[go: up one dir, main page]

WO2002058635A2 - Immunogenes a la nicotine, anticorps et leurs applications - Google Patents

Immunogenes a la nicotine, anticorps et leurs applications Download PDF

Info

Publication number
WO2002058635A2
WO2002058635A2 PCT/US2002/002146 US0202146W WO02058635A2 WO 2002058635 A2 WO2002058635 A2 WO 2002058635A2 US 0202146 W US0202146 W US 0202146W WO 02058635 A2 WO02058635 A2 WO 02058635A2
Authority
WO
WIPO (PCT)
Prior art keywords
nicotine
compound
immunogen
mmol
antibody
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2002/002146
Other languages
English (en)
Other versions
WO2002058635A3 (fr
Inventor
Kim D. Janda
Peter Wirsching
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Scripps Research Institute
Original Assignee
Scripps Research Institute
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Scripps Research Institute filed Critical Scripps Research Institute
Priority to AU2002251821A priority Critical patent/AU2002251821A1/en
Publication of WO2002058635A2 publication Critical patent/WO2002058635A2/fr
Publication of WO2002058635A3 publication Critical patent/WO2002058635A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/18—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having one double bond between ring members or between a ring member and a non-ring member
    • C07D207/22—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having one double bond between ring members or between a ring member and a non-ring member with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D207/24—Oxygen or sulfur atoms
    • C07D207/26—2-Pyrrolidones
    • C07D207/263—2-Pyrrolidones with only hydrogen atoms or radicals containing only hydrogen and carbon atoms directly attached to other ring carbon atoms
    • C07D207/27—2-Pyrrolidones with only hydrogen atoms or radicals containing only hydrogen and carbon atoms directly attached to other ring carbon atoms with substituted hydrocarbon radicals directly attached to the ring nitrogen atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/08—Antiepileptics; Anticonvulsants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/04—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D207/06—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with radicals, containing only hydrogen and carbon atoms, attached to ring carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/16—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from plants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • the field of this invention is nicotine addiction. More particularly, this invention pertains to nicotine immunogens, anti-nicotine antibodies and the use of such immunogens and antibodies for treating nicotine addiction.
  • Nicotine is the most widely used addictive drug in the world.
  • 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.
  • the present invention provides a nicotine immunogen.
  • the immunogen has the structure
  • R 1 0 or H
  • R 2 is H, CH 3 or (CH 2 ) n CONH(CH 2 ) 2 COOH
  • R 3 is H or
  • n is an integer from 1 to 10.
  • R is H
  • R is (CH 2 ) n CONH(CH 2 ) 2 COOH
  • R 3 is pyridine.
  • n is from 3 to 7 and, more preferably 5 or 6.
  • the immunogen is linked to an immunogenic carrier such as keyhole limpet hemocyanin or bovine serum albumin.
  • this invention provides a method for immunizing an animal against nicotine.
  • an animal is administered an effective immunogenic amount of the immunogen of this invention.
  • this invention provides an antibody that immunoreacts with the immunogen of this invention.
  • the antibody also immunoreacts with nicotine.
  • the antibody is a monoclonal antibody such as designated herein 3G3, 6C12, 13A3, 1B10, 5E8 or 9D9.
  • 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.
  • the invention further provides a pharmaceutical composition containing a subject immunogen or antibody together with a physiological diluent.
  • FIG. 1 shows the structure of nicotine related tobacco components and their metabolites.
  • FIG. 2 shows nicotine haptens
  • FIG. 3 shows a synthetic scheme for making a (S)-nicotine hapten: a) ethanol, cone. H 2 S0 4 , reflux; b) i) NaH, THF, 1 -vinyl-2-pyrrolidinone, reflux, ii) aq. HC1, reflux, iii) aq.
  • FIG. 4 shows a synthetic scheme for making racemic, and (S)- and (R)-nornicotine haptens: a) 20, DIEA, acetonitrile; b) H 2 , Pd/C, MeOH; c) benzyl alcohol, p-TsOH, cyclohexane; d) Nal, acetonitrile.
  • FIG. 5 shows a synthetic scheme for making a (S)-cotinine hapten: a) 27, DIEA, acetonitrile; b) Hg(OAc) 2 , aq. EDTA, pH 9, dioxane, reflux; c) i) TFA, CH 2 C1 2) ii) 29, HBTU, N-methylmorpholine, DMF; d) H 2 , Pd/C, MeOH; e) di-t-butyldicarbonate, MeOH; f) i) methanesulfonyl chloride, NEt 3 , CH 2 C1 2 , ii) Nal, acetonitrile; g) benzyl alcohol, DMAP, CH 2 C1 2 .
  • FIG. 6 shows locomotor activity data for rats actively immunized with nicotine immunogen and challenged with nicotine.
  • FIG. 7 shows locomotor activity data for rats passively immunized with mAb NIC9D9 and challenged with nicotine. Detailed Description of the Invention
  • 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.
  • Nicotine is a small, haptenic molecule and requires coupling to an immunogenic carrier protein to elicit an immune response.
  • an immunogenic carrier protein to elicit an immune response.
  • a number of reports appeared in the literature that described nicotine haptens and immunoconjugates [Abad, A.;
  • 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.
  • an optimal immunogen i.e., hapten
  • nicotinic compounds useful in the design of immunogens are shown in FIG. 1. Those compounds are (S)-(-)- nicotine, Compound 1, the structurally related nornicotine, Compound 2, present in tobacco and a metabolite, cotinine, Compound 3, the major primary metabolite of nicotine, and N- methylpyrrolidine, Compound 4, a minor tobacco component.
  • the general approach to designing nicotine immunogens is to maintain unchanged the significant structural and stereochemical features of nicotine as it occurs in tobacco. If possible, no additional functionality is imparted to the immunogen that is not in nicotine. Any changes that are made should be weakly immunogenic and a linker of appropriate length is attached in a position to present all important recognition elements.
  • implementation of the above strategy first requires an immunogen with the natural (S)-configuration. Although an immune response would be expected against both antipodes of a racemic immunogen, stereospecificity is likely enhanced using the single (S)- antipode. Only an immune response against (S)-(-)-nicotine. (R)-(+)-nicotine does not have significant pharmacological activity. Therefore, it is not necessary to bind any trace amounts of this isomer during therapy. Consequently, avoidance of even a few percent of cross- reactivity with this isomer would benefit binding of the desired target (S)-(-)-nicotine.
  • the immunogen should contain an alkyl linker at the position of the methyl group on the pyrrolidine nitrogen. In this way, all the characteristics of nicotine remain intact for display to the immune system: stereochemistry, two unaltered rings, charge, and the important pyrrolidyl methyl group mimicked as part of an alkyl linker.
  • the length and chemical composition of the linker are selected in order to afford recognition of the nicotine framework in its entirety.
  • an immunogen of this invention corresponds to formula I, below.
  • R 1 0 or H
  • R ? is a linker moiety and R ⁇ is H or a 5- or 6-membered aromatic.
  • a preferred linker moiety is (CH 2 ) n CONH(CH 2 ) 2 COOH or CO (CH 2 ) n NH(CH 2 ) 2 COOH where n is an integer from 1 to 10.
  • Exemplary and preferred aromatics are phenyl, pyridine, and other six membered aromatics.
  • haptens employed by others were derived from 2-aminonicotine (Compound A), 6-aminonicotine (Compound B), and trans-3'- hydroxymethylnicotine (Compound C), all as shown in FIG. 2. Further, all prior haptens were racemic compounds.
  • Compound 4 a constituent of tobacco smoke [Langone, J. J.; Gjika, H. B.; Van Vunakis, H., Biochem. 12, 5025-5030 (1973)].
  • racemic Compound B conjugated using a diazotized p-aminobenzoyl linker afforded antisera highly specific for natural (S)-(-)-nicotine with only 5% cross-reaction of the (R)-(+)-isomer [Matsushita, H.; Noguchi, M.; Tamaki, E. Biochem. Biophys. Res. Commun. 57, 1006-1010 (1974)]. While these data might be correct and even rather favorable, such a result is difficult to readily explain. Langone et al. prepared nicotine antisera and mAbs derived from Compound C, perhaps the most widely recognized, studied and accepted hapten for anti- nicotine antibody production [Bjercke, R.
  • a preferred immunogen is Compound 10 (NIC), which was synthesized with high enantiomeric excess.
  • NIC Compound 10
  • a literature procedure was used to prepare nomicotine with the (S)-(-) stereochemistry corresponding to natural nicotine in >98% optical purity [Jacob, P. III., J. Org. Chem. 47, 4165-4167 (1982)].
  • KLH keyhole limpet hemocyanin
  • BSA bovine serum albumin
  • 5-bromomyosmine Compound 7 occurred via the base-mediated condensation of ethyl 5-bromonicotinate Compound 6 with N- vinyl -pyrrolidinone, followed by in situ acid-catalyzed hydrolysis of the ⁇ -keto-/V-vinyllactam, decarboxylation, and cyclization.
  • the isolated imine was then reduced with sodium borohydride to afford 5-bromonornicotine Compound 8.
  • Nomicotine Compound 2b was then obtained with the (S)-(-) stereochemistry in >98% ee via a classical resolution of the racemic Compound 8.
  • the benzylic-like C-N bond adjacent to the pyridyl ring was sufficiently labile to compete with hydrogenolysis of the benzyl ester C-0 bond.
  • the reaction conditions were different and incorporated triethylamine. Since we omitted a base in the reaction of Compound 9 in order to avoid salt formation of the NIC hapten, perhaps the more acidic environment, especially after benzyl ester hydrogenolysis, activated the C-N bond towards cleavage.
  • Nomicotine 2 occurs in tobacco as a mixture of (R)- and (S)-enantiomeric forms.
  • the compound is pharmacologically active in the central nervous system similar to nicotine [Jacob, P., JJJ; Yu, L.; Liang, G.; Shulgin, A. T.;
  • Dwoskin L. P. Biochem. Pharmacol. 54, 743-753 (1996); Dwoskin, L. P.; Teng, L.; Buxton, S. T.; Ravard, A.; Deo, N.; Crooks, P. A. Eur. J. Pharmacol. 276, 195-199 (1995); Dwoskin,
  • Nomicotine also accounts for -8% of metabolized nicotine in both humans and rats and has a plasma half- life of about eight hours which is longer than the average two-hour half-life of nicotine [Curvall, M.; Kazeni, V. E. In Nicotine and Related Alkaloids: Absorption, Distribution,
  • the hapten Compound 16a (code: NOC) was synthesized starting from rac- nomicotine Compound 2a (Fig. 3). However, preliminary tests indicated the absence of a viable immune response against the hapten in accord with previous hypotheses. Therefore, separate (S)-NOC and (R)-NOC haptens were prepared in order to obtain antibodies specific for each isomer of nomicotine. Hence, linker Compound 20 was introduced onto the nomicotine framework using reaction chemistry similar to that employed for the preparation of Compound 9. Hydrogenation of Compound 16a-c afforded NOC haptens which required purification, again due to the formation of a ring-opened product analogous to Compound , 15.
  • the "tuning" of the linker length should be critical. We anticipate that the shorter and less flexible linker will afford a substantial fraction of antibodies that recognize only the pyridyl and pyrrolidyl rings and not the alkyl-chain region. Consequently, binding should be more specific for nomicotine rather than the nicotine structure bearing a methylated nitrogen.
  • Cotinine Compound 3 accounts for 70-80% of metabolized nicotine and has a long half-life of 16-20 hours [Jacob, P., HI; Benowitz, N. L. Clin. Pharmacol. Ther. 56, 483-493 (1994); Jacob, P., Ill; Benowitz, N. L.; Shulgin, A. T. Pharmacol. Biochem. Behav. 30, 249- 253 (1988); Benowitz, N. L.; Kuyt, F.; Jacob, P., HI; Jones, R. T.; Osman, A. L. Clin. Pharmacol. Ther. 34, 604-611 (1983); Benowitz, N. L.; Hall, S. M.; He ing, R. I.
  • cotinine levels can average 15-fold higher than nicotine during smoking or nicotine replacement therapy [Jacob, P., HI; Benowitz, N. L. Clin. Pharmacol. Ther. 56, 483-493 (1994)].
  • cotinine also 1) has an effect on nicotinic cholinergic receptors in vitro, 2) influences release of neurotransmitters, 3) inhibits androgen biosynthesis, and 4) possibly contributes to the lower blood pressure of smokers during nonsmoking intervals [Dwoskin, L. P.; Teng, L.; Buxton, S. T.; Crooks, P. A. J. Pharmacol. Exp.
  • the (S)-hapten Compound 24 (code: COT) was synthesized starting from Compound 2b (Fig. 4).
  • the central transformation was the Wenkert oxidation of the amine Compound 21 using conditions similar to those in the literature for the conversion of nicotine to cotinine [Wenkert, E.; Angell, E. C. Synth. Commun. 18, 1331-1337 (1988)].
  • the stereochemical integrity should not be effected, since no racemization occurs in this reaction.
  • 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.
  • KLH keyhole limpet hemocyanin
  • edestin edestin
  • thyroglobulin albumins
  • 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 toxo
  • 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.
  • 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. HI. Anti-nicotine Antibodies
  • 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.
  • 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.
  • NIC-KLH Compound 10-KLH
  • NIC-BSA Compound 10- BSA
  • the NIC-KLH was also used in rat behavioral models to study the efficacy of vaccination protocols against nicotine addiction.
  • NIC-KLH was used to immunize mice in a highly consistent fashion and thereby produce both a quality, reproducible immune response as well as mAbs with excellent affinity and specificity for nicotine. All of the mAbs were fully characterized with respect to purity, isotype, and hybridoma production yields.
  • this hapten or its immunoconjugates, might not be conducive for achieving consistent titers which would be a drawback for therapeutic purposes.
  • our anti-nicotine mAbs had lower affinities, the specificity for nicotine relative to nomicotine and cotinine was improved by several-fold compared to the Langone mAbs which lends support to aspects of our hapten design. Consequently, 2, 3, and 4 should not compete with nicotine for antibody binding sites and therefore would not pose a problem during treatment.
  • the murine mAbs can be "humanized” via several techniques well known in the art [James, K. Human monoclonal antibodies.
  • 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 (FIG. 6).
  • the initial pre-immunization nicotine injections resulted in the classic inhibitory effect on locomotor activity induced by acute treatment of the dmg (Fig. 6A). With repeated administrations of nicotine, tolerance to the initial depressant effect developed, and by the sixth nicotine injection, the activating effects of the dmg were observed (Fig. 6B).
  • the harvested cells (generally 15-20 ml) are centrifuged at 1500 rpm for 10 min.
  • the supernatant is filtered through a 0.22 ⁇ m filter and purified through a protein G column.
  • the concentration of antibody in the cell supernatant is usually -1-2 mg/ml.
  • 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.
  • 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.
  • physiological effects such as nausea, dizziness, gastric upset and the like.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 amino 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 HC1.
  • Physiologically tolerable carriers are well known in the art.
  • Exemplary of 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.
  • 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.
  • EXAMPLE 2 Ethyl 5-bromonicotinate Compound 6.
  • EXAMPLE 5 Resolution of racemic 5-bromonornicotine Compound 8 via the ⁇ - methoxy- ⁇ -(trifluoromethyl) phenylacetate (MTPA) salts.
  • a solution of (-)-MTPA (3.9 g, 16.7 mmol) in EtOAc (13 mL) was added to a solution of Compound 8 (7.57 g, 33.3 mmol) in EtOAc (52 mL) with stirring. The mixture was allowed to stand at r.t. for 15 min, after which time the crystalline product was collected by filtration to give (R)-isomer enriched crystals.
  • MTPA ⁇ - methoxy- ⁇ -(trifluoromethyl) phenylacetate
  • EXAMPLE 7 N-[l-oxo-6-[(2S)-2-(3-pyridinyl)-l-pyrrolidinyl]hexyl]- ⁇ -aIanine phenylmethyl ester Compound 9.
  • a solution of Compound 14 (136 mg, 0.338 mmol) in acetonitrile (500 ⁇ L) was added to a mixture of Compound 2b (52.1 mg, 0.338 mmol) and diisopropylethylamine (DIEA) (117 ⁇ L, 0.676 mmol) in acetonitrile (850 ⁇ L) with stirring at r.t.
  • DIEA diisopropylethylamine
  • EXAMPLE 8 /V-[l-oxo-6-[(2S)-2-(3-pyridinyl)-l-pyrrolidinyl]hexyl]- ⁇ -alanine Compound 10 (NIC).
  • NIC benzylester Compound 9
  • MeOH MeOH
  • Pd/C 10% Pd/C
  • the filtrate was evaporated and the residue purified by chromatography on silica gel eluting with CH 2 Cl 2 /MeOH (3:2) to give hapten Compound 10 as a pale yellow oil (26 mg, 66% yield).
  • EXAMPLE 11 ⁇ c-2-(3-pyridinyl)-l-pyrrolidinebutanoic acid phenylmethyl ester Compound 16a.
  • EXAMPLE 13 Benzyl 4-bromobutanoate Compound 19.
  • a solution of 4- bromobutanoic acid Compound 18 (25 g, 0.15 mol), benzyl alcohol (21 g, 0.194 mol), andp- TsOH hydrate (1.3 g, 6.87 mmol) in cyclohexane (225 ml) was heated to reflux.
  • the water was azeotropically removed with the aid of a Dean-Stark trap. After one hr, the stoichiometric amount of water was collected and the solution refluxed an additional hr. After cooling to r.t., the solution was washed with sat. sodium bicarbonate, brine, dried over Na 2 S0 and evaporated.
  • EXAMPLE 15 [6-[(2S)-2-(3-pyridinyl)-l-pyrrolidinyl]hexyl]-carbamic acid 1,1- dimethylethyl ester Compound 21.
  • a solution of Compound 27 (425 mg, 1.30 mmol) in acetonitrile (2 mL) was added to a mixture of Compound 2b (200 mg, 1.35 mmol) and DIEA (470 ⁇ L, 2.70 mmol) in acetonitrile (4 mL) with stirring at r.t.
  • 1,1-dimethyIethyl ester Compound 22 A solution of Compound 21 (265 mg, 0.764 mmol) in dioxane (30 mL) was added to a mixture of Hg(OAc) 2 (1.22 g, 3.82 mmol) and aqueous EDTA (7.66 mL, 5 mmol/10 mL, pH 9.0) in water (39 mL) and the mixture refluxed with stirring for 2 hr. After cooling, the mixture was filtered through celite and the filter cake washed with a small amount of dioxane. The filtrate was extracted with CH 2 C1 2 and the organic layer washed with brine, dried over Na 2 S0 4 and evaporated.
  • EXAMPLE 17 4-oxo-4-[[6-[(5S)-2-oxo-5-(3-pyridinyl)-l- pyrrolidinyl]]hexyl]amino]-butanoic acid phenylmethyl ester Compound 23.
  • a solution of Compound 22 (91.7 mg, 0.254 mmol) in CH 2 C1 2 (500 ⁇ L) was treated with TFA (400 ⁇ L) at 0 °C. After completion, the mixture was evaporated with toluene (3 times).
  • HBTU 111 mg, 0.292 mmol
  • EXAMPLE 18 4-oxo-4-[[6-[(55)-2-oxo-5-(3-pyridinyl)-l- pyrroIidinyl]]hexyl]amino]-butanoic acid Compound 24 (COT).
  • the benzyl ester Compound 23 (77.0 mg, 0.171 mmol) in MeOH (2 mL) was hydrogenated with 10% Pd/C (15 mg) using a balloon technique. After 40 min, Pd/C was removed by filtration washing with MeOH.
  • EXAMPLE 21 Succinic acid monobenzyl ester Compound 29.
  • a mixture of succinic anhydride Compound 28 (1.00 g, 10.0 mmol), benzyl alcohol (860 ⁇ L, 8.30 mmol) and DMAP (1.02 g, 8.30 mmol) in CH 2 C1 2 (30 mL) was stirred at r.t. for 18 hr. After this time, a solution of 5% Na 2 C ⁇ 3 was poured into the reaction mixture and the layers separated. The aqueous layer was acidified with 1 M HC1 and extracted with EtOAc.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Neurosurgery (AREA)
  • Public Health (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Engineering & Computer Science (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pain & Pain Management (AREA)
  • Neurology (AREA)
  • Veterinary Medicine (AREA)
  • Botany (AREA)
  • Immunology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention se rapporte à des immunogènes utiles dans la production d'anticorps anti-nicotine. Lesdits anticorps sont immunoréactifs vis-à-vis des immunogènes et de la nicotine. Font également l'objet de cette invention les immunogènes et les anticorps utilisés dans le traitement du tabagisme.
PCT/US2002/002146 2001-01-26 2002-01-25 Immunogenes a la nicotine, anticorps et leurs applications Ceased WO2002058635A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2002251821A AU2002251821A1 (en) 2001-01-26 2002-01-25 Nicotine immunogens and antibodies and uses thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US26457501P 2001-01-26 2001-01-26
US60/264,575 2001-01-26

Publications (2)

Publication Number Publication Date
WO2002058635A2 true WO2002058635A2 (fr) 2002-08-01
WO2002058635A3 WO2002058635A3 (fr) 2003-03-20

Family

ID=23006669

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2002/002146 Ceased WO2002058635A2 (fr) 2001-01-26 2002-01-25 Immunogenes a la nicotine, anticorps et leurs applications

Country Status (2)

Country Link
AU (1) AU2002251821A1 (fr)
WO (1) WO2002058635A2 (fr)

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6932971B2 (en) 2002-07-18 2005-08-23 Cytos Biotechnology Ag Hapten-carrier conjugates and uses thereof
WO2009068335A1 (fr) * 2007-11-29 2009-06-04 Cytos Biotechnology Ag Anticorps monoclonaux humains spécifiques à la nicotine
WO2011151807A1 (fr) 2010-06-04 2011-12-08 Pfizer Vaccines Llc Conjugués pour la prévention ou le traitement de la dépendance à la nicotine
US8344111B2 (en) 2007-11-29 2013-01-01 Cytos Biotechnology Ag Human monoclonal nicotine specific antibodies
US20150056251A1 (en) * 2012-02-28 2015-02-26 Cornell University Aav-directed persistent expression of an anti-nicotine antibody gene for smoking cessation
US20150297739A1 (en) * 2012-04-13 2015-10-22 Cornell University Development of a highly efficient second generation nicotine-conjugate vaccine to treat nicotine addiction
WO2015173685A1 (fr) 2014-05-16 2015-11-19 Pfizer Inc. Conjugués utilisables en vue de la prévention ou du traitement de la dépendance à la nicotine et leurs procédés de production
WO2019036419A1 (fr) 2017-08-15 2019-02-21 Kalnik Matthew W Nouveaux anticorps de liaison à la nicotine
WO2019236665A1 (fr) 2018-06-06 2019-12-12 Antidote Therapeutics, Inc. Composition permettant d'améliorer la circulation et de traiter une maladie cardiovasculaire
WO2022111564A1 (fr) * 2020-11-27 2022-06-02 海南凯菲士企业管理合伙企业（有限合伙） Procédé de racémisation et d'utilisation d'un dérivé de pyridine

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993023076A1 (fr) * 1992-05-20 1993-11-25 The Johns-Hopkins University Therapie utilisant des recepteurs de substitution
US5876727A (en) * 1995-03-31 1999-03-02 Immulogic Pharmaceutical Corporation Hapten-carrier conjugates for use in drug-abuse therapy and methods for preparation of same
SE9801923D0 (sv) * 1998-05-29 1998-05-29 Independent Pharmaceutical Ab Nicotine vaccine
US6232082B1 (en) * 1998-12-01 2001-05-15 Nabi Hapten-carrier conjugates for treating and preventing nicotine addiction

Cited By (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6932971B2 (en) 2002-07-18 2005-08-23 Cytos Biotechnology Ag Hapten-carrier conjugates and uses thereof
US7452541B2 (en) 2002-07-18 2008-11-18 Cytos Biotechnology Ag Hapten-carrier conjugates and uses thereof
EP2392345A2 (fr) 2002-07-18 2011-12-07 Cytos Biotechnology AG Conjugués vecteurs d'haptène comportant des particules de type viral et utilisations associées
US8187607B2 (en) 2002-07-18 2012-05-29 Cytos Biotechnology Ag Hapten-carrier conjugates and uses thereof
WO2009068335A1 (fr) * 2007-11-29 2009-06-04 Cytos Biotechnology Ag Anticorps monoclonaux humains spécifiques à la nicotine
CN101878228A (zh) * 2007-11-29 2010-11-03 赛托斯生物技术公司 尼古丁特异性人单克隆抗体
EP2319867A1 (fr) * 2007-11-29 2011-05-11 Cytos Biotechnology AG Anticorps spécifiques à la nicotine monoclonale humaine
US8344111B2 (en) 2007-11-29 2013-01-01 Cytos Biotechnology Ag Human monoclonal nicotine specific antibodies
WO2011151807A1 (fr) 2010-06-04 2011-12-08 Pfizer Vaccines Llc Conjugués pour la prévention ou le traitement de la dépendance à la nicotine
US10093947B2 (en) * 2012-02-28 2018-10-09 Cornell University AAV-directed persistent expression of an anti-nicotine antibody gene for smoking cessation
US20150056251A1 (en) * 2012-02-28 2015-02-26 Cornell University Aav-directed persistent expression of an anti-nicotine antibody gene for smoking cessation
US20150297739A1 (en) * 2012-04-13 2015-10-22 Cornell University Development of a highly efficient second generation nicotine-conjugate vaccine to treat nicotine addiction
US10004811B2 (en) * 2012-04-13 2018-06-26 Cornell University Development of a highly efficient second generation nicotine-conjugate vaccine to treat nicotine addiction
WO2015173685A1 (fr) 2014-05-16 2015-11-19 Pfizer Inc. Conjugués utilisables en vue de la prévention ou du traitement de la dépendance à la nicotine et leurs procédés de production
US9303013B2 (en) 2014-05-16 2016-04-05 Pfizer Inc. Conjugates and associated methods of producing them for the prevention or treatment of nicotine addiction
US9475793B2 (en) 2014-05-16 2016-10-25 Pfizer Inc. Conjugates and associated methods of producing them for the prevention or treatment of nicotine addiction
WO2019036419A1 (fr) 2017-08-15 2019-02-21 Kalnik Matthew W Nouveaux anticorps de liaison à la nicotine
CN111432883A (zh) * 2017-08-15 2020-07-17 安蒂多特疗法公司 新型烟碱结合抗体
US11440970B2 (en) 2017-08-15 2022-09-13 Antidote Therapeutics, Inc. Nicotine-binding antibodies
EP4624496A2 (fr) 2017-08-15 2025-10-01 BliNK Biomedical SAS Nouveaux anticorps se liant à la nicotine
WO2019236665A1 (fr) 2018-06-06 2019-12-12 Antidote Therapeutics, Inc. Composition permettant d'améliorer la circulation et de traiter une maladie cardiovasculaire
WO2022111564A1 (fr) * 2020-11-27 2022-06-02 海南凯菲士企业管理合伙企业（有限合伙） Procédé de racémisation et d'utilisation d'un dérivé de pyridine

Also Published As

Publication number Publication date
AU2002251821A1 (en) 2002-08-06
WO2002058635A3 (fr) 2003-03-20

Similar Documents

Publication Publication Date Title
Isomura et al. An immunotherapeutic program for the treatment of nicotine addiction: hapten design and synthesis
JP4270753B2 (ja) ニコチン免疫原
JPH08511028A (ja) Ｐ物質拮抗剤としてのベンジルオキシキヌクリジン
US11712467B2 (en) Methods of rational nicotine hapten design and uses thereof
EA007932B1 (ru) Азабициклопроизводные в качестве антагонистов мускариновых рецепторов
WO2002058635A2 (fr) Immunogenes a la nicotine, anticorps et leurs applications
AU2017213534B2 (en) Heroin haptens, immunoconjugates and related uses
US7446205B2 (en) Method for making nicotine hapten
AU1565897A (en) Anti-cocaine vaccine
WO1997021451A9 (fr) Vaccin anti-cocaine
US10487141B2 (en) Enantiopure haptens for nicotine vaccine development
WO2005040338A2 (fr) Immunogenes et anticorps alcaloides contraints et leurs utilisations
EP1849780A1 (fr) Vaccin contre la toxicomanie de nicotine
US20120225087A1 (en) Nicotine haptens, immunoconjugates and their uses
WO2009120954A2 (fr) Immunoconjugués de nicotine

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ OM PH PL PT RO RU SD SE SG SI SK SL TJ TM TN TR TT TZ UA UG US UZ VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP

WWW Wipo information: withdrawn in national office

Country of ref document: JP