Binding characteristics of [3H]14-methoxymetopon, a high affinity μ-opioid receptor agonist

The highly potent mu-opioid receptor agonist 14-methoxymetopon (4,5alpha-epoxy-3-hydroxy-14beta-methoxy-5beta,17-dimethylmorphinan-6-one) was prepared in tritium labelled form by a catalytic dehalogenation method resulting in a specific radioactivity of 15.9 Ci/mmol. Opioid binding characteristics of [(3) H]14-methoxymetopon were determined using radioligand binding assay in rat brain membranes. [(3) H]14-Methoxymetopon specifically labelled a single class of opioid sites with affinity in low subnanomolar range (K (i) = 0.43 nm) and maximal number of binding sites of 314 fmol/mg protein. Binding of [(3) H]14-methoxymetopon was inhibited by ligands selective for the mu-opioid receptor with high potency, while selective kappa-opioids and delta-opioids were weaker inhibitors. 14-Methoxymetopon increased guanosine-5'-O -(3-[(35) S]thio)-triphosphate ([(35) S]GTPgammaS) binding with an EC50 of 70.9 nm, thus, providing evidence for the agonist character of this ligand. The increase of [(35) S]GTPgammaS binding was inhibited by naloxone and selective mu-opioid antagonists, indicating a mu-opioid receptor-mediated action. [(3) H]14-Methoxymetopon is one of the few nonpeptide mu-opioid receptor agonists available in radiolabelled form up to now. Due to its high affinity and selectivity, high stability and extremely low nonspecific binding (<10%), this radioligand would be an important and useful tool in probing mu-opioid receptor mechanisms, as well as to promote a further understanding of the opioid system at the cellular and molecular level.