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

• Published in: European journal of pharmacology 1999-10, Vol.383 (2), p.209-214
• Main Authors: Márki, Árpád, Monory, Krisztina, Ötvös, Ferenc, Tóth, Géza, Krassnig, Roland, Schmidhammer, Helmut, Traynor, John R, Roques, Bernard P, Maldonado, Rafael, Borsodi, Anna
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; Cell receptors; Cell structures and functions; Cyprodime; Fundamental and applied biological sciences. Psychology; GTPγS binding; Molecular and cellular biology; Morphine; Neuropeptide receptors; Radioligand; μ-Opioid receptor
• ISSN: 0014-2999; 1879-0712
• DOI: 10.1016/S0014-2999(99)00610-X

The use of compounds with high selectivity for each opioid receptor (μ, δ and κ) is crucial for understanding the mechanisms of opioid actions. Until recently non-peptide μ-opioid receptor selective antagonists were not available. However, N-cyclopropylmethyl-4,14-dimethoxy-morphinan-6-one (cyprodime) has shown a very high selectivity for μ-opioid receptor in in vivo bioassays. This compound also exhibited a higher affinity for μ-opioid receptor than for δ- and κ-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 high 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 μ, δ and κ tritiated specific ligands confirmed the selective labelling of cyprodime to a μ-opioid receptor population. The μ-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 δ- ([ d-Pen 2, d-Pen 5]enkephalin (DPDPE)) and κ- (5α,7α,8β-(−)- 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 [ 35S]GTPγS binding. The EC 50 value of morphine increased about 500-fold in the presence of 10 μM cyprodime. These findings clearly indicate that cyprodime is a useful selective antagonist for μ-opioid receptor characterization.