Nicotine hapten structure, antibody selectivity and effect relationships: Results from a nicotine vaccine screening procedure
Introduction
Tobacco use is the leading preventable cause of death in the world according to the WHO [1]. The tobacco epidemic currently causes about 5.4 million deaths every year, and tobacco smoking is a risk factor in six of the eight leading causes of death in the world, including ischaemic heart disease, chronic obstructive pulmonary disease and lung cancer [2].
Nicotine, the major addictive component in tobacco, has been shown to stimulate brain mesolimbic dopamine (DA) neurons, and this effect is critically involved with the dependence producing properties of nicotine [3]. The mesolimbic DA pathway, originating in the ventral tegmental area (VTA) and projecting to limbic areas, such as the nucleus accumbens (NAC), has been shown to be involved in both natural and drug induced reward-related behaviour (for review see [4]). Thus, nicotine increases the firing of mesolimbic DA neurons [5] as well as DA synthesis [6] and DA release in the NAC [7] in the rat. Nicotine preferentially increases DA in the shell region of the NAC [8], which is considered to be specifically involved in emotional and motivational processes [9], [10]. In humans, nicotine from tobacco smoking increases the dopamine release in the ventral caudate/NAC and this increase is correlated with mood improvement [11].
The current pharmacological aids in smoking cessation have significant clinical effects. Nicotine replacement therapy and the antidepressant drug bupropion approximately double the otherwise very low chance of remaining abstinent from cigarette smoking [12], [13], and the recently introduced varenicline, a partial agonist at α4β2 and a full agonist at α7 nicotinic receptors [14], approximately triples the abstinence rates compared to placebo (for review see [15]). Still, even with these pharmacological aids, the majority of the smokers will relapse within a year. Nicotine dependence is a chronically relapsing disorder [16], and at present there are no approved pharmacological treatments that have shown effectiveness in long term relapse prevention.
A novel approach to treat nicotine dependence is active immunisation against nicotine, which might provide a safe treatment with few side-effects, provided a high nicotine-selectivity of the antibodies formed. The so-called nicotine vaccines may have the potential to provide long-lasting protection against relapse, i.e. months to years. To date there are four nicotine vaccines in clinical phase II trials, TA-NIC by Celtic Pharma (company home page: www.celticpharma.com), NicVAX [17], [18] by Nabi Biopharmaceuticals (company home page: www.nabi.com), Niccine by Independent Pharmaceutica AB (company home page: www.independentpharma.com) and NIC002 [19], [20] (formerly NicQb) by Cytos Biotechnology (company home page: www.cytos.com) which recently reported that the primary endpoint was not achieved in an interims analysis of their ongoing study. So far all of the nicotine vaccines seem to be well tolerated, and in addition, two of the clinical studies report clinical proof-of-concept in the high antibody responder subgroups.
The rationale behind this treatment is to generate endogenous anti-nicotine antibodies that bind nicotine in the blood, thus preventing it from entering the brain were it exerts its reinforcing effects. Nicotine in itself is too small to be recognised by the immune system; therefore it has to be coupled to a larger carrier protein via a linker. In order to generate antibodies that are highly selective for nicotine, the nicotine part of the immunogen should optimally be as similar as possible to nicotine regarding its three-dimensional structure as well as its physical–chemical properties. Since the structure of the linker should theoretically influence the properties of the nicotine molecule, the choice of linker may be of considerable importance. The nicotine-linker, i.e. the nicotine hapten, is coupled to different sites on the carrier protein depending on the functional group of the linker, and this may subsequently affect the amount of haptens that are coupled to the carrier protein, as well as their accessibility by the immune system.
The present study reports results obtained with seven different nicotine immunogens from our initial screening process of novel nicotine immunogens [21] with a putative therapeutic potential for the treatment of nicotine dependence. This work includes a series of linkers of different lengths and flexibility that were coupled to the 5- or 6-position of the nicotine molecule and conjugated to keyhole limpet hemocyanin (KLH, Fig. 1). The pyridine ring was chosen for linker attachment in order to increase the antigenicity of the pyrrolidine ring, in which nicotine is first metabolised in the body, and thereby minimising cross-reactivity of generated antibodies with the metabolites of nicotine [22]. Since in tobacco, (S)-nicotine is the natural occurring enantiomer, some immunogens comprising the (S)-enantiomer of nicotine were also prepared and tested.
Enzyme-linked immunosorbent assay (ELISA) was used to determine the titers of elicited nicotine antibodies in rats after immunisation with the immunogens in Freund's adjuvant and competitive ELISA was used to study the selectivity of the antibodies. We subsequently used in vivo voltammetry to investigate whether active immunisation with the nicotine immunogens might alter the nicotine-induced DA release in the shell region of the nucleus accumbens (NACshell). Finally, the potential correlations between antibody selectivity and their effect on the dependence producing actions of nicotine were investigated.
Section snippets
Nicotine haptens
Nicotine haptens were synthesised at the Department of Organic Pharmaceutical Chemistry, Uppsala University, Uppsala, Sweden. The linkers were attached to the 5- or 6-position of nicotine (racemate) or (S)-nicotine (see Table 1).
ELISA nicotine antibody titers
All tested nicotine immunoconjugates generated nicotine antibodies in rats as measured by ELISA (Table 2). There were significant differences in antibody titers (Kruskal–Wallis test; H(6) = 15.67, P = 0.016) between the group with the lowest (GK81-KLH) and the two groups showing the highest antibody titers (IP31-KLH, P = 0.006 and IB87-KLH, P = 0.04). Experiments were performed 3–7 days post-immunisation although the titers measured at this time point may probably not reflect the maximum levels of IgG,
Discussion
When we begun our screening of a vaccine for the treatment of nicotine dependence, we expected that immunisation with any of the nicotine immunogens would cause a decrease in the nicotine-induced dopamine release compared to controls, as measured by in vivo voltammetry, as long as anti-nicotine antibodies were generated. However, this study of a set of structurally different nicotine immunogens demonstrates that the structure of the linkers, as well as the site of their coupling on the nicotine
Disclosure statement
Sabina de Villiers and Torgny Svensson are consultants for Independent Pharmaceutica AB, Sweden. Nina Lindblom and Torgny Svensson own stocks in Independent Pharmaceutica AB, Sweden.
Role of the funding source
This work was supported by the Swedish Research Council, grant no. 4747, The Karolinska Institutet, Stockholm and Independent Pharmaceutica AB, Sweden.
Acknowledgements
We thank Annika Olsson and Ann-Chatrine Samuelsson for providing valuable technical assistance in this study.
References (36)
- et al.
Nicotine preferentially stimulates dopamine release in the limbic system of freely moving rats
Eur J Pharmacol
(1986) - et al.
Pharmacological characterization of dopamine systems in the nucleus accumbens core and shell
Neuroscience
(1992) - et al.
Radioimmunoassay of nicotine
Biochem Biophys Res Commun
(1975) Nonlinear relationship between impulse flow and dopamine released by rat midbrain dopaminergic neurons as studied by in vivo electrochemistry
Neuroscience
(1988)- et al.
Differential actions of typical and atypical antipsychotic drugs on dopamine release in the core and shell of the nucleus accumbens
Eur Neuropsychopharmacol
(1996) - et al.
Disconnection between activation and desensitization of autonomic nicotinic receptors by nicotine and cotinine
Neurosci Lett
(2007) - et al.
Development of an anti-cotinine vaccine to potentiate nicotine-based smoking cessation strategies
Vaccine
(2007) The World health report: 2003 shaping the future
(2003)Report on the global tobacco epidemic, 2008: the MPOWER package
(2008)- et al.
The mesolimbic dopaminergic system is implicated in the reinforcing effects of nicotine
Psychopharmacology (Berl)
(1992)
Reward, motivation and cognition: Psychobiology of the mesotelencephalic dopamine systems
Nicotinic effects on the firing pattern of midbrain dopamine neurons
Acta Physiol Scand
Selective stimulation of limbic dopamine activity by nicotine
Acta Physiol Scand
Differential effects of acute and chronic nicotine on dopamine output in the core and shell of the rat nucleus accumbens
J Neural Transm
Prefrontal cortical dopamine systems and the elaboration of functional corticostriatal circuits: implications for schizophrenia and Parkinson's disease
J Neural Transm Gen Sect
Ventral striatal dopamine release in response to smoking a regular vs a denicotinized cigarette
Neuropsychopharmacology
A controlled trial of sustained-release bupropion, a nicotine patch, or both for smoking cessation
N Engl J Med
Nicotine replacement therapy for smoking cessation (Cochrane Review)
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2017, BiomaterialsCitation Excerpt :Together with our previously reported physiologically-based-pharmacokinetic-modeling data [15], these clinical findings suggest that while the basic concept of using immunotherapy to promote smoking cessation is solid, future vaccines have to be able to elicit high titers of antibodies to be effective. A variety of approaches to strengthen the immunogenicity of traditional conjugate nicotine vaccines have been investigated, including the design of hapten structure [16,17], the modulation of the linker position and composition [8], the selection of carrier proteins [10], the use of different adjuvants [18], the application of multivalent vaccines [19–22], and the optimization of administration routes [23]. However, as the immune system is relatively poor at recognizing small soluble protein antigens [24,25], traditional conjugate nicotine vaccines bear a serious innate shortcoming, poor recognition and internalization by immune cells.