In vivo imaging of nicotinic receptor upregulation following chronic (-)-nicotine treatment in baboon using SPECT
Introduction
Neuronal nicotinic acetylcholine receptors (nAChRs) are the principal biological targets of the tobacco alkaloid (-)-nicotine, and are thought to play an important role in nicotine addiction of smokers [2], [11]. nAChRs are members of the multisubunit neurotransmitter-gated superfamily of ion channels. Thus far, numerous subunits have been identified: α2 to α9 and β2 to β4 which are expressed in the autonomic nervous system and CNS and α1, β1, γ, ϵ, and δ which are expressed in muscle [7]. Different combinations of α and β subunits can form different nAChR subtypes whose activation can elicit a number of functional responses. Two classes of nAChRs identified in the brain using radioligand binding assays include those with high affinity for (-)-nicotine (α4β2) [43] and those with high affinity for α-bungarotoxin (α-BgT) (α7) [9]. The pairing of α4 and β2 subunits represents the major nAChR subtype found in the brain [27].
Chronic treatment with agonists for most neurotransmitter receptor systems results in a decrease in receptor number. However, it has been demonstrated that chronic (-)-nicotine treatment in mice [4] and rats [15] elicits a dose-dependent increase in α4β2 binding sites in the brain [29]. This observed upregulation in the number of binding sites represents an increase in receptor density (Bmax) rather than a change in receptor affinity (KD). The upregulation of the α4β2 subtype is not permanent, with binding returning to control levels within 7–10 days in mice [30] and 15–20 days in rats [10] after cessation of (-)-nicotine treatment. The significance of (-)-nicotine induced receptor changes in rodent brain is underscored by the finding that nAChRs labeled with [3H](-)-nicotine are increased in homogenates of autopsied brain samples from people who smoked cigarettes compared to non-smokers [5], [6]. Smokers who had stopped smoking at least two months before death had nAChR levels that were similar to those found in non-smokers. These findings suggest that upregulation of nAChRs in the brain plays an important role in (-)-nicotine tolerance and addiction in smokers.
All previous efforts to demonstrate nAChR upregulation used in vitro binding assays and in one case ex vivo assays were used in mice [33]. The upregulation of nAChRs with chronic (-)-nicotine treatment and return to baseline levels following treatment cessation has yet to be observed and quantified in the living brain. Initial attempts to study nAChRs using Positron Emission Tomography (PET) focused on carbon-11 labeled forms of (-)-nicotine. However, despite interesting clinical results obtained with this ligand, its pharmacological characteristics were far from ideal. Lack of specificity, rapid metabolism and egress of the radioligand from the brain limit its application in quantification of nAChRs in vivo [39]. The discovery of (±)epibatidine, a potent nAChR agonist, stimulated development of nAChR radioligands with favorable properties for PET and Single Photon Emission Computed Tomography (SPECT) studies. These ligands displayed exquisite binding properties toward the α4β2 subtype but also a narrow safety margin which severely limited their use in humans [13].
A series of 3-pyridyl ether compounds possessing subnanomolar affinity for brain nAChRs with an ability to differentially activate subtypes of neuronal nAChRs have been reported [1]. A lead member of this series is 3-(2(S)-azetidinylmethoxy)pyridine (A-85380) that displays high affinity for α4β2 nAChRs (Ki = 0.05 nM vs. [3H]Cytisine). Recently, halogenated derivatives have been synthesized with the iodinated analogue 5-iodo-A-85380 being the most potent nAChR ligand of the series in vitro (Ki = 0.01 nM) [23]. 5-Iodo-A-85380 has been labeled with iodine-123 and demonstrated high selectivity and specificity for nAChRs in mouse brain in vivo, with relatively low acute toxicity [18], [34]. 5-[123I]Iodo-A-85380 has also been used in SPECT experiments with Rhesus monkey and Papio anubis baboon and showed great promise as a radioligand for nAChR imaging [8], [35].
The ability to quantify receptor binding in vivo has been predominantly the domain of PET. However, recent advances in instrumentation, corrections for photon interactions in the body and image reconstruction have made quantitative kinetic studies feasible with SPECT [17], [21]. In this paper we describe our methodology for imaging nAChR binding using 5-[123I]iodo-A-85380 and SPECT and report the first in vivo imaging of nAChR upregulation in response to chronic (-)-nicotine treatment in baboon.
Section snippets
Radiochemistry
5-Iodo-A-85380 and 5-tributyltin-A-85380 where synthesized according to literature procedures [35]. 5-[123I]Iodo-A-85380 was synthesized with slight modification of the literature method [35]. Briefly, Na123I (200 μL of a 0.1 M NaOH solution) was passed through a maxi-clean IC-H cartridge and washed with water (0.5 mL) into a 5 mL pear shaped flask. The aqueous solution was evaporated to dryness using rotary evaporation. To this was added the tin precursor, 5-tributyltin-A-85380, (100 μg in 10
Chronic (-)-nicotine treatment
The concentration of nicotine and cotinine in plasma for both animals are given in Table 2. For the (-)-nicotine treated animal (Evan), plasma nicotine levels of 27 ng/mL were detected during the 15 days while the osmotic pump was implanted in the animal. The levels of the major nicotine metabolite, cotinine, ranged from 238 ng/mL 2 days after pump implantation to 191 ng/mL at day 15. Levels of nicotine and cotinine returned to baseline seven days after removal of the osmotic pump. In the
Discussion and conclusion
The aim of this preliminary study was to determine whether nAChR upregulation in baboon following chronic (-)-nicotine treatment could be observed and quantified using [123I]iodo-A-85380 SPECT and appropriate tracer kinetic modeling. Previous studies have demonstrated gender differences in nAChR upregulation following chronic (-)-nicotine treatment in rodents [24], [33]. Therefore baboons of the same sex were chosen in this study. The classical method for inducing nAChR upregulation in animal
Acknowledgements
We would like to thank Prof Neal Benowitz for determining the nicotine and cotinine levels in plasma and for his helpful discussions. Part of this work was supported by CA 77349 (JLM) and The Smokeless Tobacco Research Council (DFW)
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