[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

Receptor binding was characterized for [H-3](1S,4S)-2,2-dimethyl5-(6-phenylpyridazin-3-yl)-5-aza-2-azoniabicyclo[2.2.1]heptane([3H]A-585539), a selective high-affinity alpha 7 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 1/2 = 8.0 min); dissociation was slower (t 1/2 = 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, [H-3]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 [H-3] methyllycaconitine ([H-3]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 [H-3] MLA, and in general, a 5 to 10-fold higher affinity was observed for [H-3] A-585539 binding. There was also a good correlation of K-i values between [H-3] A-585539 binding to rat brain and human cortex. The use of a alpha 7/5-hydroxytryptamine type-3 chimera revealed that the N-terminal domain of alpha 7 nAChR was sufficient to faithfully reproduce the pharmacology of [H-3]A-585539 binding. Autoradiographic studies comparing [H-3]A-585539 and [I-125]alpha-bungarotoxin revealed a similar pattern of labeling in the rat. In summary, [H-3]A-585539 was shown to have excellent binding characteristics in rat and human brain and represents the first high-affinity alpha 7 agonist radioligand with utility in the characterization of this important nAChR subtype that is targeted toward ameliorating cognitive deficits underlying neuropsychiatric and neurodegenerative disorders.