Elsevier

Vaccine

Volume 28, Issue 10, 2 March 2010, Pages 2161-2168
Vaccine

Nicotine hapten structure, antibody selectivity and effect relationships: Results from a nicotine vaccine screening procedure

https://doi.org/10.1016/j.vaccine.2009.12.051Get rights and content

Abstract

The aim of the present study was to synthesise and screen a set of novel nicotine hapten immunogens used for the treatment of nicotine dependence. In the screening process we studied the amount of antibodies generated and their selectivity, using ELISA techniques, and their effects on nicotine-induced dopamine release in the NACshell of the rat, assessed by in vivo voltammetry. We conclude that even small changes such as the linker attachment on the nicotine molecule as well as the structure of the linker may greatly influence the selectivity of the antibodies and the central neurobiological effects of nicotine that are considered critical for its dependence producing properties.

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.

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