In vitro and in vivo characterization of JNJ-31020028 (N-(4-{4-[2-(diethylamino)-2-oxo-1-phenylethyl]piperazin-1-yl}-3-fluorophenyl)-2-pyridin-3-ylbenzamide), a selective brain penetrant small molecule antagonist of the neuropeptide Y Y2 receptor

The lack of potent, selective, brain penetrant Y-2 receptor antagonists has hampered in vivo functional studies of this receptor. Here, we report the in vitro and in vivo characterization of JNJ-31020028 (N-(4-{4-[2-(diethylamino)-2-oxo-1-phenylethyl]piperazin-1-yl}-3-fluorophenyl)-2-pyridin-3-ylbenzamide), a novel Y-2 receptor antagonist. The affinity of JNJ-31020028 was determined by inhibition of the PYY binding to human Y-2 receptors in KAN-Ts cells and rat Y-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-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. JNJ-31020028 bound with high affinity (pIC(50) = 8.07 +/- 0.05, human, and pIC(50) = 8.22 +/- 0.06, rat) and was > 100-fold selective versus human Y-1, Y-4, and Y-5 receptors. JNJ-31020028 was demonstrated to be an antagonist (pK(B) = 8.04 +/- 0.13) in functional assays. JNJ-31020028 occupied Y-2 receptor binding sites (similar to 90% at 10 mg/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. These results suggest that Y-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.