In vitro and in vivo characterization of JNJ-31020028 (N-(4-{4-[2-(diethylamino)-2-oxo-1-phenylethyl]piperaz in-1-yl}-3-fluorophenyl)-2-pyridin-3-ylbenzamide), a selective brain penetrant small molecule antagonist of the neuropeptide Y Y sub(2) receptor

Rationale: The lack of potent, selective, brain penetrant Y sub(2) receptor antagonists has hampered in vivo functional studies of this receptor. Objective: Here, we report the in vitro and in vivo characterization of JNJ-31020028 (N-(4-{4-[2-(diethylamino)-2-oxo-1-phenylethyl]piperaz in-1-yl}-3-fluorophenyl)-2-pyridin-3-ylbenzamide), a novel Y sub(2) receptor antagonist. Methods: The affinity of JNJ-31020028 was determined by inhibition of the PYY binding to human Y sub(2) receptors in KAN-Ts cells and rat Y sub(2) receptors in rat hippocampus. The functional activity was determined by inhibition of PYY-stimulated calcium responses in KAN-Ts cells expressing a chimeric G protein Gqi5 and in the rat vas deferens (a prototypical Y sub(2) bioassay). Ex vivo receptor occupancy was revealed by receptor autoradiography. JNJ-31020028 was tested in vivo with microdialysis, in anxiety models, and on corticosterone release. Results: JNJ-31020028 bound with high affinity (pIC sub(50)=8.07c0.05, human, and pIC sub(50)=8.22c0.06, rat) and was >100-fold selective versus human Y sub(1), Y sub(4), and Y sub(5) receptors. JNJ-31020028 was demonstrated to be an antagonist (pK sub(B)=8.04c0.13) in functional assays. JNJ-31020028 occupied Y sub(2) receptor binding sites (~90% at 10mg/kg) after subcutaneous administration in rats. JNJ-31020028 increased norepinephrine release in the hypothalamus, consistent with the colocalization of norepinephrine and neuropeptide Y. In a variety of anxiety models, JNJ-31020028 was found to be ineffective, although it did block stress-induced elevations in plasma corticosterone, without altering basal levels, and normalized food intake in stressed animals without affecting basal food intake. Conclusion: These results suggest that Y sub(2) receptors may not be critical for acute behaviors in rodents but may serve modulatory roles that can only be elucidated under specific situational conditions.