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Relative efficacies of δ-opioid receptor agonists at the cloned human δ-opioid receptor

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Abstract

The present study was conducted to determine the relative efficacies of the selective δ-opioid receptor agonists SNC80 ((+)-4-[(αR)-α-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide), pCl-DPDPE (cyclic[d-Pen2,4′-ClPhe4,d-Pen5]enkephalin) and (−)-TAN67 ((−)-2-methyl-4aα-(3-hydroxyphenyl)-1,2,3,4,4a,5,12,12aα-octahydro-quinolino-[2,3,3-g]isoquinoline). Experiments compared the abilities of the three drugs to competitively inhibit [3H]naltrindole binding and also stimulate [35S]GTPγS binding in membranes prepared from stably transfected Chinese hamster ovary (CHO) cells that express the cloned human δ-opioid receptor. Efficacy was determined according to the formula: efficacy=(Emax-A/Emax)(A′/A+1)×0.5. Results show that SNC80 and pCl-DPDPE had efficacy values that were about 6–7 times greater than that of (−)-TAN67.

Introduction

It has long been recognized that drugs with similar receptor affinities are not necessarily equally efficacious in evoking behavioral or neurochemical effects. For example, the nonpeptidic δ-opioid receptor agonist (±)-TAN67 ((±)-2-methyl-4aα-(3-hydroxyphenyl)-1,2,3,4,4a,5,12,12aα-octahydro-quinolino-[2,3,3-g]isoquinoline) has high affinity for the δ-opioid receptors in the rat brain (Ki=0.7 nM) and is also highly selective for the δ-opioid receptors (1600–2000 times greater affinity than at the μ- or κ-opioid receptors) (Suzuki et al., 1995). Yet (±)-TAN67 possesses no antinociceptive activity in the 51°C warm plate antinociceptive test (Suzuki et al., 1995).

The present study was conducted to ascertain the efficacy of the high-affinity stereoisomer (−)-TAN67 relative to two other selective δ-opioid receptor drugs, the nonpeptidic compound SNC80 ((+)-4-[(αR)-α-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide) and the peptidic agent pCl-DPDPE (cyclic[d-Pen2,4′-ClPhe4,d-Pen5]enkephalin), by comparing their ability to competitively inhibit [3H]naltrindole binding (Yamamura et al., 1992) and to stimulate [35S]GTPγS binding (Sim et al., 1995).

Previous work in this laboratory resulted in the initial cloning of the human δ-opioid receptor (Knapp et al., 1994) and its subsequent stable transfection and expression by Chinese hamster ovary (CHO) cells (Malatynska et al., 1995). The use of a recombinant cell line expressing a homogeneous population of the human δ-opioid receptor permits the study of the receptor in isolation without the potentially confounding interference of other opioid receptors.

Section snippets

Preparation of membranes from CHO cells

Crude membranes were prepared from CHO cells that express the human δ-opioid receptor. The recombinant CHO cells were produced by stable transfection of the human δ-opioid receptor cDNA (Malatynska et al., 1995). These cells were determined to express human δ-opioid receptor at a density of 968±170 fmol/mg protein (Malatynska et al., 1995). The cell line was grown in 162 cm2 culture flasks using Ham's F-12 with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, and 500 μg/ml

Results

SNC80, pCl-DPDPE and (−)-TAN67 all competitively inhibited [3H]naltrindole binding and stimulated [35S]GTPγS binding in membranes prepared from CHO cells that stably express the human δ-opioid receptor. In the [3H]naltrindole binding study, results demonstrate that the affinity of (−)-TAN67 for the human δ-opioid receptor is approximately 20–25-times greater than those of SNC80 and pCl-DPDPE which are approximately equal (Fig. 1A). In the [35S]GTPγS binding study (Fig. 1B), findings show that

Discussion

The concept of efficacy was introduced by Stephenson (1956)in an effort to explain how drugs might evoke effects of equal magnitude while occupying different proportions of target receptors. Efforts have been made to determine the efficacy of various agonist drugs in vivo and in vitro using a variety of paradigms. For example, many of these approaches are based on the assumptions that opioid receptors in the mouse vas deferens or guinea pig ileum are identical with comparable opioid receptors

Acknowledgements

This research was supported in part by grants from the Arizona Disease Control Research Commission and N.I.D.A. We also thank Dr. Frederick J. Ehlert (University of California, Irvine, CA, USA) for his helpful discussion of the results.

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1

Present address: Department of Biomedical Sciences, University of Illinois College of Medicine at Rockford, Rockford, IL 61107, USA.

2

Present address: Cortex Pharmaceuticals, Irvine, CA 92718, USA.

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