Characterization of the bifunctional opioid UFP505

Abstract

Morphine is a gold standard analgesic acting at μ-opioid receptors (MOP) with analgesia accompanied by side-effects, for example, tolerance. If δ-opioid receptors (DOP) are blocked at the same time that MOP is activated, then analgesia with reduced tolerance results. As part of an ongoing programme to design mixed MOP agonist/DOP antagonist drugs (bifunctional) we have characterized the prototype pseudopeptide H-Dmt-Tic-Gly-NH-CH2-Ph (UFP505). We have measured ability to interact with MOP and DOP receptors with differential agonism/antagonism and the effects of 1 h pretreatment on cell surface MOP receptor numbers. We have used Chinese Hamster Ovary cells expressing human recombinant MOP, DOP KOP (κ), and NOP (nociceptin/orphanin FQ) receptors (CHOhMOP/hDOP/hKOP/ hNOP). Receptor binding was measured using the radioligand 3]diprenorphine (3]DPN) in saturation format to determine receptor density (Bmax) and affinity (pKD) or displacement format to determine the affinity (pKi) of UFP505. Functional activity at MOP and DOP was assessed by measuring agonist stimulated GTPγ35S] binding. Loss of cell surface receptors was measured using 3]DPN in saturation format after 1 h treatment of CHOhMOP with 10 µM UFP505. UFP505 displaced 3]DPN binding to MOP and DOP receptor with pKi 7.79 (0.18) and 9.82 (0.06) mean (SEM), n=5], respectively. Affinity at KOP and NOP was negligible. At MOP in a GTPγ35S] binding assay, UFP505 behaved as a full agonist (compared with MOP agonist endomorphin-1) with a pEC50 (potency) and Emax (efficacy:stimulation factor) of 6.23 (0.15) and 2.24 (0.15) (n=3), respectively. At DOP, UFP505 (10 µM) reversed the effects of the DOP agonist DPDPE in the GTPγ35S] assay with potency (pKB) of 9.81 (0.18) (n=3), in agreement with pKi determined in 3]DPN binding assays. Pretreatment of CHOhMOP cells with 10 µM UFP505 for 1 h produced substantial loss of cell surface receptors with no change in DPN pKD. Bmax and pKD values for control and 1 h UFP505 treated cells were 332 (53) fmol mg-1 protein and 9.20 (0.11) and 155 (12) fmol mg-1 protein and 9.15 (0.10), respectively (n=5). Here we show that UFP505 is a bifunctional MOP agonist/ DOP antagonist capable of producing tolerance like actions in a single MOP expression system. The next stage is to examine the effects of this ligand in a double (MOP and DOP) expression system. ABSTRACT FROM AUTHOR]; Copyright of BJA: The British Journal of Anaesthesia is the property of Oxford University Press / USA and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)