CONSTRAINED ALKALOID IMMUNOGENS AND ANTIBODIES AND USES THEREOF By Kim D. Janda Peter Wirsching TECHNICAL FIELD OF THE INVENTION [0001] The field of this invention is methods of treating drug addiction. More particularly, this invention pertains to alkaloid immunogens, anti-alkaloid antibodies and the use of such immunogens and antibodies for treating alkaloid addiction. In one embodiment, the alkaloid is nicotine. The present invention also relates to methods of enhancing the immunogenicity of small haptens by constraining their conformation.

BACKGROUND OF THE INVENTION [0002] Nicotine is the most widely used addictive drug in the world. As an alkaloid, [(S)-(-)-1-methyl-2-(3-pyridyl) pyrrolidine] derived from tobacco leaves, nicotine is available in several forms such as cigarettes, cigars, pipe tobacco, and chewing tobacco.

Hence, the drug is intimately linked with cigarette smoking, the leading preventable cause of death in the United States [Nelson, D. E. ; Kirkendall, R. S.; Lawton, R. L.; Chrismon, J.

H.; Merritt, R. K.; Arday, D. A.; Giovino, G. A. Morbid. Mortal. Wkly. Rep. 43,1-8 (1994); US Dep. Health Hum. Serv. Reducing the health consequences ofsmoking. 25 years of progress. A report of the Surgeon General ; Public Health Services: Rockville, MD, 1989]. Smoking contributes to coronary heart disease, stroke, vascular disease, peptic ulcers, chronic lung diseases and lung cancer, and fetal brain damage and morbidity.

Although the dangers of smoking are well known, people continue to smoke.

[0003] A great deal of evidence supports the view that people continue to smoke because of the addictive effects of nicotine [Benowitz, N. L. Annu. Rev. Pharmacol.

Toxicol. 36,597-613 (1996); Rose, J. E. Annu. Rev. Med. 47,493-507 (1996) ]. However, since nicotine is legally and widely accessible there is relatively little stigma associated with its use, unlike cocaine, heroin and other illicit drugs. Although a large percentage of addicted smokers have expressed a desire to stop smoking and most who quit do so without treatment, less than 5% of unaided attempts lead to successful long-term abstinence [Hughes, J. R.; Gulliver, S. B.; Fenwick, J. W. et al. Health Psycho. 11,331- 334 (1992); Gritz, E.; Marcus, A.; Carr, C. J. Psychosoc. Oncol. 6,217-234 (1988) ].

Similarly, the two most popular therapies, nicotine gum and transdermal nicotine patches used to slowly wean the user off the drug, have afforded inadequate long-term success rates of <20% [Henningfield, J. E. New Engl. J. Med. 333,1196-1203 (1995); Hughes, J.

R. In Integrating Behavior Theories with Medication in the Treatment ofDrug Dependence (Eds. Onken, L. S.; Blaine, J. D.; Boren, J. J. ) ; NIDARes Monogr. US GPO: Washington, DC, 1995, pps. 92-109; Palmer, K. J., Buckle, M. M.; Faulds, D. Drugs 44, 498-529 (1992) ]. Perhaps because relatively little is known about the specific neuropharmacologic mechanisms underlying nicotine addiction and the response to smoking cessation treatment, no highly effective therapy has been developed.

[0004] There is a need to develop a treatment approach to nicotine addiction which does not depend solely on unaided compliance or on the administration of nicotine itself for rehabilitation. One alternative might rely on immunological reagents and the immune system. Recently, the efficacy of immunological strategies with regard to the cocaine abuse problem has been demonstrated [Carrera, M. R. A.; Ashley, J. A.; Wirsching, P.; Koob, G. F.; Janda, K. D. Proc. Natl. Acad. Sci. USA 98,1988-1992 (2001); Carrera, M.

R. A.; Ashley, J. A.; Zhou, B.; Wirsching, P.; Koob, G. F, Janda, K. D. Proc. Natl. Acad.

Sci. USA 97,6202-6206 (2000) ; Carrera, M. Rocio A.; Ashley, J. A.; Parsons, L. H.; Wirsching, P.; Koob, G. F.; Janda, K. D. Nature 378,727-730 (1995) ]. These same strategies would also be useful to treat nicotine abuse. However, nicotine is not as immunogenic as cocaine.

10005] The immune-mediated binding of nicotine impedes its passage into the central nervous system and results in a suppression of its characteristic actions.

Accordingly, under the right conditions, both active immunization (immunoconjugate vaccine) and passive immunization (monoclonal antibodies, mAbs) protocols would be useful to provide therapies to counteract nicotine addiction.

[0006] Although the preceding discussion has focused on the treatment of nicotine addiction, the present invention also relates to immunogenic treatment of other alkaloid- based drugs of abuse. Unlike cocaine, which has a rigid tropine-based ring structure, nicotine and other alkaloids have less rigid structures. This flexibility makes them less effective at eliciting a strong immunogenic response. Hence, there is a need to develop methods of enhancing the immunogenicity of alkaloid haptens to improve the efficacy of immunogenic-based therapies for drug addiction.

BRIEF SUMMARY OF THE INVENTION [0007] In one aspect, the present invention provides a constrained nicotine immunogen. The immunogen has the structure shown in FIG. 1 and designated as CNA or CNI. FIG. 1 shows a synthetic scheme for making CNA and CNI. In one embodiment, the immunogen is linked to an immunogenic carrier such as keyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA).

[0008] In another aspect, this invention provides a method for immunizing an animal against nicotine. In accordance with that method, an animal is administered an effective immunogenic amount of the immunogen of this invention.

[0009] In another aspect, this invention provides an antibody that immunoreacts with the immunogen of this invention. Preferably, the antibody also immunoreacts with nicotine. Preferably, the antibody is a monoclonal antibody. An anti-nicotine antibody is made by a process that comprises the step of immunizing an animal with the immunogen of this invention. This invention further provides a method of immunizing an animal against nicotine that includes the step of administering to the animal an effective amount of a subject antibody.

[0010] The invention further provides a pharmaceutical composition containing a subject immunogen or antibody together with a physiological diluent.

[0011] In yet another aspect, the invention provides a method of enhancing the immunogenicity of an alkaloid hapten by constraining its conformation.

BRIEF DESCRIPTION OF THE DRAWINGS [0012] In the drawings that form a portion of the specification: [0013] FIG. 1 shows nicotine haptens CNA and CNI and a scheme for making CNA and CNI.

[0014] FIGS. 2 and 3 illustrate the results of Example 2.

Detailed Description of the Invention I. The Invention [0015] This invention provides nicotine immunogens, anti-nicotine antibodies and the use of such immunogens and antibodies for immunizing animals against nicotine and treating nicotine addiction. This invention also provides methods of treating addictions to other alkaloids by constraining the conformation of alkaloid haptens.

II. Nicotine Immunogens [0016] Nicotine is a small, haptenic molecule and requires coupling to an immunogenic carrier protein to elicit an immune response. In the past 30 years, a number of reports appeared in the literature that described nicotine haptens and immunoconjugates [Abad, A.; Manclus, J. J.; March, C.; Montoya, A., Anal. Chem. 65, 3227-3231 (1993): Bjercke, R. J.; Cook, G.; Rychlik, N.; Gjika, H. B.; Van Vunakis, H.; Langone, J. J. J.

Immunol. Methods 90, 203-213 (1986); Castro, A.; McKennis Jr. , H.; Monji, N.; Bowman, E. R. Biochem. Arch. 1,205-214 (1985); Castro, A.; Monji, N.; Hacer, A.; Bowman, E.

R.; McKennis Jr. , H. Biochem. Arch. 1,173-183 (1985); Langone, J. J.; Van Vunakis, H.

Methods Enzymol. 84,628-640 (1982); Matsushita, H.; Noguchi, M.; Tamaki, E.

Biochem. Biophys. Res. Commun. 57,1006-1010 (1974); and Langone, J. J.; Gjika, H. B.; Van Vunakis, H., Biochem. 12,5025-5030 (1973) ].

[00171 That work was aimed primarily at the development of enzyme-linked immunosorbent assays (ELISA) for more convenient detection of nicotine in a variety of media suchas blood, urine, and smoke residue. The majority of these studies utilized polyclonal immunoglobulins (antisera) from rabbits/goats. In a few examples, murine monoclonal antibody (mAb) preparations were examined. While the data in most cases were acceptable with regard to antibody affinity and specificity, erratic and variable results were noted. In light of some of these observations, there is clearly room for improvement with regard to both hapten design and the quality of anti-nicotine immune responses and antibody preparations. Notably, even slight improvements would afford enhanced performance in immunopharmacotherapy protocols.

[00181 For optimum results, the design and synthesis of a nicotine hapten requires attention to stereochemistry, ionic/acid-base properties, and the site of attachment and characteristics of a linker moiety. Nicotine, which occurs as the (S) -configuration in nature (e. g. tobacco), contains two rings with an asymmetric center. There are two sites of basicity: the pyridyl and pyrrolidyl nitrogens. Nicotine is expected to carry a positive charge at physiological pH. Hence, an optimal immunogen (i. e. , hapten) should incorporate a linker when coupled as the immunoconjugate in such a way as to present both ring nitrogens and have the proper stereochemistry and charge characteristics.

Exemplary and preferred nicotinic compounds useful in the design of immunogens are shown in FIG. 1. Those compounds are CNA and CNI.

[0019] CNA and CNI were designed and synthesized based on reported conformationally constrained nicotine analogues 1 and 2 (FIG. 1). The N-Me nicotine-like derivative of 1 had analgesic effects, but the receptor remains unidentified.

Recently, other constrained analogues were developed, and shown to bind with low nanomolar affinity to the nicotinic acetylcholine receptor. The present invention focuses on 1 and 2, since they are readily prepared and provide a reasonable mimic of the two trans confolmers of nicotine that is supported by crystal-structure data. The aim was for CNA and CNI to elicit high antibody titers that could bind (5)-nicotine in solution.

[0020] An immunogen is typically operatively linked to an immunogenic carrier before immunization of an animal. Useful carriers are well known in the art and are generally proteins themselves. Exemplary of such carriers are keyhole limpet hemocyanin (KLH), edestin, thyroglobulin, albumins such as bovine serum albumin or human serum albumin (BSA or HSA, respectively), red blood cells such as sheep erythrocytes (SRBC), tetanus toxoid, cholera toxoid as well as polyamino acids such as poly (D-lysine: D- glutamic acid), and the like. The choice of carrier is more dependent upon the ultimate intended use of the antigen than upon the determinant portion of the antigen, and is based upon criteria not particularly involved in the present invention. For example, if the conjugate is to be used in laboratory animals, a carrier that does not generate an untoward reaction in the particular animal should be selected. The carrier-hapten conjugate is dissolved or dispersed in an aqueous composition of a physiologically tolerable diluent such as normal saline, PBS, or sterile water to form an inoculum. An adjuvant such as complete or incomplete Freund's adjuvant or alum can also be included in the inoculum.

The inoculum is introduced as by injection into the animal used to raise the antibodies in an amount sufficient to induce antibodies, as is well known.

E. Anti-nicotine Antibodies [0021] The present invention provides antibodies that immunoreact with an immunogen of this invention. An antibody can be a polyclonal or monoclonal antibody or an immunoreactive fragment thereof. Means for making polyclonal and monoclonal antibodies are well known in the art. Still further an antibody of this invention can be a recombinant antibody. Means for making recombinant antibodies are well known in the art. By way of example, immunoglobulin mRNA can be cloned from specific hybridomas and expessed using phage display. A recombinant antibody can be manipulated or mutated so as to improve its binding ability to an antigen such as nicotine. Means for such manipulation/mutation are also well known in the art. An antibody of this invention also cross reacts with nicotine. Preferably, the antibody cross reacts with S- (-), but not R- (+) nicotine.

[00221 Murine mAbs can be"humanized"via several techniques well known in the art [James, K. Human monoclonal antibodies. In : Handbook of Experimental Pharmacology : The Pharmacology of Monoclonal Antibodies, Rosenberg, M.; Moore, G.

P. , eds. New York: Springer-Verlag, vol. 113, pp. 3-19,1994 ; Padlan, E. A., Mol.<BR> <P>Immunol. 28,489-498 (1991); Daugherty, B. L.; DeMartino, J. A.; Law, M. -F. ; Kawka, D.

W.; Singer, 1. 1. ; Mark, G. E., Nucleic Acids Res. 19,2471-2476 (1991) ; and Riechmann, L.; Clark, M.; Waldmann, H.; Winter, G., Nature 332,323-327 (1988) ]."Humanization" of anti-cocaine antibodies can be accomplished in which the binding properties are similar to the starting murine mAb. In addition, a methodology for selecting antibodies of desired specificity from combinatorial libraries makes human mAbs (scFvs or Fabs) directly available. If desired, protein engineering can be utilized to prepare human IgG constructs.

Access to humanized or fully human mAbs makes clinical application feasible.

[0023] The constrained haptens utilize a linker for coupling to a carrier protein, such as keyhole limpet hemocyanin (KLH). The same linker is used to prepare native nicotine-hapten conjugates (NIC-KLH) for use as controls. After standard immunization protocols, the NIC-KLH immunoconjugate provides low titers having a mean value of about 3,200. Competition ELISA and equilibrium dialysis measurements yield the serum affinity for (5)-nicotine as a Kd-avg, ~1. 7, uM + 0.20 u. M. Other groups reported titers for nicotine-based haptens of 10,000 or less and affinities in the micromolar range. On the other hand, immunizations using the constrained haptens (CNA-KLH and CNI-KLH) resulted in antisera with greatly increased titers of about 25,000, comparable with those that were observed using cocaine vaccination studies. Significantly, the Kd-avg-1. 0 M t 0. 10, M and 0.60 uM 0. 10 uM, respectively, were nearly two-and three-fold improved. Also, the antisera showed >10 : 1 specificity for (5) -nicotine versus the major<BR> metabolite (5) -cotinine, similar to NIC-KLH antiserum. Interestingly, CNA-KLH and CNI-KLH antisera had low cross-reactivity in binding CNI-bovine serum albumin (BSA) (1: 15) and CNA-BSA (1: 6) conjugates used for titering, respectively, suggesting an important role for the pyridyl nitrogen in the antibody-hapten interactions and that recognition of the distinct trans species of nicotine is possible.

[0024] Constraining nicotine conformations yields an enhanced immune response compared to a flexible nicotine hapten. The improved effect is likely caused by a narrowing in the range of antibody heterogeneity skewed towards a population of increased affinity. The titers are in the range of the necessary minimum in mice/rats to model the development of a clinically useful nicotine vaccine. In addition, this example of augmenting immunogenicity using conformational constrain of other alkaloid haptens provides a general route towards vaccination strategies for other pharmacologically important small molecules. t0025] Some alkaloid haptens, such as nicotine, have more than one conformation, which means each conformation is"sampled"less often by the immune system. To enhance the immunogenicity of these small haptens, others have focused on changes to the hapten-carrier linkage. See, for example, U. S. Patent No. 6,455, 047 which describes enhanced immunogenicity of cotinin using special linkers to attach it to a carrier. l00261 Unlike this linker-based approach, the present invention relates to a constraining structural modification of the hapten itself to improve immunogenicity.

This approach may involve closing a ring around the chiral center of the hapten to"lock" it into one conformation or the other. However, any approach that would result in a more conformationally constrained hapten structure is also applicable.

IV. Pharmaceutical Composition [0027] The present invention further provides a pharmaceutical composition. The pharmaceutical composition includes a compound of this invention (immunogen, antibody) together with a physiologically tolerable carrier.

[0028] As used herein, the terms"pharmaceutically acceptable", "physiologically tolerable"and grammatical variations thereof, as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration to or upon a mammal without the production of undesirable physiological effects such as nausea, dizziness, gastric upset and the like.

[0029] The preparation of a pharmacological composition that contains active ingredients dissolved or dispersed therein is well understood in the art and need not be limited based on formulation. Typically such compositions are prepared as injectables either as liquid solutions or suspensions, however, solid forms suitable for solution, or suspensions, in liquid prior to use can also be prepared. The preparation can also be emulsified.

[0030] The active ingredient can be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient and in amounts suitable for use in the therapeutic methods described herein. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like and combinations thereof. In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like which enhance the effectiveness of the active ingredient.

[0031] The therapeutic composition of the present invention can include pharmaceutically acceptable salts of the components therein. Pharmaceutically acceptable salts include the acid addition salts (formed with the free arnino groups of the polypeptide) that are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, tartaric, mandelic and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-aminoethanol, histidine, procaine and the like.

Particularly preferred are the salts of TFA and HCI.

10032] Physiologically tolerable carriers are well known in the art. Exemplary liquid carriers are sterile aqueous solutions that contain no materials in addition to the active ingredients and water, or contain a buffer such as sodium phosphate at physiological pH value, physiological saline or both, such as phosphate-buffered saline. Still further, aqueous carriers can contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, polyethylene glycol and other solutes. Liquid compositions can also contain liquid phases in addition to and to the exclusion of water. Exemplary of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, and water- oil emulsions.

EXAMPLES [00331 The Examples that follow illustrate preferred embodiments of the present invention and are not limiting of the specification and claims in any way. In particular, the embodiments focus on nicotine as a model system for conformationally constraining haptens to enhance immunogenicity.

Example 1 : Methods of Immunization A. Active Immunization [00341 One goal in an immunological approach aimed at the abatement of nicotine addiction is the de novo design of a vaccine. In principle, vaccination imparts to the host's immune system the ability to defend against the acute psychostimulant and toxic effects of nicotine and its powerful reinforcing properties. Importantly, an active immunization against nicotine offers a means of blocking the actions of the drug by preventing it from entering the central nervous system and should have fewer side effects than treatments based on manipulation of central neurotransmitter function. Thus, immunopharmacotherapy offers a nontoxic, substance-specific strategy that should not affect normal neurochemical physiology, presenting a solid scientific approach for the treatment of nicotine dependence. l0035] The effect of immunization with NIC-KLH on acute nicotine-induced locomotor activity was investigated. Male Wistar rats were tested in photocell cages after s. c. administration of nicotine (0.50 mg/kg; 1 ml/kg) to determine pre-immunization drug response (baseline). Experimental animals were immunized i. p. three days later with NIC-KLH in an emulsion with an adjuvant (Ribi). Control animals were injected with a similar emulsion containing KLH solution only. This treatment was followed by boosts at 21 and 35 days. Rats were then challenged with systemic nicotine and their locomotor responses were again measured.

[0036] The initial pre-immunization nicotine injections resulted in the classic mhibitory effect on locomotor activity induced by acute treatment of the drug. With repeated administrations of nicotine, tolerance to the initial depressant effect developed, and by the sixth nicotine injection, the activating effects of the drug were observed. The first nicotine challenge resulted in a non-significant difference in locomotor activity [F (1, 14) = 3. 125; P < 0. 099]. This result may be interpreted as a re-emergence of the initial depressant effect of nicotine in control but not experimental rats. The last nicotine challenge produced a significant difference between groups in locomotor activity [F (1, 14) = 6.392 ; P < 0.024]. Compared to baseline values, experimental rats as a group showed a 45% decrease in the ambulatory measure (crossovers) (NIC-KLH : 542.63 59.85 ; 307.38 49.13), whereas controls showed a decrease of 16% (KLH: 516.63 64: 81; 437.50 61.48). These results indicate that inoculation with NIC-KLH significantly suppressed the psychosmotor effects of nicotine as compared to controls.

B. Passive Immunization [0037] Large amounts of anti-nicotine mAbs elicited with NIC-KLH have been accumulated for in vivo experimentation in rats. Integra Biosciences CELLine flasks are utilized for in vitro production of mAb NIC9D9. Each CL-1000 flask produces 100-200 mg of mAb per month. To produce-3 grams of antibody, 6 CL-1000's are set up and cultures are maintained for-3 months. The cell chamber is innoculated with 1-1. 5 x 108 hybridoma cells (generally 200 ml of between 5-7 x 105 cells/ml) in 15 ml of complete media (RPMI with 2 mM L-glutamine, 10 mM Hepes, 1 mM Na pyruvate, 100 Fg/ml gentamycin sulfate, 500 units penicillin, 500 units streptomycin supplemented with 20% fetal calf serum). The basal chamber has one liter of complete media without serum.

After one week, the cell density and viability is checked. The cells are harvested and fed the cell compartment, keeping the viability over 50% and the density 5 x 106 cells/ml, and change the basal media. The harvested cells (generally 15-20 ml) are centrifuged at 1500 rpm for 10 min. The supernatant is filtered through a 0.22 pm filter and purified through a protein G column. The concentration of antibody in the cell supernatant is usually-1-2 mg/ml. As the culture gets more established in the second and third month, we can harvest two times per week with 15-20 ml/harvest and obtain 400 mg per CL-1000.

10038) The administration of antibodies to suppress the effects of nicotine can be accomplished using known methods of cocaine immunopharmacotherapy [Carrera, M. R. <BR> <BR> <P>A.; Ashley, J. A.; Wirsching, P.; Koob, G. F. ; Janda, K. D. , Proc. Natl. Acad. Sci. USA 98, 1988-1992 (2001); Carrera, M. R. A.; Ashley, J. A.; Zhou, B.; Wirsching, P.; Koob, G. F, Janda, K. D., Proc. Natl. Acad. Sci. USA 97,6202-6206 (2000); and Carrera, M. Rocio A. ; Ashley, J. A.; Parsons, L. H.; Wirsching, P.; Koob, G. F. ; Janda, K. D., Nature 378,727- 730 (1995) ]. The passive administration of anti-nicotine antibodies is expected to be beneficial to reduce serum levels and attenuate"toxic" (cardiovascular, metabolic, endocrine) effects, or as (bi) weekly pharmacotherapy during smoking cessation programs.

The latter is accomplished by self-injection of mAb to maintain a high circulating level of antibody. Significantly, in a more user-palatable approach, it is possible to establish passive mucosal protection against nicotine in the respiratory tract through the use of aerosolized immunoglobulin [Crowe, J. E. , Jr.; Murphy, B. R.; Chanock, R. M.; Williamson, R. A.; Barbas, C. F., III ; Burton, D. R., Proc. Natl. Acad. Sci. USA 91,1386- <BR> <BR> 1390 (1994) ]. This method is particularly applicable to the nicotine dependence problem since the vast majority of users obtain nicotine by smoking.

[0039] In a preliminary experiment, locomotor activity data for rats passively immunized with mAb IVIC9D9 was acquired. Male Wistar rats were prepared with intrajugular catheters and allowed a 7-day recovery period. All animals were treated with <BR> <BR> a subcutaneous (s. c. ) nicotine (0.28 mg/kg; 1 ml/kg body weight) for five consecutive days and observed in photocell cages after administration to determine pre-immunization drug response (baseline). On testing days, experimental animals were infused i. v. with 5,25 or 50 mg/kg of NIC9D9 in a volume of approximately 1.5 ml/kg. Control rats were treated with an equivalent volume of physiological saline (i. v.). Approximately 30 min after infusions, all animals received s. c. nicotine injection (0.28 mg/kg) and their locomotor responses were assessed for the next 90 min. All locomotor activity test sessions were preceded by a 90 min habituation session following s. c. saline injection (1 ml/kg). The dose-response to NIC9D9 was produced by testing each dose two weeks apart, in order to avoid carry-over effects. All locomotor data reported refer to the ambulatory measure or crossovers.

[00401 The initial pre-immunization nicotine injections resulted in the classic inhibitory effect on locomotor activity induced by acute treatment of the drug. With repeated administrations of nicotine, tolerance to the initial depressant effect developed, and by the sixth nicotine injection, the activating effects of the drug were observed. This last pre-immunization nicotine challenge resulted in a nonsignificant difference in locomotor activity [F (1, 14) = 0.18 ; P < 0.895]. Passive transfer with NIC9D9 (5 mg/kg) in the experimental group resulted in a significant decrease in locomotor activity compared to controls during the first 20 min of the session [F (1, 14) = 6.757 ; P < 0.020]. This effect was optimized by increasing doses of the mAb. After i. v. infusion of NIC9D9 (25 mg/kg), differences in locomotion between groups were observed throughout the first 40 min of the session [F (1, 14) = 18.214 ; P < 0. 0008]. The maximum effect was obtained upon treatment with the highest mAb dose (50 mg/kg), where differences in the ambulatory measure persisted throughout the first 40 min of the session [F (1. 14) = 37. 376 ; P < 0.000].

In addition, a dramatic decrease during the initial 20 min of testing of the ambulatory locomotor responses were observed. Compared to baseline values, experimental rats as a group showed a 66.9% decrease in locomotor activity (NIC9D9 : 703.38 + 111.88 ; 308.75 + 57.51), whereas controls showed a decrease of 3.4% (SAL: 688.13 + 111.10 ; 740 + 117.62). l0041 l These results indicate that passive transfer with the mAb NIC9D9 dose- dependently suppressed the psychosmotor effects of nicotine as compared to controls.

Accordingly, this approach is highly amenable to the treatment of nicotine dependence in the human condition.

[0042] In humans, a variation on the passive protection scenario is also applicable.

From the standpoint of scientific rationale and user compliance, inhalation of anti-nicotine antibodies via compact, portable inhalers may constitute the single most effective means to treat the problem of nicotine addiction. More particularly, binding nicotine in the systemic circulation using a vaccine or with passive antibodies should be an effective therapy.

However, a direct blockade in the pulmonary system, together with systemic protection, or even as a stand-alone treatment, would greatly suppress the concentrations of nicotine that rapidly reach the brain during the action of smoking. Hence, a passive immunization approach is likely effective by keeping nicotine below a threshold concentration required for reinforcement.

[0043] Hence, while both a nicotine vaccine and an anti-nicotine mAb are part of the present invention, passive immunization protocols are as well. Certainly, the binding and blockade of nicotine is of paramount importance for therapy. However, the additional treatment with a"cocktail"of mAbs specific for nicotine and cotinine may also serve to reduce reinforcement and relapse potential, especially for some individuals. The haptens described provide the essential elements in a program of immunopharmacotherapy that offers a new avenue in the challenging battle against nicotine addiction.

Example 2: Preparation and Testing of CNA and CNI [0044] General procedures. Unless otherwise stated, all reactions were performed under an inert atmosphere with dry reagents, solvents, and flame-dried glassware. All starting materials were purchased from Aldrich, Sigma, Fisher, or Lancaster and used as received. All flash column chromatography was performed using silica gel 60 (230-400 mesh). Analytical and preparative thin-layer chromatography (TLC) was performed using Merck Kieselgel 60 F254 silica gel plates (0.25, 0.5, or 1 mm).'H NMR spectra were recorded on Bruker AMX-600 (600 MHz), AMX-500 (500 MHz), or AMX- 400 (400 MHz) spectrometer, and 13C NMR spectra were recorded on a Bruker AMX- 500 (125.7 MHz) or AMX-400 (100.6 MHz) spectrometer. Chemical shifts are reported in parts per million (ppm) on the 8 scale from an internal standard. Mass spectra were recorded on a VG ZAB-VSE instrument.

[00451 The synthesis of constrained nicotine analogues 1 and 2 proceeded using slight modifications of the literature procedures (Glassco et al., J. Med Chem.

1993, 36, 3381-3385; Chavdarian et al., J. Org. Chem., 48, 492-494) as follows: a) The oxidation of tetrahydroisoquinoline was performed using an elevated reaction temperature (40 °C) ; b) Instead of Kugelrohr distillation for purification of intermediates, flash chromatography was used, providing better yields than those reported; c) The low- yield Michael addition was improved upon using multiple equivalents (4-5 eq) of nitroethylene. Compounds 1 and 2 were purified using flash chromatography with a mixture of CHCl3/MeOH/NH40H (85: 15: 1.5). For the preparation of the constrained haptens CNA and CNI, the linker was synthesized as we previously reported (Isomura et al., J. Org. Chem., 66, 4115-4121).

10046] N- [6- (2, 3,3a, 4,5, 9b-hexahydro-IH-pyrrolo [3, 2-blisoquinolin-l- yl) hexanoyl]-ß-alanine (CNA).

[0047] A solution of 3 (340 mg, 1.0 mmol) in acetonitrile (0.5 mL) was added to a mixture of 1 (70 mg, 0.4 mmol) and diisopropylethylamine (DEA) (0.104 mL, 0.6 mmol) in acetonitrile (0.85 mL) with stirring at room temperature. After 18 h, the mixture was evaporated and the residue was purified by preparative TLC with CH2Ck/MeOH (19 : 1) to give CNA-ethyl ester as a pale yellow oil (80 mg, 51% yield).'H-NMR (500 MHz, CDC13) : S 8.44 (d + s, 2H), 7.14 (d, J= 4.8 Hz, 1H), 6.28 (br s, 1H), 4.14 (q, J = 7.2 Hz, 2H), 3.50-3. 45 (m, 3H), 3.23 (t, J= 8.2 Hz, 1H), 3.1 (m, 1H), 2. 57 (t, J= 5.9 Hz, 2H), 2.3 (m, 1H), 2.11 (t, J = 7.2 Hz, 2H), 2.02 (m, 2H), 1.9-1. 5 (m, 6H), 1.29-1. 23 (m, 2H). 13C NMR (500 MHz, CDC13): 8 173. 2,173. 0,151. 6,147. 9,147. 2,133. 3,123. 7,100. 0,61. 8,54. 8,53. 6,52. 0,43. 0, 36.3, 3$. 6, 35. 1,34. 3,29. 4,26. 8,26. 6,25. 0,14. 3,12. 2.

10048] CNA-ethyl ester (50 mg, 0.13 mmol) was dissolved in MeOH (2 mL) and 1 N NaOH (0.26 mL, 0.26 mmol) was added. After 60 min, the solution was concentrated in vacuo and water (5 mL) was added. The aqueous solution was acidified to pH 2 and extracted with CH2CI2 and EtOAc. The organic solvent was evaporated and the residue was purified by preparative TLC with CHCI3lMeOH (5: 1) to give the CNA hapten as a pale yellow oil (41 mg, 87% yield). H NMR (500 MHz, MeOD) : 8 8.36 (s, 1H), 8. 33 (br d, 1H), 7.24 (d, J = 4.8 Hz, 1H), 3.73 (d, J = 8.4 Hz, 1H), 3.33-3. 25 (m, 3H), 2.95-2. 90 (m, 2H), 2.75-2. 45 (m, 4H), 2.50-2. 43 (m, 1H), 2.30-2. 20 (m, 3H), 2.08 (t, J = 7.2 Hz, 2H), 1.86-1. 76 (m, 2H), 1.65-1. 45 (m, 4H), 1.30-1. 22 (m, 2H). 13C NMR (500 MHz, MeOD) : 8 160.1, 148.6, 139.2, 122.3, 100.0, 67.2, 54.3, 51.9, 36.8, 35.8, 35.7, 29.4, 28.7, 26.6, 26.2, 26.1, 25.1. HRMS (MALDI-FTMS) : calculated for C20H29N303 (Mt) 360.2282, found 360.2279. l0049] N- [6- (2,3, 3a, 4,5, 9b-hexahydro-1H-pyrrolol2, 3-0quinolin-1- yl) hexanoyl]-p-alanine (CNI).

[0050] A solution of 3 (340 mg, 1.0 mmol) in acetonitrile (500 mL) was added to a mixture of 2 (70 mg, 0.4 mmol) and diisopropylethylamine (DIEA) (0.104 mL, 0.6 mmol) in acetonitrile (0.85 mL) with stirring at room temperature. After 18 h, the mixture was evaporated and the residue was purified by preparative TLC with CH2Cl2/MeOH (19: 1) to give CNI-ethyl ester as a pale yellow oil (87 mg, 55% yield).'H NMR (400 MHz, CDC13) : 8 8.41 (d, J = 4.1 Hz, 2H), 7.5 (br s, 1H), 7.12 (t, J = 4.1 Hz, 1H), 6.07 (br s, 1H), 4.14 (q, J = 7.2 Hz, 2H), 3.49-3. 45 (q, J= 7.0 Hz, 2H), 3.4 (m, 1H), 3.17 (t, J = 8.2 Hz, 1H), 3.1 (m, 1H), 2.76 (m, 1H), 2. 6-2.5 (m, 4H), 2.3-2. 1 (m, 5H), 2.02 (m, 2H), 1.90-1. 65 (m, 3H), 1.55- 1.40 (m, 4H) 1. 30-1. 20 (m +1, 5H). 13C NMR (500 MHz, CDC13) : S 173. 3,173. 0,161. 6, 148.3, 138.2, 132.3, 121.9, 100.0, 61.2, 55.0, 53.8, 52.2, 43.2, 36.9, 36.5, 36.0, 35.3, 35.1, 34.5, 30.7, 29.7, 26.9, 25.2, 14.6, 12.5.

[0051] CNI-ethyl ester (50 mg, 0.13 mmol) was dissolved in MeOH (2 mL) and 1 N NaOH (0.26 mL, 0.26 mmol) was added. After 60 min, the solution was concentrated in vacuo and water (5 mL) was added. The aqueous solution was acidified to pH 2 and extracted with CH2CI2 and EtOAc. The organic solvent was evaporated and the residue was purified by preparative TLC with CHC13/MeOH (5: 1) to give the CNI hapten as a pale yellow oil (38 mg, 81 % yield). H NMR (500 MHz, MeOD) : 8 8.43 (d, J = 4.8 Hz, 1H), 7.80 (d, J = 7.2 Hz, 1H), 7. 38 (dd, J = 4.8, 7.2 Hz, 1H), 3.67 (d, J = 8.4 Hz, 1H), 3.46-3. 32 (m, 3H), 3.15-3. 09 (m, 1H), 2.95-2. 90 (m, 2H), 2.85-2. 70 (m, 2H), 2.51-2. 47 (m, 2H), 2.40 (t, J= 6.0 Hz, 2H), 2.27-2. 20 (m, 3H), 1.97-1. 93 (m, 1H), 1.86-1. 76 (m, 2H), 1.69-1. 60 (m, 2H), 1.58-1. 50 (m, 1H), 1.40-1. 32 (m, 2H). I3C NE (500 MHz, MeOD) : 8 160. 0,148. 6,139. 7, 122.2, 67.2, 54.2, 51.9, 36.6, 35.8, 35.7, 29.2, 28.7, 26.7, 26.5, 26.2, 25.1. HRMS (MALDI- FTMS) : calculated for C2oH29N303 (MH) 360.2282, found 360.2288.

[0052] Immunoconjugates. For each hapten, KLH and BSA conjugates were prepared. CNA and CNI were activated for 24 h at 4 °C using a standard EDC/sulfo-NHS (1.3 eq. each) coupling procedure in DMF/water (90/10). A 100-fold excess of the activated haptens were then added to the proteins (5 mg/mL) in 50 mM phosphate buffer, pH 7.5 and allowed to stand for 24 h at 4 °C. Coupling levels were determined for BSA conjugates and afforded 16 CNA and 13 CNI molecules per molecule of BSA, respectively, as determined by MALDI-TOF.

[0053] Active immunization protocols. 129G1XF mice (6-8 weeks, 23-28 g) were immunized i. p. on days 1,7, and 14 with a suspension (0.2 mL) of either CNA-KLH, CNI- KLH or NIC-KLH (0.1 mg) in phosphate buffered saline (PBS) with RIBI adjuvant. On day 21, serum (0.1 mL) was collected via retroorbital puncture and titers were measured by ELISA (see below).

[0054] ELISA of serum titers. CNA-BSA, CNI-BSA, NIC-BSA and a KLH control (0.1 mL, 0.1 g/mL) were added to 96-well microtiter plates (Fischer Biotech) and allowed to stand for 2 h at room temperature. The plates were washed with PBS and then milk solution (1% w/v in PBS, 0.1 mL) was added and allowed to stand at room temperature for 2 h. The plates were washed with PBS and the mouse serum (serially diluted with PBS) was added (50 tL) and allowed to stand overnight at 4 °C.

[0055] The plates were washed with PBS and a goat anti-mouse horseradish peroxidase conjugate (0.01 ug, 50 pL) was added and incubated at 37 °C for 2 h. The plates were washed and substrate solution (50 uL) 3,3', 5, 5'-tetramethylbenzidine [ (0. 1 mg in 10 mL of 0.1 M sodium acetate, pH 6.0 and hydrogen peroxide (0.01% w/v) ] was added.

The plates were developed in the dark for 30 min. Sulfuric acid (1.0 M, 50 pL) was added to quench the reaction and the OD was measured at 450 nm. The reported titer is the serum dilution that corresponds to 50% of the maximum OD.

[0056] Competition ELISA was performed using NIC-BSA as the plate antigen with serum dilutions ranging from 1: 800 (CNA) to 1: 6400 (NIC) and varying concentrations of competing agent (nicotine, cotinine, acetylcholine or N- methylpyrrolidine) were added. Competing agents were compared with respect to the concentration of agent that reduced serum affinity to the plate bound antigen by 50%.

The results are shown in FIG. 2 for the experiment that compares serum specificities for nicotine with those for cotinine.

[0057] Equilibrium dialysis. The experiments were performed using ['H]- nicotine as ligand and four high titer serum samples from mice immunized with the following immunoconjugates : CNA-KLH, CNI-KLH or NIC-KLH and a conjugate based on nomicotine coupled via glutaric anhydride, TD1-KLH. Serum samples were diluted 1 : 50 (NIC-KLH) or 1: 100 (the other samples) in PBS and added to 10 wells in a 96-well micro titer plate (60 0 well). Wells (10 per sample) in a second microtiter plate were filled (40 p1L) with serially diluted [3H]-nicotine in PBS, starting with 10 I1M and 20 zL PBS was added to each well. The two plates were very tightly connected with filled wells facing each other and separated with a dialysis membrane (cutoff 6000-8000 Da).

The plates were attached vertically to a shaker and were shaken at high frequency for 6-10 h at 4 °C after which they were carefully separated. The membrane was discarded and 50 iL from each well was transferred to a scintillation vial containing 5 mL of scintillation fluid. The samples were counted for 5 min. The experiment was repeated twice for each serum sample. The average in differences in DPM between partnered wells was determined for each concentration of [3H]-nicotine and the Kd-avg value calculated.

These results are shown in FIG. 3.

[0058] Results of the equilibrium dialysis assays; Columns B, D, F and H in FIG. 3 show the dpm counts in wells without antiserum, whereas columns C, E, G and I in FIG. 3 show the corresponding counts for wells with antiserum added. The differences are also shown in columns B-E and they are plotted against log [3H-nicotine]. The Kd-avg is calculated by dividing the estimated saturation interval by 2 and determining the corresponding nicotine concentrations for those values.

[0059] The examples set forth above are provided to give those of ordinary skill in the art with a complete disclosure and description of how to make and use the preferred embodiments of the compositions, and are not intended to limit the scope of what the inventors regard as their invention. Modifications of the above-described modes for carrying out the invention that are obvious to persons of skill in the art are intended to be within the scope of the following claims. All publications, patents, and patent applications cited in this specification are incorporated herein by reference as if each such publication, patent or patent application were specifically and individually indicated to be incorporated herein by reference.