Rapid identification of a novel phosphodiesterase 7B tracer for receptor occupancy studies using LC─MS/MS

Abstract

Phosphodiesterase 7B (PDE7B) inhibition has been considered as a therapeutic target for the treatment of several neurological disorders. Currently, there are no radio-labeled tracers available to determine receptor occupancy (RO) of this target. Developing such a tracer could greatly facilitate the identification of viable PDE7B inhibitors. In the current study, a liquid chromatography tandem mass spectrometry (LC─MS/MS) method was utilized to evaluate the brain distribution of unlabeled tracer candidates following intravenous micro-dosing. This novel approach resulted in an accelerated identification of a potential novel RO tracer for PDE7B. The identified molecule, Compound 30, showed reasonable target-tissue specificity (striatum/cerebellum ratio of 2.2) and suitable uptake (0.25% of the injected dose/g brain tissue) as demonstrated in rats dosed with the unlabeled compound. Compound 30 was subsequently labeled with tritium (³H). In vitro characterization of ³H-Compound 30 demonstrated that this compound possessed a high target affinity with a subnanomolar K_d (0.8 nM) and a B_max of 58 fmol/mg of protein using rat brain homogenate. Intravenous microdosing of ³H-Compound 30 showed preferential binding in the rat striatum, consistent with the mRNA distribution of PDE7B. In vitro displacement study with other structurally distinct PDE7B target-specific inhibitors using rat brain homogenate indicated that ³H-Compound 30 is an ideal tracer for K_i analysis. This is the first report of a preclinical tracer for PDE7B. With further characterization, Compound 30 may ultimately show the appropriate properties required to be further developed as a PDE7B PET ligand for clinical studies.

Keywords: CNS; LC─MS/MS; Phosphodiesterase 7B; Positron emission tomography (PET) ligand; Receptor occupancy tracer screening.