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

A. Márki, Krisztina Monory, F. Ötvös, Géza Tóth, Roland Krassnig, Helmut Schmidhammer, John R. Traynor, Bernard P. Roques, Rafael Maldonado, A. Borsodi

Research output: Article

32 Citations (Scopus)

Abstract

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-Ala2,N-MePhe4,Gly5-ol]enkephalin (DAMGO) was readily displaced by cyprodime (K(i) values in the low nanomolar range) while the competition for δ- ([D-Pen2,D-Pen5]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 EC50 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. Copyright (C) 1999 Elsevier Science B.V.

Original languageEnglish
Pages (from-to)209-214
Number of pages6
JournalEuropean Journal of Pharmacology
Volume383
Issue number2
DOIs
Publication statusPublished - okt. 27 1999

Fingerprint

Narcotic Antagonists
Opioid Receptors
Biological Assay
Morphine
Morphinans
D-Penicillamine (2,5)-Enkephalin
Membranes
Tritium
Enkephalins
Brain
cyprodime
In Vitro Techniques
Benzene
Radioactivity
Opioid Analgesics
Ligands
Population

ASJC Scopus subject areas

  • Cellular and Molecular Neuroscience
  • Pharmacology

Cite this

μ-Opioid receptor specific antagonist cyprodime : Characterization by in vitro radioligand and [35S]GTPγS binding assays. / Márki, A.; Monory, Krisztina; Ötvös, F.; Tóth, Géza; Krassnig, Roland; Schmidhammer, Helmut; Traynor, John R.; Roques, Bernard P.; Maldonado, Rafael; Borsodi, A.

In: European Journal of Pharmacology, Vol. 383, No. 2, 27.10.1999, p. 209-214.

Research output: Article

Márki, A. ; Monory, Krisztina ; Ötvös, F. ; Tóth, Géza ; Krassnig, Roland ; Schmidhammer, Helmut ; Traynor, John R. ; Roques, Bernard P. ; Maldonado, Rafael ; Borsodi, A. / μ-Opioid receptor specific antagonist cyprodime : Characterization by in vitro radioligand and [35S]GTPγS binding assays. In: European Journal of Pharmacology. 1999 ; Vol. 383, No. 2. pp. 209-214.
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AU - Ötvös, F.

AU - Tóth, Géza

AU - Krassnig, Roland

AU - Schmidhammer, Helmut

AU - Traynor, John R.

AU - Roques, Bernard P.

AU - Maldonado, Rafael

AU - Borsodi, A.

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N2 - 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-Ala2,N-MePhe4,Gly5-ol]enkephalin (DAMGO) was readily displaced by cyprodime (K(i) values in the low nanomolar range) while the competition for δ- ([D-Pen2,D-Pen5]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 EC50 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. Copyright (C) 1999 Elsevier Science B.V.

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