Abstract
The novel nonpeptide orphanin FQ/nociceptin (OFQ/N) ligand {(1S,3aS)-8-(2,3,3a,4,5,6-hexahydro-1H-phenalen-1-yl)-1-phenyl-1,3,8-triaza-spiro[4.5]decan-4-one} (Ro 64-6198) was characterized in vitro and in vivo for its agonistic potential. Ro 64-6198 was 130- to 3500-fold selective for the OFQ/N receptor (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 opiate receptor. When human embryonic kidney 293 cells stably expressing the human ORL1 receptor were pre-exposed (30 min) to either OFQ/N or Ro 64-6198, the ability of both agonists to inhibit forskolin-mediated cAMP accumulation was strongly reduced, indicating a functional desensitization of the second messenger cascade. However, acidic washes of OFQ/N-exposed cells fully restored the sensitivity of the ORL1 receptor for agonists. In contrast, the cAMP response in Ro 64-6198-exposed cells remained impaired after acidic washes, suggesting sustained receptor internalization at 30 min. In agreement with this finding, the number of cell-surface ORL1 receptors was significantly reduced after Ro 64-6198 pre-exposure, and this effect could be blocked with high sucrose concentrations. When Ro 64-6198 was chronically administered to rats, no signs of tolerance to its anxiolytic-like effects were 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 at1/2 of ∼5.5 h. These data show that nonpeptide agonists at the ORL1 receptor have a good clinical potential as anxiolytics without causing tolerance.
Footnotes
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This study was supported by F. Hoffmann-La Roche AG.
- Abbreviations:
- ORL1
- OFQ/N receptor
- HEK293
- human embryonic kidney 293
- DAMGO
- [d-ala2,N-methyl-phe4, glyol5][tyrosyl-3,5]-enkephalin
- OFQ/N
- orphanin FQ/nociceptin
- OP1
- delta opiate receptor
- OP2
- kappa opiate receptor
- OP3
- mu opiate receptor
- Ro 64-6198
- {(1S,3aS)-8-(2,3,3a,4,5,6-hexahydro-1H-phenalen-1-yl)-1-phenyl-1,3,8-triaza-spiro[4.5]decan-4-one}
- BSA
- bovine serum albumin
- BHK
- baby hamster kidney
- CHO
- Chinese hamster ovary
- PBS
- phosphate-buffered saline
- ANOVA
- analysis of variance
- Received December 21, 2000.
- Accepted May 1, 2001.
- The American Society for Pharmacology and Experimental Therapeutics
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