.52 43 3’AmNic-SucTT 824.09 186.50 47 a copy number determined by the difference in mass analysis of conjugated and unconjugated protein.


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8000

Treatment: N4N-SucFliC 3'AmNic-SucFliC

6000

4000

2000

3 le ed

B

1

le ed B

B

le ed B

le ed

3

2

1

le ed B

le ed

N4N-SucBSA

2

0

B

Average Titer

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(-)-3'AmNic-SucBSA

Coating Antigen Figure 6: Average titer of anti-hapten antibody in pooled serum measured by ELISA. Average values ± SEM. Difference between individual bleeds is not significant (P > 0.5).



\

*

0

cF liC Sa lin e

60

N4 NSu

cF liC

80

Nic -Su

%MPE (Hot Plate)

Treatment

b.)

100

3'A m

a.)

40

****

-2

-4

N

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-3

'- A

m

N

4N -S

uc Fl ic iC -S uc Fl iC Sa lin e

0

∆T (ºC)

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(- )

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Treatment Figure 7: a) Antinociceptive response 15 minutes after challenge with 2 mg/kg nicotine. *P < .05. ****P < .0001. b) Nicotine induced hypothermia measured 30 minutes after treatment with 2 mg/kg nicotine. *P < .05.


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1.0×10-07

8.0×10-08

[Nicotine] (M)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


6.0×10-08

4.0×10-08

2.0×10-08

0 N4N Serum

N4N Brain

3AM Serum

3AM Brain Saline Serum Saline Brain

N4N 3-AmNic Saline Figure 8: Concentrations of nicotine measured in serum and brain samples from vaccinated and unvaccinated mice taken 10 minutes after challenge with nicotine at a dose of 2 mg/kg. *Outlier excluded from N4N group by Chauvenet’s and Dixon’s methods.

Table 2: Average brain and serum nicotine concentrations. Forumlation [nicotine] serum (nM) [nicotine] brain (nM) N4N-SucFliC 51.9 (± 13.1) 13.3 (± 3.03) 3’AmNic-SucFliC 49.8 (± 15.0) 13.6 (± 3.75) Saline 48.1 (± 6.48) 16.4 (± 4.80)



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Table of Contents Graphic:


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Investigations of Enantiopure Nicotine Haptens Using an Adjuvanting

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