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

Abstract 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 α7 neuronal nicotinic receptor with sub-nanomolar affinity for the human and rat α7 [ 125 I]α-bungarotoxin binding sites. In a search for novel agonist radioligands and imaging ligands for the α7 nicotinic receptor, [ 3 H]AZ11637326 was synthesized and its in vitro membrane binding properties were characterized. [ 3 H]AZ11637326 bound to hα7-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 α7 antagonist [ 125 I]α-bungarotoxin, indicating that [ 3 H]AZ11637326 bound to hα7-HEK membranes with an α7 nicotinic-like pharmacology. The K i values for the standards were on average 2.3-fold lower affinity than determined using the prototypical α7 nicotinic antagonist [ 125 I]α-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]α-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 α7 nicotinic pharmacology. Overall, these studies indicate that [ 3 H]AZ11637326 is a useful new in vitro probe of the α7 nicotinic receptor agonist site and support its potential utility for in vivo receptor occupancy studies.