[³H]A-585539 [(1S,4S)-2,2-Dimethyl-5-(6-phenylpyridazin-3-yl)-5-aza-2-azoniabicyclo[2.2.1]heptane], a Novel High-Affinity α7 Neuronal Nicotinic Receptor Agonist: Radioligand Binding Characterization to Rat and Human Brain

Abstract

Receptor binding was characterized for [³H](1S,4S)-2,2-dimethyl-5-(6-phenylpyridazin-3-yl)-5-aza-2-azoniabicyclo[2.2.1]heptane ([³H]A-585539), a selective high-affinity α7 nicotinic acetylcholine receptor (nAChR) agonist with rapid kinetics, low nonspecific binding, and high specific activity. At 4°C, the association was monophasic and rapid (t_½ = 8.0 min); dissociation was slower (t_½ = 64.2 min). The K_d in rat brain at 4°C was 0.063 nM, whereas at 22 and 37°C, the K_d values were 0.188 and 0.95 nM, respectively. In contrast, the B_max (34 fmol/mg protein) was unaffected by temperature. In human cortex, [³H]A-585539 bound with a K_d of 0.066 nM and a B_max of 5.8 fmol/mg protein at 4°C, whereas under similar conditions, specific [³H]methyllycaconitine ([³H]MLA) binding was not measurable. A number of agonist and antagonist nAChR ligands displaced binding to rat brain membranes with rank order of affinity similar to that for [³H]MLA, and in general, a 5 to 10-fold higher affinity was observed for [³H]A-585539 binding. There was also a good correlation of K_i values between [³H]A-585539 binding to rat brain and human cortex. The use of a α7/5-hydroxytryptamine type-3 chimera revealed that the N-terminal domain of α7 nAChR was sufficient to faithfully reproduce the pharmacology of [³H]A-585539 binding. Autoradiographic studies comparing [³H]A-585539 and [¹²⁵I]α-bungarotoxin revealed a similar pattern of labeling in the rat. In summary, [³H]A-585539 was shown to have excellent binding characteristics in rat and human brain and represents the first high-affinity α7 agonist radioligand with utility in the characterization of this important nAChR subtype that is targeted toward ameliorating cognitive deficits underlying neuropsychiatric and neurodegenerative disorders.