Roland Krassnig

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 (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 (Kd 3.8±0.18 nM) to membranes of rat brain affording a Bmax 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 (Ki 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.

Signal transduction efficacy of the highly potent mu opioid agonist 14-methoxymetopon.

In search of a truly high-efficacy (i.e., tau > 100) mu opioid analgesic, we determined the efficacy (tau) and apparent in vivo affinity (KA) of the high-potency alkoxymorphinan 14-methoxymetopon. However, in the present study, 14-methoxymetopons efficacy proved to be only 1.5-fold higher than that of morphine (tau, 19 vs. 12). KA values were 2,900 nmol/kg for 14-methoxymetopon and 46,000 nmol/kg for morphine (Ki for [3H]DAMGO binding, 0.33 vs 3.4 nmol/l). Thus, the 24-fold higher potency of methoxymetopon could be fully accounted for by its 16-fold higher apparent in vivo affinity and its only 1.5-fold higher efficacy. Furthermore, the 10-fold higher affinity of 14-methoxymetopon for the mu opioid receptor - as previously determined in radioligand binding assays - was confirmed in the present behavioral tests of thermal antinociception.

Opioid peptide receptor studies. 9. Identification of a novel non-μ- non-δ-like opioid peptide binding site in rat brain

Abstract The two binding sites had lower (δ ncx-2 , Ki = 96.6 nM) and higher (δ ncx-1 , Ki = 1.55 nM) affinity for DPDPE. The ligand-selectivity profile of the δ ncx-1 site was that of a classic δ binding site. The ligand-selectivity profile of the δ ncx-2 site was neither μ- or δ-like. The Ki values of selected agents for the δ ncx-2 site were: [pCl]DPDPE (3.9 nM), DPLPE (140 nM), and DAMGO (2.6 nM). Under these assay conditions, [ 3 H][ d -Ala 2 , d -Leu 5 ]enkephalin binding to the cells expressing the cloned μ receptor is very low and pretreatment of cell membranes with BIT almost completely inhibits [ 3 H]DAMGO and [ 3 H][ d -Ala 2 , d -Leu 5 ]enkephalin binding. Intracerebroventricular administration of antisense DNA to the cloned delta receptor selectively decreased [ 3 H][ d -Ala 2 , d -Leu 5 ]enkephalin binding to the δ ncx-1 site. Administration of buprenorphine to rats 24 h prior to preparation of membranes differentially affected μ, δ ncx-1 , and δ ncx-2 binding sites. Viewed collectively, these studies have identified a novel non-μ- non-δ-like binding site in rat brain.

A New and Efficient Synthesis of the m-Selective Opioid Antagonist Cyprodime

The μ-selective opioid antagonist cyprodime has been prepared in a six- step sequence starting from naltrexone. The 3-hydroxy group of naltre- xone was removed via tetrazolyl ether (3) which was hydrogenated cata- lytically to give 17-cyclopropylmethyl-4,5α-epoxy-14-hydroxymorphinan- 6-one (4). Methylation gave 17-cyclopropylmethyl-6,7-didehydro-4,5α-epo- xy-6,14-dimethoxymorphinan (5) and hydrolysis of it 17-cyclopropylme- thyl-4,5α-epoxy-14-methoxymorphinan-6-one (6). Reductive opening of the 4,5-epoxy bridge and methylation of the resulting phenol (7) yielded cyprodime (1)