A series of novel high affinity opioid receptor ligands have been made whereby the phenolic-OH group of nalbuphine, naltrexone methiodide, 6-desoxonaltrexone, hydromorphone and naltrindole was replaced by a carboxamido group and the furan ring was opened to the corresponding 4-OH derivatives. These furan ring 'open' derivatives display very high affinity for mu and kappa receptors and much less affinity for delta. The observation that these target compounds have much higher receptor affinity than the corresponding ring 'closed' carboxamides significantly strengthens our underlying pharmacophore hypothesis concerning the bioactive conformation of the carboxamide group.

The role of the opioid system in controlling pain, reward and addiction is well established, but its role in regulating other emotional responses is poorly documented in pharmacology. The mu-, delta- and kappa- opioid receptors (encoded by Oprm, Oprd1 and Oprk1, respectively) mediate the biological activity of opioids. We have generated Oprd1-deficient mice and compared the behavioural responses of mice lacking Oprd1, Oprm (ref. 6) and Oprk1 (ref. 7) in several models of anxiety and depression. Our data show no detectable phenotype in Oprk1-/- mutants, suggesting that kappa-receptors do not have a role in this aspect of opioid function; opposing phenotypes in Oprm-/- and Oprd1-/- mutants which contrasts with the classical notion of similar activities of mu- and delta-receptors; and consistent anxiogenic- and depressive-like responses in Oprd1-/- mice, indicating that delta-receptor activity contributes to improvement of mood states. We conclude that the Oprd1-encoded receptor, which has been proposed to be a promising target for the clinical management of pain, should also be considered in the treatment of drug addiction and other mood-related disorders.

Opioid peptides have been shown to modulate various parameters of both the humoral and cellular arms of the immune system. The modulatory capacity of the peptides can often be substantially reduced in the presence of naloxone, an opioid receptor antagonist, indicating a classical ligand-receptor interaction. In order to characterize these interactions further, we investigated the characteristics of opioid receptors on a macrophage cell line, P388d1. A delta-class opioid receptor was found with an Mr of 58,000. We also identified opioid receptors on MOLT-4 (T-cell) and IM-9 (B cell) cell lines as well as thymocytes and T cell-and B cell-enriched populations. Using the central (brain) kappa-selective agonist, U-69,593, it was also determined that P388d1 cells possess kappa-like opioid receptors. Scatchard analysis of the binding of [3H]U-69,593 revealed a single population of sites with a dissociation constant of 17 +/- 3 (S.E.M.) nmol/l and a total number of binding sites of 53.8 +/- 1.0 (S.E.M.) fmol/10(6) cells. Moreover, the racemic kappa-selective agonist U-50,488H was able to displace 50% of [3H]U-69,593 binding at 8.0 nmol/l, whereas other opioid ligands such as [Met]-enkephalinamide (delta-selective) and [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (mu-selective) were ineffective displacers of [3H]U-69,593 except at high concentrations.

This study was to identify specific regions in kappa opioid receptors that accounted for binding selectivity of kappa ligands. Six chimeric mu/kappa receptors were constructed from cloned rat kappa and mu opioid receptors and transiently expressed in COS-1 cells. All six chimeric mu/kappa receptors bound [3H] diprenorphine with high affinities, indicating that these chimeras retain opioid receptor conformation. Binding affinities of three peptide ligands (dynorphin A, alpha-neo-endorphin, and dynorphin B) and three nonpeptide ligands (norbinaltorphimine, U50,488H, and U69,593) for chimeras were determined and compared to those for mu and kappa opioid receptors. The second extracellular loop and the adjoining C-terminal portion of the fourth transmembrane helix were essential for the high affinity binding of dynorphin A, alpha-neo-endorphin, and dynorphin B to the kappa receptor. The third extracellular loop and the sixth and seventh transmembrane helices played an important role in determining the selectivity of nor-binaltorphimine for the kappa over the mu receptor. U50,488H and U69,593 appeared to require the whole kappa receptor except the second extracellular loop to attain high affinity binding. Thus, the kappa opioid receptor has differential binding domains for peptide and non-peptide ligands.

The three agonists, methionine-enkephalin, leucine-enkephalin and beta-endorphin have different pharmacological patterns. It may be of particular importance that they vary in their relative affinities to the enkephalin and naltrexone binding sites in the brain; the former are probably related to delta-receptors prevalent in the mouse vas deferens and the later to mu-receptors prevalent in the guinea-pig. It is possible that mu-receptors are more important for the mediation of analgesic effects than delta-receptors. An understanding of the pharmacokinetics of the opioid peptides will be of basic importance for the design of enkephalin analogues suitable for use as analgesics in man.

To determine the duration of blockade of mu-opiate receptors by naltrexone, we measured the binding of [11C]carfentanil in the brain of five normal volunteers with a positron radiation detection system before and 1, 48, 72, 120, and 168 hr after naltrexone administration. The half-time of blockade by naltrexone in the brain ranged from 72 to 108 hr which is greater than the fast plasma clearance components (4-12 hr) of naltrexone or its metabolite, beta-naltrexol, but corresponds well to the half-time of the terminal phase of plasma naltrexone clearance (96 hr). These results are consistent with the duration of the pharmacologic effects of naltrexone in response to heroin administration and indicate that 50 mg/day of oral naltrexone results in plasma levels in excess of that needed to saturate opiate receptors. This is the first example of the use of a simple dual-detector system with positron-emitting radioactive drugs to provide information regarding the duration of action of the drug on its specific receptor site. The plasma clearance half-time of a drug may not give an accurate reflection of the duration of action of the drug on a specific neuroreceptor site. Direct measurement of drug effects on recognition sites greatly extend current studies of pharmacokinetics.

Nalmefene (6-methylene-naltrexone) is a potent, orally active, opiate antagonist. IC50's were obtained for nalmefene, naloxone and naltrexone using radiolabelled prototype ligands for mu, kappa and delta receptors in homogenates of rat brain minus cerebellum. Nalmefene antagonized the bindings of [3H]-dihydromorphine,[3H]-ethylketocyclazocine and [3H]-D-ala-D-leu enkephalin with IC50's in the low nanomolar range. At the central mu receptor, nalmefene bound with an IC50 of 1.0 nM, equal to that of naltrexone and approximately four times lower than that of naloxone. At central kappa and delta sites the IC50's for nalmefene were somewhat lower than those of naltrexone and considerably lower than those of naloxone. All three antagonists had sodium indices less than 1.0. These results indicate that nalmefene is a universal opiate antagonist, has no agonist character at the central mu site and binds more effectively to central opiate receptors than either naloxone or naltrexone.

Guinea pig brain membranes depleted of mu and delta receptors by pretreatment with the site-directed acylating agents, 2-(4-ethoxybenzyl)-1- diethylaminoethyl-5-isothiocyanatobenzimidazole.HCl (BIT) and N-phenyl-N-[1-(2-(4-isothiocyanato)phenethyl)-4- piperidinyl]-propanamide.HCl (FIT), were used in this study to test the hypothesis that guinea pig brain possesses subtypes of kappa receptors. Pretreatment of membranes with either (-)-(1S,2S)-U50,488 or the kappa selective acylating agent,(1S,2S)-trans-2-isothiocyanato-N-methyl-N-[2-(1- pyrrolidinyl)cyclohexyl]benzeneacetamide, caused a wash-resistant inhibition of kappa 1 binding sites labeled by [3H]U69,593 binding, but not kappa 2 binding sites labeled by [3H]bremazocine. Binding surface analysis of [3H]bremazocine binding resolved two binding sites, termed kappa 2 and kappa 2b, present at densities of 212 and 225 fmol/mg protein, which had low affinity for (-)-(1S,2S)-U50,488 and U69,593. The kappa 2b site had high affinity for beta-endorphin(1-31)(Kd = 5.5 nM) and [D-Ala2,D-Leu5]enkephalin (Kd = 14 nM), and lower affinity for [D-Ala2-MePhe4,Gly-ol5]enkephalin (Kd = 147 nM) and [Leu5]enkephalin (Kd = 46.0 nM). Binding surface analysis of [3H]U69,593 binding also resolved two binding sites, termed kappa 1a and kappa 1b, present at densities of 6.0 and 40.0 fmol/mg protein. The kappa 1a binding site was characterized by very high affinity for alpha-neoendorphin. Quantitative autoradiographic studies demonstrated that kappa 2a and kappa 2b binding sites are heterogeneously distributed in guinea pig brain, and that the anatomical distribution of kappa 1 binding sites reported in the literature is different from that observed in this study for the kappa 2 binding sites. Viewed collectively, these data provide evidence for four kappa receptor subtypes in guinea pig brain.

It is unclear how receptor/ligand families that are evolutionarily closely related achieve functional separation. To address this question, we focus here on the newly discovered Orphanin FQ, a peptide homologous to the opioid peptide Dynorphin, and its receptor, the Orphanin FQ receptor, which is highly homologous to the opioid receptors. In spite of this high degree of homology in terms of both ligands and receptors, there is little direct cross-talk between the Orphanin FQ system and the endogenous opioid system. Thus, the opioid peptides show either relatively low affinity or no affinity toward the Orphanin FQ receptor; conversely, Orphanin FQ has no affinity toward any of the opioid receptors. We sought to investigate the molecular basis of such discrimination by attempting to reverse it and endowing the Orphanin FQ receptor with the ability to bind opioids. We report that by mutating as few as four amino acids, we can produce a receptor that recognizes pro-Dynorphin products with very high affinity and yet still binds Orphanin FQ as well as the wild-type receptor. This suggests that the Orphanin FQ receptor has developed features that specifically exclude the opioids and that these features are distinct from those required for the high affinity binding of its own endogenous ligand.

The structural determinants for the selective binding of the nonpeptide opioid receptor antagonist nor-binaltorphimine (nor-BNI) to the kappa-opioid receptor were characterized using a systematic series of chimeras between the kappa receptor and the homologous mu-opioid receptor. All 10 chimeric constructs bound the nonselective antagonists (-)-naloxone and diprenorphine with similar affinities, as did the two wild-type receptors. Introduction of amino-terminal segments of increasing length, extending to and including transmembrane segment VI, from the mu receptor into the kappa receptor did not impair the high affinity binding of nor-BNI, and neither did introduction of the intracellular carboxyl-terminal extension of the mu receptor. In contrast, nor-BNI binding was impaired > or = 600-fold in constructs in which extracellular loop 3 and transmembrane segment VII originated from the mu receptor. The exchange of a single residue within this region, Glu297, for lysine, the corresponding residue from the mu receptor, reduced the binding affinity of nor-BNI 142-fold, without affecting the binding the nonselective compounds (-)-naloxone and diprenorphine. It is concluded that the selective binding of nor-BNI to the kappa-opioid receptor is determined by nonconserved residues located in extracellular loop 3 and transmembrane segment VII and that Glu297, located just outside transmembrane segment VI, plays a major role in the kappa-selective binding characteristics of nor-BNI.