Pharmacological characterization of the novel nonpeptide orphanin FQ/nociceptin receptor agonist Ro 64-6198: Rapid and reversible desensitization of the ORL1 receptor in vitro and lack of tolerance in vivo

The novel nonpeptide orphanin FQ/nociceptin (OFQ/N) ligand {(1 S,3aS)-8-(2, 3,3a,4,5,6-hexahydro-1H-phenalen-1-yl)-1-phenyl-1,3,8-triaza-spiro[4.5]deca n-4-one} (Ro 64-6198) was characterized in vitro and in vivo for its agonis tic potential. Ro 64-6198 was 130- to 3500-fold selective for the OFQ/N rec eptor (ORL1) compared with opiate receptors. In the CAMP inhibition assay, Ro 64-6198 was a full agonist at the ORL1 and a partial agonist at the mu o piate receptor. When human embryonic kidney 293 cells stably expressing the human ORL1 receptor were pre-exposed (30 min) to either OFQ/N or Ro 64-619 8, the ability of both agonists to inhibit forskolin-mediated CAMP accumula tion was strongly reduced, indicating a functional desensitization of the s econd messenger cascade. However, acidic washes of OFQ/N-exposed cells full y restored the sensitivity of the ORL1 receptor for agonists. In contrast, the CAMP response in Ro 64-6198-exposed cells remained impaired after acidi c washes, suggesting sustained receptor internalization at 30 min. In agree ment with this finding, the number of cell-surface ORL1 receptors was signi ficantly reduced after Ro 64-6198 pre-exposure, and this effect could be bl ocked with high sucrose concentrations. When Ro 64-6198 was chronically adm inistered to rats, no signs of tolerance to its anxiolytic-like effects wer e detected following 15 days of daily drug exposure. In agreement with the behavioral results, Ro 64-6198 was able to reduce brain ORL1 binding sites in both acutely and chronically treated rats. Full recovery of ORL1 binding sites was observed 24 h after Ro 64-6198 administration with a t(1/2) of s imilar to5.5 h. These data show that nonpeptide agonists at the ORL1 recept or have a good clinical potential as anxiolytics without causing tolerance.