Patent Application
20090196886
The present invention provides a vaccine against nicotine addiction. More particularly, the present invention provides a hapten in the form of a novel nicotine derivative that may suitably be conjugated with an appropriate carrier to yield an effective vaccine against nicotine addiction.
According to the World Health Organisation, there are more than a billion smokers worldwide. It is estimated that about four million die each year because of cancer, heart disease and other smoking related illnesses.
It is widely accepted that people continue to smoke because tobacco contains nicotine, which is an addictive chemical. Many smoking cessation strategies, in fact, provide cigarette addicts with nicotine from sources other than tobacco, such as patches or gum.
An alternative cessation strategy is to provide a vaccine that stimulates the immune system to clear nicotine from the system by producing antibodies against nicotine. Thus, if following immunisation with a nicotine vaccine, an individual smokes a cigarette, the antibodies will clear the nicotine from the system before it reaches the brain. As a result, the expected stimulating effect of nicotine will not be experienced or be significantly reduced. Because the smoker will experience a lessening or cessation of the stimulating effect of nicotine, he/she will lose the desire to smoke.
Nicotine vaccines for use in smoking cessation strategies have been described in a number of patent publications:
WO 99/61054, for instance, describes a nicotine immunogen comprising a 5- or 6-nicotinyl-linker-carrier protein.
Furthermore, U.S. Pat. No. 6,232,082 describes nicotine-carrier conjugates in which the carrier is bonded to the nicotine residue in the 3′, 4′ or 5′-position. The haptenes of U.S. Pat. No. 6,232,082 contain a nicotine moiety derivatised at the 3′, 4′ or 5′-position with a (CH2)n-Z moiety, wherein Z is NH2, COOH, CHO or SH. These haptenes are conjugated to the carrier protein using a homobifunctional or a heterobifunctional cross-linker. In formula (I) of this document, X and Y represent the groups of linkage of the cross-linker with the haptene and the carrier respectively. However, the immunogenicity studies described in the U.S. patent made use of complete and incomplete Freund's adjuvant to boost the immune response. This adjuvant is not allowed for use in humans.
WO 2004/09116 describes a method of producing a nicotine-Qβ-conjugate comprising the steps of synthesising the N-hydroxysuccinimide ester of O-succinyl-trans-3′-hydroxymethylnicotine and allowing the nicotine derivative to react with lysines on the surface of Qβ under formation of an amide bond. An important drawback of these conjugates resides in their poor in vivo stability.
WO 02/49667 describes a cotinine conjugate wherein the cotinine is conjugated to a carrier via the 1, 2, 5, 6 or 4′-position. WO 02/49667 discloses a conjugate of a a hapten comprising a cotinine moiety derivatised with a —CO—NH—CH2—CH2—SH group, which hapten is conjugated with ovalbumin using gamma-maleimidobutyryloxy-succinimide ester (GMBS). In the international application it is observed that cotinine can antagonise the effects of nicotine in vivo. Furthermore, it is hypothesised that an immune reaction against cotinine can reduce the dampening effect of cotinine on the nicotine neurological effect, thereby resulting in the subject craving a lesser amount of nicotine. This hypothesis is not plausible, especially not since it is not substantiated by any experimental evidence.
The present inventors have developed a novel hapten that can advantageously be used in the production of highly effective nicotine vaccines.
The hapten according to the present invention is a nicotine derivative of the following formula:
wherein either the five-membered ring or the six-membered ring is substituted with one R—SH radical, wherein R represents: CH2—Y-Z:
The aforementioned nicotine derivatives may be used to produce an immunogenic nicotine-carrier conjugate that can advantageously be used in the therapeutic or prophylactic treatment of nicotine addiction, said treatment comprising administration of the conjugate to an individual suffering from nicotine addiction or being at risk of becoming nicotine addicted. The nicotine-carrier conjugates of the present invention are highly stable and can be stored for several months or even years without significant loss of immunogenic efficacy. Also, in vivo stability of the present conjugates is very high, especially if the nicotine derivative is bound to the conjugate by a succinimide crosslinker and the nicotine derivative and crosslinker are linked by a thioether bond. Furthermore, the present nicotine-carrier conjugates can be produced using not more than a couple of relatively simple synthesis steps, yielding a well-defined conjugate product in high yield.
Accordingly, one aspect of the invention relates to a nicotine derivative of the following formula (I):
wherein either the five-membered ring or the six-membered ring is substituted with one R—SH radical, wherein R represents: CH2—Y-Z:
According to another, particularly preferred embodiment, in formula I, Y represents NH or NH—CO. Most preferably, Y represents NH.
According to further preferred embodiment, Z in formula (I) C1-C4 alkylene, most preferably ethylene.
The R—SH residue in formula (I) may be bonded to the 3′, 4′ or 5′ position of the five-membered ring of the nicotine molecule. Preferably, R is bonded to the 3′- or 4′-position, most preferably to the 3′-position of said five-membered ring.
According to another particularly preferred embodiment, the nicotine derivative of formula (I) is trans-substituted with the R—SH residue. In another particularly preferred embodiment, the nicotine derivative of the present invention is a L-nicotine derivative.
Especially preferred nicotine derivatives of the present invention are 3′-(2-mercaptoethylaminomethyl)nicotine, 3′-(3-mercaptopropionamidomethyl)nicotine and 3′-(mercaptoacetamidomethyl)nicotine.
Another aspect of the present invention relates to a precursor of the nicotine derivative of the present invention, which is represented by the following formula (Ia):
wherein either the five-membered ring or the six-membered ring is substituted with one R—S—CO—R′ moiety, R having the same meaning as defined herein before and wherein R′—CO represents a lower acyl group, preferably a branched or linear acyl group comprising 1 to 8 carbon atoms, more preferably 1 to 6 carbon atoms, still more preferably 1 to 5 carbon atoms. In a particularly preferred embodiment R′—CO in the above formula represents lower acyl selected from formyl, acetyl, propionyl, butyryl, valeryl and pivaloyl, most preferably acetyl and propionyl.
In a particularly preferred embodiment of the invention, said precursor is 3′-(S-acetylmercaptacetamido-methyl)nicotine. These precursors can be easily converted to a corresponding nicotine derivative conjugate of the present invention by S-deacetylation and subsequent conjugation, preferably in a two-step one-pot procedure. Suitable S-deactylation reactions are generally known by the skilled person. In addition, the inventors found that these S-acetylmercapto compounds are rather insensitive to oxidation, i.e. formation of disulfide dimers, and the precursor is much more convenient to handle during purification and storage than the corresponding nicotine derivatives.
Another aspect of the present invention relates to a hapten-carrier conjugate of the following formula (II):
wherein either the five-membered ring or the six-membered ring is substituted with one R—SH radical; p is 1 to 500; and R has the same meaning as defined herein before in relation to formula (I).
The carrier employed in the hapten-carrier conjugate of the present invention may be any immunogenic material that can safely be used in humans and that can be linked chemically to the present nicotine derivative. In a preferred embodiment, the carrier is selected from the group consisting of immunogenic substances, viruses, virus-like particles, protein complexes, proteins, polypeptides, liposomes and immuno-stimulating complexes (ISCOM). According to a particularly preferred embodiment, the carrier is an immunogenic protein or polypeptide. Particularly suitable examples of immunogenic proteins include tetanus toxoid, diphtheria toxoid, keyhole limpet hemocyanin (KLH), hemocyanin, albumine, non-toxic mutant diphtheria toxoid CRM197, outer membrane protein complex (OMPC) from Neisseria meningitidis, the B subunit of heat-labile Escherichia coli, recombinant exoprotein A from Pseudomonas aeruginosa (rEPA) and a virus-like particle assembled from recombinant coat protein of bacteriophage Qb, the most preferred examples being tetanus toxoid, diphtheria toxoid, keyhole limpet hemocyanin (KLH), hemocyanin, albumine, non-toxic mutant diphtheria toxoid CRM197, outer membrane protein complex (OMPC) from Neisseria meningitidis, the B subunit of heat-labile Escherichia coli and a virus-like particle assembled from recombinant coat protein of bacteriophage Qb. In a preferred embodiment of the invention a hapten-carrier conjugate as defined herein is provided, wherein the carrier is a protein and wherein the hapten:protein molar ratio exceeds 1.
The spacer in the present immunoconjugate covalently links the nicotine derivate to the carrier. Spacers that can be used to crosslink haptens and carriers (especially protein/polypeptide components) are well-known in the art. Preferably, in the present invention, the spacer is a residue of a heterobifunctional crosslinker, notably a succinimidyl crosslinker, like:
A further aspect of the present invention relates to the use of a hapten-carrier conjugate as defined herein before in the preparation of a vaccine composition, especially a vaccine composition for use in a method of preventing or treating nicotine addiction, said method comprising administering a therapeutically effective amount of the hapten-carrier conjugate. The conjugates of the present invention offer the advantage that they are highly stable under in vivo conditions. Furthermore, the present hapten-carrier conjugates can be prepared in a well-defined form which means that the risk of undesired side-effects is effectively minimised.
Suitable modes of administration include subcutaneous, intramuscular, transmucosal and intravenous administration. Most preferably, the vaccine composition is administered subcutaneously or intramuscularly.
The addictive effect of nicotine is believed to be associated with the capability of nicotine to cross the blood-brain barrier. The present method of treating or preventing nicotine addiction relies on preventing nicotine from crossing the blood brain barrier. In particular, administration of a nicotine hapten-carrier conjugate to a person will generate antibodies against nicotine, in the bloodstream of the person. If the person smokes, the nicotine in his blood will be bound by the circulating anti-nicotine antibodies, preventing the nicotine from reaching the brain. Therefore, the antibodies will prevent the physiological and psychological effects of nicotine that originate in the brain. Because the smoker will experience a lessening or cessation of these effects, he/she will lose the desire to smoke. The same therapeutic effects are expected if a person uses smokeless tobacco, after being immunized with a nicotine hapten-carrier conjugate of the invention. Additionally, the conjugates and antibodies of the invention may exert their effects by affecting the ability of nicotine to stimulate the peripheral nervous system.
The conjugates of the invention are suitable for treating and preventing nicotine addiction. For treating nicotine addiction, the nicotine-carrier conjugate of the invention is administered to a person suffering from nicotine addiction. For preventing nicotine addiction, persons at risk for developing nicotine addiction are treated with the conjugate according to the invention.
A vaccine composition of the present invention comprises at least one nicotine hapten-carrier conjugate in an amount sufficient to elicit an immune response thereto. Initial vaccination with the nicotine hapten carrier conjugate of the present invention creates high titers of antibodies that are specific to nicotine. The therapeutically effective amount of a conjugate which is administered to a person in need of treatment for nicotine addiction is readily determined by the skilled artisan. Suitable dosage ranges are 1-1000 μg/dose. It generally takes a person one to several weeks to generate antibodies against a foreign antigen. The production of antibodies in a person's blood can be monitored by using techniques that are well-known to the skilled artisan, such as ELISA, radioimmunoassay, surface plasma resonance, and Western blotting methods. Therapeutic effectiveness also can be monitored by assessing various physical effects of nicotine, such as blood pressure.
It will be understood that initial administration of the present immunogenic conjugate may be followed by subsequent administration of one or more “boosters” of conjugate. Such a booster will increase the production of antibodies against the nicotine hapten-carrier conjugate of the invention.
The vaccine compositions of the present invention may contain at least one adjuvant. The adjuvant used in the present invention will be selected so that the effect of the carrier protein is not inhibited. Adjuvants used in the present invention are those which are physiologically acceptable to humans, these include, but are not limited to aluminium hydroxide, aluminium phosphate, oil/surfactant based emulsion adjuvants such as Montanide™ in which different surfactants (especially mannityl oleate) are combined with a mineral oil, squalene-containing emulsions such as MF59™, monophosphoryl lipid A, or Neisseriae mutant lipopolysaccharide (as described in PCT/NL98/0063).
The vaccine compositions of the present invention may optionally contain one or more pharmaceutically acceptable excipients. Suitable excipients include sterile water, salt solutions and buffers. According to a preferred embodiment, the vaccine composition the present hapten-carrier conjugate solubilised in an aqueous, saline solution at a pharmaceutically acceptable pH. Alternatively, the vaccine composition comprises a suspension of the hapten-carrier conjugate.
Additionally, the vaccine composition may optionally contain at least one auxiliary agent, such as dispersion media, coatings, microspheres, liposomes, microcapsules, lipids, surfactants, lubricants, preservatives and stabilizers.
The vaccine composition of the present invention preferably is sterile. Furthermore, the composition may contain components that preserve against infestation with, and growth of, micro-organisms.
It is preferred that the vaccine composition is manufactured in the form of a sterile aqueous liquid which is ready for immediate administration.
The nicotine derivates according to the present invention can advantageously be linked to carriers using synthesis routes that do not produce protein-protein conjugates. Because such protein-protein conjugates adversely affect the solubility as well as the efficacy of the vaccine composition, another aspect of the invention relates to a vaccine composition comprising a hapten-carrier conjugate according to the present invention and a pharmaceutically acceptable excipient, wherein the vaccine composition contains no protein-protein conjugates.
The invention is further illustrated by means of the following examples.
Trans-3′-nicotine-N-(2-mercaptoethyl)carboxamide (1) was synthesised as follows:
N,N′-Bis-(trans-4′-cotininylcarbonyl)cystamine (A)
A mixture of 1.00 g (4.54 mmol) trans-4′-cotininecarboxylic acid, 0.45 g (2.00 mmol) cystamine dihydrochloride and 0.68 g (4.44 mmol) N-hydroxybenzotriazole was concentrated from 25 ml dry N,N-dimethylformamide twice. The residue was dissolved in 25 ml dry N,N-dimethylformamide and 0.80 ml (4.59 mmol) N,N-diisopropylethylamine and 0.68 ml (4.39 mmol) N,N′-diisopropylcarbodiimide were added. The mixture was stirred at room temperature. After 20 h, 4 ml (0.2 mol) water was added and stirring was continued for 1 h. Then, the mixture was concentrated in vacuo. The residue was taken up in 100 ml 0.5 M KHSO4 and washed with chloroform (3×25 ml). Solid Na2CO3.H2O (8.0 g) was added slowly to the aqueous layer which was then extracted with chloroform (3×25 ml). The organic layers were combined, dried (MgSO4), filtered and concentrated to give compound A as a syrup, which was used in the next step without purification
A small sample of compound A in chloroform was acidified with trifluoroacetic acid and then concentrated. The residue was concentrated from methanol (2×10 ml); 1H NMR (D2O), δ 8.84-8.86 (m, 2×2H), 8.59-8.64 (m, 2×1H), 8.17 (dd, 2×1H), 5.14 (d, 2×1H), 3.45-3.85 (m, 2×2H), 3.17-3.24 (m, 2×1H), 2.93-3.02 (m, 2×1H), 2.76-2.86 (m, 2×3H), 2.71 (s, 2×3H). 13C NMR (D2O), δ 180.0, 176.4, 149.2, 145.5, 143.9, 142.9, 131.8, 68.0, 50.1, 41.6, 40.1, 37.5, 32.0. Ion-spray mass spectrometry: MH+ (found/calculated) 557/557.
Trans-4′-cotinine-N-(2-mercaptoethyl)carboxamide (B)
Compound A (≈2 mmol, free base) was dissolved in 20 ml methanol and concentrated. The residue was redissolved in 20 ml methanol/water, 99/1, v/v, and 0.60 g (2.10 mmol) tris-(2-carboxyethyl)phosphine hydrochloride was added. The mixture was stirred at room temperature for 3 h, in which period the phosphine gradually dissolved. Then, the mixture was diluted with 100 ml chloroform and washed with 50 ml 1 M NaHCO3. The aqueous layer was extracted with chloroform (2×25 ml). The combined organic layers were washed with 50 ml 1 M NaHCO3 once more and again the aqueous layer was extracted with chloroform (2×25 ml). The organic layers were combined, dried (MgSO4), and filtered. The filtrate was concentrated to give B as a syrup of approximately 97% purity, as determined by TLC and HPLC.
HPLC-analysis was carried out using a Waters Alliance 2695 HPLC which was equipped and operated as follows:
Flow: 0.2 ml/min, splitter na UV detector
Gradient: in 20 min from 5% to 67% eluens B
Ion-spray mass spectrometry: MH+ (found/calculated) 280/280. 1H NMR (CDCl3), δ 8.61 (dd, 1H), 8.52 (d, 1H), 7.58 (dt, 1H), 7.34-7.39 (m, 1H), 6.04 (br, 1H), 4.77 (dd, 1H), 3.33-3.50 (m, 2H), 2.72-2.88 (m, 3H), 2.60-2.66 (m, 2H), 2.65 (s, 3H) 1.26 (t, 1H). 13C NMR (CDCl3), δ 173.2, 170.8, 150.2, 148.7, 135.3, 134.6, 124.3, 65.0, 48.6, 42.6, 34.6, 28.6, 24.6.
Trans-3′-(2-mercaptoethylaminomethyl)nicotine (1):
Crude compound B (≈2 mmol) was concentrated from dry tetrahydrofuran (2×5 ml) and dissolved in 10 ml dry tetrahydrofuran. The solution was added slowly to 10 ml 1M borane-tetrahydrofuran complex in tetrahydrofuran under a nitrogen atmosphere. The reaction mixture was stirred at room temperature for 20 h. Dry methanol (5 ml) was added and the mixture was concentrated. The residue was concentrated from dry methanol (3×10 ml) and dissolved in 10 ml 1 N HCl. Then, 50 ml 1 M NaHCO3 was added slowly and the mixture was extracted with chloroform (3×25 ml). The organic layers were combined, dried (MgSO4) and filtered. Trifluoroacetic acid (0.45 ml, 6 mmol) was added to the filtrate which was then concentrated. The residue was concentrated from methanol (2×10 ml) and further purification using HPLC was performed.
Immunoconjugates are prepared starting from the thiol-containing hapten described in Example 1, using the methodology described by J. W. Drijfhout and P. Hoogerhout: “Methods of preparing peptide-carrier conjugates” In: Fmoc Solid Phase Peptide Synthesis: A Practical Approach (W. C. Chan and P. D. White, eds.). Oxford University Press, 2000, pp. 229-241.
The carrier is dissolved in 0.1 M sodium phosphate buffer, pH 8, at a concentration of 3.0 mg/ml for TTd and 3.0 mg/ml for BSA. The cross-linker is dissolved freshly in N,N-dimethylacetamide at a concentration of 0.20 M or 80 mM. An aliquot of 50 μl of 0.20 M cross-linker is added to 1.75 ml TTd solution, whereas 50 μl of 80 mM cross-linker is added to 1.75 ml BSA solution. The solutions are mixed and left to stand at room temperature for 1 h. Then, the reaction mixture is applied to a PD-10™ column equilibrated in 0.1 M sodium phosphate buffer, containing 5 mM EDTA, pH 6 (de-aerated with helium). Elution is effected with the same buffer. The modified protein is collected in a volume of 3.0 ml and used immediately for conjugation of the thiol-containing haptens.
The purified thiol-containing hapten (trifluoroacetate) (3-4 μmol) is dissolved in 250 μl water and added to 2.0 ml of a freshly prepared solution of the sulfhydryl-reactive carrier in 0.1 M sodium phosphate buffer, containing 5 mM EDTA, pH 6. The solutions are mixed and left to stand at room temperature for 16-24 h. Then, the reaction mixture is applied to a PD-10™ column equilibrated in phosphate buffered saline, pH 7.2 (PBS). Elution is effected with the same buffer. The immunoconjugate (≈3.5 mg) is collected in a volume of 3.5 ml and stored at 2-8° C. until the preparation of a vaccine.
Conjugates prepared from the thiol-containing haptens and pyridyldithiopropionyl-modified proteins showed hapten/protein molar ratios of approximately 60 and 30, for TTd and BSA respectively.
Vaccines are prepared by appropriate dilution of the stock solutions (1 mg/ml) of the conjugates prepared from compounds 1 and bromoacetylated TTd and (bromoacetamido)propionylated TTd with a suspension of aluminium phosphate (0.5-1.5 mg/ml) in PBS, pH 7. Plain PBS is diluted with aluminium phosphate suspension for obtaining a mock vaccine. Groups of eight mice are immunized on days 0 and 28 with vaccine compositions containing 50 μg of immunoconjugate and 75 μg AlPO4 in 0.3 ml PBS. Sera are taken on day 42 and stored at −20° C. until use.
Antibody titers in the sera of Example 3 are determined by enzyme-linked immunosorbent assay (ELISA). The hapten-BSA conjugate of Example 2, prepared by using the NHS-PEG2-maleimide cross-linker, was used as coating antigen. Nicotine, cotinine, or acetylcholine were used as inhibitors in an inhibition ELISA.
The conjugate tested was found to induce satisfactory antibody titers in mice. The response is nicotine-specific. The antibodies do not cross-react significantly with cotinine or acetylcholine.
Trans-3′-(3-mercaptopropionamidomethyl)nicotine (2) was synthesised as follows:
A mixture of 90 mg (0.47 mmol) 3′-aminomethylnicotine [Pentel et al. 2000], 0.42 g (2.0 mmol) 3,3′-dithiodipropionic acid, and 144 mg (1.0 mmol) hydroxybenzotriazole hydrate was concentrated from 5 ml dry N,N-dimethylformamide twice, redissolved in 5 ml dry N,N-dimethylformamide, and 155 μl (1.0 mmol) N,N′-diisopropylcarbodiimide was added. The reaction mixture was stirred overnight at room temperature. Then, 100 μl water was added and stirring was continued for 1 h. Finally, 0.43 g (1.5 mmol) tris-carboxyethylphosphine hydrochloride was added and stirring was continued for 3 more hours. The reaction mixture was concentrated. The residue was taken up in 10 ml 0.5 M KHSO4 and washed with chloroform (4×3 ml). The aqueous layer was neutralized by slow addition of solid Na2CO3 and extracted with chloroform (4×3 ml). The organic layer was dried (MgSO4), filtered, and concentrated to give 2 as the major product (MH+=279.9). The pure compound was obtained by preparative HPLC.
Trans-3′-(S-Acetylmercaptacetamidomethyl)nicotine (precursor of 3) was prepared as follows:
90 mg (0.47 mmol) 3′-Aminomethylnicotine [Pentel et al. 2000] was dissolved in 5 ml acetonitrile and 144 mg (1.0 mmol) hydroxybenzotriazole hydrate and 165 mg (0.55 mmol) pentafluorophenyl S-acetylmercaptoacetate were added. After 2 h, 77 μl (1.0 mmol) trifluoroacetic acid was added and the reaction mixture was concentrated. 3′-(S-acetylmercaptacetamidomethyl)nicotine was purified by preparative HPLC and showed MH+=308.0 by mass spectrometrical analysis. The purified compound was stored at −20° C. until use in conjugation. Compound 3 was generated from 3′-(S-acetylmercaptacetamidomethyl)nicotine by S-deacetylation with e.g. hydroxylamine. It is most convenient to perform the deacetylation in the presence of a sulfhydryl-reactive protein [Drijfhout and Hoogerhout 2000] to give a nicotine conjugate without intermediate isolation of 3.
Vaccines of the invention are prepared from tetanus toxoid conjugates of compounds 1, 2 and 3 in accordance with the methods described in examples 2 and 3. Each vaccine was tested on a group of eight mice by immunization on days 0 and 28 with vaccine composition containing 50 μg of immunoconjugate and 75 μg AlPO4 in 0.3 ml PBS. Serum was taken on day 42, the antibody titers of the sera were determined by Direct ELISA using the homologues hapten-BSA conjugates. Subsequently the sera taken from the four groups of mice were tested on a hapten 1-BSA conjugate (“1-BSA”) by Direct ELISA. Inhibition ELISA's using nicotine and acetylcholine respectively on hapten 1-BSA conjugate were used to assess nicotine binding affinity and specificity of the anti-nicotine antibodies of the sera taken from the four groups of mice.
Wells of microliter plates were incubated with 100 μl of solutions of hapten-BSA conjugates (0.5 μg/ml) in PBS overnight at room temperature. The plates were washed with tap water containing 0.04% Tween 80. The wells were incubated with 100 μl of serial dilutions of individual sera in 0.1% Tween 80 in PBS for 60 min. at 37° C. The plates were washed with tap water containing 0.04% Tween 80 and the wells were incubated with 100 μl of a solution of a 1:5,000 dilution of horseradish peroxidase-labeled goat anti-mouse IgG in 0.1% Tween 80 in PBS for 60 min. at 37° C. The plates were washed again with tap water containing 0.04% Tween 80. The solution of the substrate was freshly prepared by successive addition of 100 μl of 3,3′,5,5′-tetramethylbenzidine (10 mg/ml) in 96% ethanol and 4 μl of 30% hydrogen peroxide to 10 ml of 0.11 M sodium acetate/citric acid buffer, pH 5.5. Each well was incubated with 100 μl of substrate solution for 10 min. at room temperature. Finally, the reaction was quenched by addition of 100 μl 2 M sulfuric acid and the optical density at 450 nm was read. The titer is calculated as the logarithm of serum reciprocal dilution at 50% of the maximum optical density (OD50).
Equal aliquots of the sera of the eight mice from each group were mixed and diluted 1:400,000 in 0.1% Tween 80 in PBS. The inhibitor (nicotine, cotinine, or acetylcholine) was dissolved at a concentration of 10−1 M in the 1:400,000 diluted pooled serum. Serial tenfold dilutions (up to 10−12 M) of the inhibitor were made in the fixed 1:400,000 serum dilution.
Plates were coated with hapten-BSA conjugate and washed as described above. In the second step, wells were incubated with 100 μl of inhibitor solution for 60 min. at 37° C. and, thereafter, washed with tap water containing 0.04% Tween 80. Incubation with horseradish peroxidase-labeled goat anti-mouse IgG and, thereafter, the substrate was performed as described above. The titer is calculated as the negative 10logarithm of the inhibitor concentration (mol/L) at 50% of the maximum optical density (OD50). IC50 values are determined from a graph wherein the OD (at 450 nm) is plotted against the negative 10logarithm of the inhibitor concentration (in mol/L).
Average antibody titers of the sera determined by Direct ELISA on homologues hapten-BSA conjugate are shown in table 1.
Average antibody titers of the sera determined by Direct ELISA on 1-BSA conjugate coatings are shown in table 2.
The results of the inhibition ELISA's on 1-BSA conjugate coating with nicotine as inhibitor are shown in
The graph shows that on 1-BSA, the nicotine IC50 is largest for the antibody from hapten 1 conjugate immunized mice and the smallest for the antibodies from mice immunized with hapten 3 conjugate.
The results of the inhibition ELISA's on 1-BSA conjugate coating with acetylcholine as inhibitor are shown in
The results of the inhibition ELISA using acetylcholine show that the nicotine antibody binding is specific.
Based on these results it was concluded that conjugates of haptens 1, 2 and 3 with tetanus toxoid carrier in accordance with the present invention, can suitably be used to induce anti-nicotine antibody responses in mice and thus provide suitable vaccines for therapeutic or prophylactic treatment of nicotine addiction.
| Number | Date | Country | Kind |
|---|---|---|---|
| 06112873.2 | Apr 2006 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/NL07/50173 | 4/20/2007 | WO | 00 | 2/10/2009 |
