5-Iodo-A-85380, an α4β2 Subtype-Selective Ligand for Nicotinic Acetylcholine Receptors

Abstract

In an effort to develop selective radioligands for in vivo imaging of neuronal nicotinic acetylcholine receptors (nAChRs), we synthesized 5-iodo-3-(2(S)-azetidinylmethoxy)pyridine (5-iodo-A-85380) and labeled it with 125 I and 123 I. Here we present the results of experiments characterizing this radioiodinated ligand in vitro. The affinity of 5-[ 125 I]iodo-A-85380 for ␣4␤2 nAChRs in rat and human brain is defined by K d values of 10 and 12 pM, respectively, similar to that of epibatidine (8 pM). In contrast to epibatidine, however, 5-iodo-A-85380 is more selective in binding to the ␣4␤2 subtype than to other nAChR subtypes. In rat adrenal glands, 5-iodo-A-85380 binds to nAChRs containing ␣3 and ␤4 subunits with 1/1000th the affinity of epibatidine, and exhibits 1/60th and 1/190th the affinity of epibatidine at ␣7 and muscle-type nAChRs, respectively. Moreover, unlike epibatidine and cytisine, 5-[ 125 I]iodo-A-85380 shows no binding in any brain regions in mice homozygous for a mutation in the ␤2 subunit of nAChRs. Binding of 5-[125 I]iodo-A-85380 in rat brain is reversible, and is characterized by high specificity and a slow rate of dissociation of the receptor-ligand complex (t 1/2 for dissociation ϳ2 h). These properties, along with other features observed previously in in vivo experiments (low toxicity, rapid penetration of the blood-brain barrier, and a high ratio of specific to nonspecific binding), suggest that this compound, labeled with 125 I or 123 I, is superior to other radioligands available for in vitro and in vivo studies of ␣4␤2 nAChRs, respectively.Nicotinic acetylcholine receptors (nAChRs) are excitatory ligand-gated cation channels that are widely distributed in mammalian organisms, appearing in the central and peripheral nervous systems, neuromuscular junctions, and adrenal glands. The nAChR channel complex is composed of five protein subunits, which form a pore that is permeable to Na ϩ , K ϩ , and Ca 2ϩ (Lindstrom, 1995;Holladay et al., 1997).To date, ␣, ␤, ␥, ␦, and ⑀ subunits have been isolated and cloned from mammalian and avian tissues, with nine varieties of ␣ and four varieties of ␤ subunits identified. The ␣1, ␤1, ␥, ␦, and ⑀ subunits form the neuromuscular junction receptor, the very first nAChR to be characterized. The other subunits (␣2-␣9 and ␤2-␤4) are found predominantly throughout the nervous system (Lindstrom, 1995;Holladay et al., 1997). This subunit diversity affords a large potential for a variety of nAChR subtypes, exhibiting distinct cationconducting properties and pharmacological heterogeneity. Based on binding properties and pharmacological sensitivity, major nAChR subtypes in mammalian brain can be categorized as ␣-bungarotoxin-sensitive (␣7) and ␣-bungarotoxin-insensitive (e.g., ␣4␤2) (Lindstrom, 1995;Holladay et al., 1997). Accordingly, 125 I-␣-bungarotoxin has been the radioligand of choice for in vitro characterization of the ␣7 subtype of nAChR, whereas tritiated agonists, such as nico-

Nicotinic Acetylcholine Receptor Distribution in Alzheimer's Disease, Dementia with Lewy Bodies, Parkinson's Disease, and Vascular Dementia: In Vitro Binding Study Using 5-[125I]-A-85380

“…In vitro visualization of 5-[ 125 I]-A-85380 receptor distribution has previously been carried out in rats (Mukhin et al, 2000) and mice (Musachio et al, 1998;Horti et al, 1999). In the present study, the binding distribution of 5 (Court et al, 1998(Court et al, , 2000a …”

“…For total binding, sections were incubated in the same buffer as prewash and 22 pM 5-[ 125 I]-A-85380 for 4 h at room temperature. The Kd of 5-[ 125 I]-A-85380 has been reported to be 12 pM in the human brain (Mukhin et al, 2000); therefore, at the above concentration binding approaches B max . A measure of 1 mM epibatidine was added to at least one adjacent section from each case to give a measure of nonspecific binding.…”

“…5-Iodo-3-[2(S)-2-azetidinylmethoxy]pyridine (5-I-A-85380) is a high-affinity nAChR ligand that demonstrates high subtype selectivity for a4b2 nAChRs (Mukhin et al, 2000). A recent study has also shown 5-I-A-85380 binding to aconotoxin MII-sensitive (a6b2) nAChRs in the rodent and monkey striatum (Kulak et al, 2002).…”

Functional brain imaging of tobacco use and dependence

“…Radiolabeling of A-85380 was a major advance in imaging nAChRs, because administration of radiolabeled nicotine (used for previous imaging studies) results in high non-specific binding and short drug-receptor interaction times (Sihver et al, 2000). In recent years, 2-[ 18 F]F-A-85380 or simply 2-FA and related compounds (Chefer et al, 1999;Horti et al, 1998;Koren et al, 1998) have been developed for PET imaging, and 5-[ 123/125 I] iodo-A85380 has been used for SPECT imaging (Chefer et al, 1998;Horti et al, 1999;Mukhin et al, 2000) of α 4 β 2 nAChRs.…”

5-123I-A-85380 binding to the α4β2-nicotinic receptor in mild cognitive impairment

“…Attention has therefore turned to SPECT with radioligands for acetylcholine receptor subtypes. (S)-5-[123I]iodo-3-(2-azetidinylmethoxy)pyridine (5-IA-85380 or 5-IA) is a nicotine-based radioligand with high affinity for the 42-receptor, to a lesser extent to the 7-receptor (Mukhin, et al, 2000). High affinity 5-IA binding is seen in thalamus, striatum and temporal regions, with some moderate in vitro binding in white matter of cingulate, occipital, and temporal areas (Pimlott, et al, 2004).…”