Separation of Binding Affinity and Intrinsic Activity of the Potent -Opioid 14-Methoxymetopon

Receptor binding studies of 5,14-O-dimethyloxymorphone (14methoxymetopon) in brain membranes have established its high affinity for -binding sites, but its analgesic potency far exceeds the modest increase in binding affinity relative to other opioids. The current study has established the selectivity of [H]14-methoxymetopon for sites in calf striatal membranes and for a number of full-length splice variants of the cloned murine -opioid receptor 1 (mMOR-1) in transfected cell lines. The binding affinity of [H]14-methoxymetopon for the variants expressed in Chinese hamster ovary cells was quite high, with KD values around 0.2 nM for all of the variants with the exception of mMOR-1F (KD of 1.2 nM). The affinity for most of the expressed variants was greater than that seen in the brain membranes (KD of 0.99 nM). Functionally, in guanosine 5 -O(3-[S]thio)triphosphate ([S]GTP S) binding assays with the MOR-1 variants, 14-methoxymetopon and the -opioid peptide [D-Ala,N-Me-Phe,Gly-ol]-enkephalin (DAMGO) showed similar efficacies, as determined by maximal stimulation, but 14-methoxymetopon was up to 65-fold more potent than DAMGO. The greatest difference was seen with mMOR-1E and the least with mMOR-1C, which displayed only a 10-fold difference. These potency differences in the stimulation of [S]GTP S binding far exceeded the differences in binding affinity. The differences between 14-methoxymetopon and DAMGO remained after normalizing the potency shifts based upon receptor binding affinities and varied from 1.2-fold with mMOR-1C to 21-fold for mMOR-1 and 42-fold with mMOR-1F. Thus, 14-methoxymetopon is a potent agonist against all of the mMOR-1 splice variants, but its potency ranged widely despite similar binding affinities for most of the variants and may give insight into its unusual pharmacological profile. 5,14-O-Dimethyloxymorphone (14-methoxymetopon) is a highly potent and selective -opiate agonist (Schmidhammer et al., 1990; Fürst et al., 1993; Freye et al., 2000; Zernig et al., 2000; King et al., 2003; Bileviciute-Ljungar et al., 2006) with a unique pharmacology and extraordinary analgesic potency that sets it apart from traditional agonists. Despite its high analgesic potency, it displays little respiratory depression, bradycardia, or sedation compared with sufentanil. It decreases gastrointestinal transit far less than morphine and reportedly develops lower levels of tolerance and physical dependence and a diminished propensity to cause convulsions in mice. Supraspinal 14-methoxymetopon analgesia is antagonized by 3-O-methylnaltrexone at a dose that antagonizes both heroin and MG6, but not morphine (King et al., 2003). Antisense mapping of MOR-1 also distinguishes 14methoxymetopon from other opioids, with 14-methoxymetopon analgesia blocked by MOR-1 antisense probes against exons 1, 2, and 8, a pattern, however, different from that of either morphine or M6G (King et al., 2003). Yet, the selectivity of 14-methoxymetopon for -opioid receptors is well established, based on receptor binding experiments and its sensitivity in vivo to -selective opioid antagonists, such as -funaltrexamine and naloxonazine, but not or antago-