Rat thalamic α₄β₂ neuronal nicotinic acetylcholine receptor occupancy assay using LC-MS/MS

In vivo brain receptor occupancy has been the key assay in driving preclinical drug discovery program and there is a need to hasten this screening step. Radiolabeled methods, which are time consuming and expensive, are most widely employed to measure receptor occupancy. Thus we sought to develop and validate an alternative novel approach for measuring rat brain α₄β₂ neuronal nicotinic acetylcholine receptor occupancy using high performance liquid chromatography combined with tandem mass spectrometric detector (LC-MS/MS).

Tracer optimization studies like in vivo dose and time dependent brain regional distribution; saturation binding and blocking study with nicotine and atropine were carried out for ZW-104 in rats. Assay validity was tested by pretreatment with potent α₄β₂ ligands; TC-1734, cytisine, ABT-089, ABT-594 and A-366833. Receptor occupancy along with plasma and brain exposure levels of α₄β₂ ligand was measured in the same set of animals.

The regional distribution of ZW-104 in rat was found to be, thalamus>frontal cortex>striatum>hippocampus>cerebellum, and is in accordance with the distribution and regional densities of α₄β₂ nAChRs measured using [¹⁸F]ZW-104 in mice and baboons. Pretreatment with nicotine and α₄β₂ ligands dose dependently reduced the binding of ZW-104 in the thalamus. Non-nicotinic antagonist atropine did not alter the binding of ZW-104 in the thalamus, indicating the tracer specificity. The ED₅₀ values calculated for occupancy were found to be 3.01, 0.83, 14.81, 0.001 and 0.11 mg/kg for TC-1734, cytisine, ABT-089, ABT-594, and A-366833, respectively.

These findings demonstrate that non-radiolabeled ZW-104 is suitable for determining the α₄β₂ receptor occupancy in rat brain. The LC-MS/MS based receptor occupancy assay is a rapid method and allows the generation of occupancy data along with the brain and plasma concentration in the same group of animals.