In vitro binding characteristics of [3H]AZ11637326, a novel α7-selective neuronal nicotinic receptor agonist radioligand

AZ11637326 (5'-(2-fluoro[3,4,5(-3)H3]phenyl)-spiro[1-azabicyclo [2.2.2]octane-3,2'(3'H)-furo[2,3-b]pyridine) is a potent partial agonist at the human alpha7 neuronal nicotinic receptor with sub-nanomolar affinity for the human and rat alpha7 [(125)I]alpha-bungarotoxin binding sites. In a search for novel agonist radioligands and imaging ligands for the alpha7 nicotinic receptor, [(3)H]AZ11637326 was synthesized and its in vitro membrane binding properties were characterized. [(3)H]AZ11637326 bound to halpha7-HEK membranes with high specificity (95%), high affinity (230 pM) and a B(max) of 460 fmol/mg. The rank order of affinity of nicotinic standards determined with [(3)H]AZ11637326 strongly correlated with those determined using the classical alpha7 antagonist [(125)I]alpha-bungarotoxin, indicating that [(3)H]AZ11637326 bound to halpha7-HEK membranes with an alpha7 nicotinic-like pharmacology. The K(i) values for the standards were on average 2.3-fold lower affinity than determined using the prototypical alpha7 nicotinic antagonist [(125)I]alpha-bungarotoxin. Because [(3)H]AZ11637326 specific binding is rapid and reversible, the K(i) values determined using this ligand are more accurate estimates of affinity than those determined using the kinetically sluggish [(125)I]alpha-bungarotoxin. [(3)H]AZ11637326 also bound to a high affinity (510 pM), nicotine-sensitive site on rat hippocampal membranes with an average B(max) of 55 fmol/mg. With rat hippocampal membranes, the nicotine-sensitive fraction of total binding was sub-optimal for a radioligand (~50%), yet the potencies and rank order of the K(i) values for standards were consistent with an alpha7 nicotinic pharmacology. Overall, these studies indicate that [(3)H]AZ11637326 is a useful new in vitro probe of the alpha7 nicotinic receptor agonist site and support its potential utility for in vivo receptor occupancy studies.