In-vivo rat striatal 5-HT4 receptor occupancy using non-radiolabelled SB207145

Objectives The objective of the current investigation was to develop a simple, rapid method for determining in‐vivo 5‐hydroxytryptamine type 4 receptor (5‐HT4R) occupancy in rat brain using non‐radiolabelled SB207145 as a tracer for accelerating the drug discovery process. Methods In‐vivo tracer optimization studies for tracer dose, survival intervals and brain distribution profile were carried out in rats. The tracer was pharmacologically validated using potent well‐characterized 5‐HT4R ligands. The brain regional concentrations of tracer (SB207145); plasma and brain concentrations of 5‐HT4R ligands were quantified using high‐performance liquid chromatography coupled with a tandem mass spectrometric detector (LC‐MS/MS). Key findings SB207145 showed a higher specific binding in striatum (1.96 ng/g) and lower binding in cerebellum (0.66 ng/g), which is consistent with findings of other published 5‐HT4R expression studies. Pretreatment with potent 5‐HT4 ligands dose‐dependently reduced striatal SB207145 concentration and the effective dose to achieve 50% receptor occupancy (ED50) values were 4.8, 2.0, 7.4, 9.9, 3.8 and 0.02 mg/kg for GR113808, piboserod, prucalopride, RS67333, TD8954 and PF04995274, respectively. Conclusions Results from the mass spectrometry approach to determine 5‐HT4R occupancy in rat brain are comparable with those reported using radiolabelled scintillation spectroscopy methods. In conclusion, the LC‐MS/MS characterization permits use of tracer at a preclinical stage in high‐throughput fashion as well as characterization of target expression.