μ-Opioid receptor specific antagonist cyprodime:: characterization by in vitro radioligand and [35S]GTPγS binding assays

The use of compounds with high selectivity for each opioid receptor (mu, delta and kappa) is crucial for understanding the mechanisms of opioid actions, Until recently non-peptide mu-opioid receptor selective antagonists were not available. However, N-cyclopropylmethyl-4,14-dimethoxy-morphinan-6-one (cyprodime) has shown a very high selectivity for mu-opioid receptor in in vivo bioassays. This compound also exhibited a higher affinity for mu-opioid receptor than for delta- and kappa-opioid receptors in binding assays in brain membranes, although the degree of selectivity was lower than in in vitro bioassays. Cyprodime has recently been radiolabelled with tritium resulting in kish specific radioactivity (36.1 Ci/mmol). We found in in vitro binding experiments that this radioligand bound with high affinity (K-d 3.8 +/- 0.18 nM) to membranes of rat brain affording a B-max of 87.1 +/- 4.83 fmol/mg, Competition studies using mu, delta and kappa tritiated specific ligands confirmed the selective labelling of cyprodime to a mu-opioid receptor population. The mu-opioid receptor selective agonist [D-Ala(2), N-MePhe(4), Gly(5)-ol]enkephalin (DAMGO) was readily displaced by cyprodime (K-i values in the low nanomolar range) while the competition for delta-([D-Pen(2), D-Pen(5)]enkephalin (DPDPE)) and kappa-(5 alpha, 7 alpha, 8 beta-(-)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro(4,5)dec-8-yl]-benzene-acetamide (U69,593)) opioid receptor selective compounds was several orders of magnitude less. We also found that cyprodime inhibits morphine-stimulated [S-35]GTP gamma S binding. The EC50 value of morphine increased about 500-fold in the presence of 10 mu M cyprodime. These findings clearly indicate that cyprodime is a useful selective antagonist for mu-opioid receptor characterization. (C) 1999 Elsevier Science B.V. All rights reserved.