Pyrrolo- and pyridomorphinans: non-selective opioid antagonists and delta opioid agonists/mu opioid partial agonists.

Opioid ligands have found use in a number of therapeutic areas, including for the treatment of pain and opiate addiction (using agonists) and alcohol addiction (using antagonists such as naltrexone and nalmefene). The reaction of imines, derived from the opioid ligands oxymorphone and naltrexone, with Michael acceptors leads to pyridomorphinans with structures similar to known pyrrolo- and indolomorphinans. One of the synthesized compounds, 5e, derived from oxymorphone had substantial agonist activity at delta opioid receptors but not at mu and/or kappa opioid receptors and in that sense profiled as a selective delta opioid receptor agonist. The pyridomorphinans derived from naltrexone and naloxone were all found to be non-selective potent antagonists and as such could have utility as treatments for alcohol abuse.

Endogenous regulators of G protein signaling differentially modulate full and partial mu-opioid agonists at adenylyl cyclase as predicted by a collision coupling model.

Regulator of G protein signaling (RGS) proteins accelerate the endogenous GTPase activity of Galpha(i/o) proteins to increase the rate of deactivation of active Galpha-GTP and Gbetagamma signaling molecules. Previous studies have suggested that RGS proteins are more effective on less efficiently coupled systems such as with partial agonist responses. To determine the role of endogenous RGS proteins in functional responses to mu-opioid agonists of different intrinsic efficacy, Galpha(i/o) subunits with a mutation at the pertussis toxin (PTX)-sensitive cysteine (C351I) and with or without a mutation at the RGS binding site (G184S) were stably expressed in C6 glioma cells expressing a mu-opioid receptor. Cells were treated overnight with PTX to inactivate endogenous G proteins. Maximal inhibition of forskolin-stimulated adenylyl cyclase by the low-efficacy partial agonists buprenorphine and nalbuphine was increased in cells expressing RGS-insensitive Galpha(o)(CIGS), Galpha(i2)(CIGS), or Galpha(i3)(CIGS) compared with their Galpha(CI) counterparts, but the RGS-insensitive mutation had little or no effect on the maximal inhibition by the higher efficacy agonists DAMGO and morphine. The potency of all the agonists to inhibit forskolin-stimulated adenylyl cyclase was increased in cells expressing RGS-insensitive Galpha(o)(CIGS), Galpha(i2)(CIGS), or Galpha(i3)(CIGS), regardless of efficacy. These data are comparable with predictions based on a collision coupling model. In this model, the rate of G protein inactivation, which is modulated by RGS proteins, and the rate of G protein activation, which is affected by agonist intrinsic efficacy, determine the maximal agonist response and potency at adenylyl cyclase under steady state conditions.

A comparison of the physiological, behavioral, neurochemical and microglial effects of methamphetamine and 3,4-methylenedioxymethamphetamine in the mouse.

3,4-Methylenedioxymethamphetamine (MDMA) and methamphetamine (METH) are amphetamine analogues with similar persistent neurochemical effects in the mouse which some have described as neurotoxicity. We attempted to identify dose regimens of MDMA and METH with similar effects on behavioral and physiological variables in the mouse, then quantified the effects of these dose regimens on neurochemistry and microglial markers. Four discrete injections of saline, MDMA (10, 20, or 30 mg/kg), or METH (5 or 10 mg/kg) were administered to mice at 2 h intervals. Body weight was quantified immediately before each injection, and 2 h after the last injection, while core temperature and locomotor activity were continuously monitored via radiotelemetry. Mice were killed 72 h after the final injection and brains were rapidly dissected on ice. Dopamine content in various brain regions was quantified via high pressure liquid chromatography (HPLC), and microglial activation was assessed by saturation binding of the peripheral benzodiazepine receptor (PBR) ligand 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline carboxamide ([(3)H]PK11195). Specific dose regimens of MDMA and METH induced similar reductions in body weight, depletions of dopamine and its metabolites, and similar hyperthermic and locomotor stimulant effects, but only METH activated microglia in striatum. These results suggest that repeated high doses of MDMA and METH that produce hyperthermia, locomotor stereotypy, weight loss and neurochemical depletion are not consistently accompanied by microglial activation. The finding that METH, but not MDMA, induces microglial effects in the striatum consistent with neurotoxicity might imply different mechanisms of toxic action for these two psychostimulants.

Inhibition of rat liver cholesterol esterase by local anaesthetics.

1. A number of local anaesthetics was shown to inhibit rat liver cholesterol esterase activity towards radioactively labelled cholesterol oleate. The anaesthetics inhibited in the order dibucaine greater than chlorpromazine greater than tetracaine greater than benzocaine greater than procaine greater than lidocaine greater than cocaine. 2. The mode of inhibition was seen to be non-competitive with respect to the substrate and is probably independent of any involvement of Ca2+. 3. The inhibition by tetracaine is partially reversed by sodium deoxycholate. However, all ionic and non-ionic detergents studied, sodium deoxycholate, sodium taurocholate, Triton X-100, and cetyltrimethylammonium bromide are capable of inhibiting the rat liver cholesterol esterase in a concentration dependent manner. Only sodium taurocholate stimulates enzymic activity.

Phospholipase A2 activity of lysosomal origin secreted by polymorphonuclear leucocytes during phagocytosis or on treatment with calcium.

1. Peritoneal neutrophil leucocytes, derived from the rabbit, release phospholipase A (EC 3.1.1.4) activity during phagocytosis of complement-coated zymosan particles, or during treatment with Ca2+. This enzyme is able to release [1-14C]oleate from the membrane phospholipids of Escherichia coli. 2. The release of phospholipase A paralleled that of the known lysosomal marker enzymes beta-glucuronidase and lysozyme. The phospholipase A would thus appear to be derived from the lysosomal granules of the cells. 3. The released enzyme is of A2 specificity, has an absolute requirement for divalent cations, and is active over a broad pH range (pH 6-9).

Effects of local anaesthetics on the lipase of Rhizopus arrhizus.

1. A number of local anaesthetics were shown to inhibit the hydrolytic activity of a partially purified lipase from the mold Rhizopus arrhizur towards both triacylglycerol and phospholipid substrates. 2. Irrespective of whether triacylglycerol or phospholipid substrates were used, the inhibition was uncompetitive with respect to the substrate, independent of Ca2+ concentration, but pH dependent. 3. The inhibitory activity of the local anaesthetics studied closely paralleled the anaesthetic potency of the compounds.

Effects of local anaesthetics on phospholipases.

1. The effects of six local anaesthetics have been studied on the activities of soluble phospholipases A2 (EC 3.1.1.4) and lysophospholipase (EC 3.1.1.5). 2. Phospholipase A2 activity in human seminal plasma towards sonicated radioactively-labelled phosphatidylethanolamine was slightly stimulated a low and inhibited at high concentrations of all anaesthetic compounds employed. The order of decreasing potency was chlorpromazine, dibucaine, tetracaine, lidocaine, cocaine and procaine. In line with previous findings, the mode of inhibition was seen to be competitive with respect to Ca2+. 3. Phospholipase A2 activity in crude venom of Crotalus adamanteus was not affected or slightly stimulated by local anaesthetics up to 10(-2) M concentrations, when egg yolk was used as substrate. However, with sonicated radioactively-labelled phosphatidylcholine or phosphatidylethanolamine as substrate, stimulation of phospholipase activity was seen with all local anaesthetics up to 10(-2) M, the order of decreasing potency again being chlorpromazine, dibucaine, tetracaine, lidocaine, cocaine and procaine. The mode of stimulation was seen to be un-competitive with respect to substrate and probably independent of any involvement of Ca2+. 4. As in seminal plasma phospholipase A2, the activity in crude Naja naja venom towards sonicated radioactively labelled phosphatidylcholine was stimulated at low and inhibited at high concentrations of dibucaine and chloropromazine, for example. The mode of inhibition was seen to be competitive with respect to Ca2+, whereas stimulation by the anaesthetic drugs was independent of Ca2+. Binding between drug and enzyme was demonstrated by equilibration filtration of purified phospholipase A2 of Naja naja venom through a Sephadex G 25-fine column, previously equilibrated with 0.5 mM radioactively labelled chlorpromazine. 5. Lysophospholipase activity in rat liver cytosol towards radioactively labelled lysophosphatidylcholine was inhibited by all local anaesthetics used; the order of decreasing potency was chlorpromazine, dibucaine, tetracaine, cocaine, lidocaine and procaine. The inhibition was un-competitive with respect to substrate. 6. The inhibitory and stimulatory potencies of the local anaesthetics employed closely parallel their lipid solubilities and anaesthetic potencies.

Positive allosteric modulators of the µ-opioid receptor: a novel approach for future pain medications.

UNLABELLED: Morphine and other agonists of the µ-opioid receptor are used clinically for acute and chronic pain relief and are considered to be the gold standard for pain medication. However, these opioids also have significant side effects, which are also mediated via activation of the µ-opioid receptor. Since the latter half of the twentieth century, researchers have sought to tease apart the mechanisms underlying analgesia, tolerance and dependence, with the hope of designing drugs with fewer side effects. These efforts have revolved around the design of orthosteric agonists with differing pharmacokinetic properties and/or selectivity profiles for the different opioid receptor types. Recently, µ-opioid receptor-positive allosteric modulators (µ-PAMs) were identified, which bind to a (allosteric) site on the µ-opioid receptor separate from the orthosteric site that binds an endogenous agonist. These allosteric modulators have little or no detectable functional activity when bound to the receptor in the absence of orthosteric agonist, but can potentiate the activity of bound orthosteric agonist, seen as an increase in apparent potency and/or efficacy of the orthosteric agonist. In this review, we describe the potential advantages that a µ-PAM approach might bring to the design of novel therapeutics for pain that may lack the side effects currently associated with opioid therapy. LINKED ARTICLES: This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2.

Characterisation of kappa-opioid binding sites in rat and guinea-pig spinal cord.

The binding of radiolabelled ligands with high affinity for kappa-opioid binding sites has been studied in homogenates of lumbo-sacral spinal cord from the rat. The unselective ligands [3H]bremazocine and [3H]diprenorphine labelled a large number of sites which could not be fully resolved in terms of mu-, delta- and kappa-types by displacement assays. In particular binding at the kappa-site appeared anomalous in that sites which could be identified as high affinity kappa-type represented only 40% of total kappa-binding, defined using the unselective [3H]ligands. This was confirmed by the low levels of binding seen with the kappa-agonists [3H]dynorphin A(1-9) and [3H]U-69593. In guinea-pig cord, under conditions in which binding to mu- and delta-sites was suppressed, [3H]dynorphin A(1-9) and [3H]U-69593 labelled only 60% of the kappa population, defined by the [3H]unselective ligands. The reasons for the observed discrepancies are discussed.

3,17 beta-Dihydroxy-20,21-epoxy-19-norpregna-1,3,5(10)-trienes: synthesis, rearrangement, cytotoxicity, and estrogen-receptor binding.

Diastereoisomers of 3,17 beta-dihydroxy-20,21-epoxy-19-norpregna-1,3,5(10)-triene have been prepared as potential antitumor agents. Both isomers undergo the base-catalyzed Payne rearrangement. The isomers were cytotoxic to mammalian cells in culture and were able to displace [3H]estradiol from binding sites in rat uterine cytosols with 1/7 and 1/70 the potency of estradiol. The reasons for this difference are discussed.

Steroidal alpha-methylene delta-lactones as potential antitumor agents.

Four novel steroidal alpha-methylene delta-lactones have been synthesized and shown to be active against human nasopharyngeal carcinoma (KB) cells in culture. The syntheses involved the use of known alpha-methylenation procedures. In addition, the lactone 6 was directly methylenated by reaction with CH2O/KOH or Et2NH/CH2O/Et2NH.HCl. The formation of a cysteine adduct (15) with the alpha-methylene lactone 10 is reported.

Synthesis and in vitro and in vivo activity of (-)-(1R,5R,9R)- and (+)-(1S,5S,9S)-N-alkenyl-, -N-alkynyl-, and -N-cyanoalkyl-5, 9-dimethyl-2'-hydroxy-6,7-benzomorphan homologues.

Two of the synthesized (-)-(1R,5R,9R)-N-homologues (N-but-3-enyl- and N-but-3-ynyl-5,9-dimethyl-2'-hydroxy-6,7-benzomorphan (9, 13)) were found to be about 20 times more potent than morphine in the mouse tail-flick assay (ED(50) = 0.05 mg/kg), and (-)-(1R,5R, 9R)-N-but-2-ynyl-5,9-dimethyl-2'-hydroxy-6,7-benzomorphan ((-)-(1R, 5R,9R)-N-but-2-ynylnormetazocine, 12) was about as potent as the opioid antagonist N-allylnormetazocine (AD(50) in the tail-flick vs morphine assay = 0.3 mg/kg). All of the homologues examined had higher affinity for the kappa-opioid receptor than the mu-receptor except (-)-N-but-2-ynyl-normetazocine (12), which had a kappa/mu ratio = 7.8 and a delta/mu ratio = 118. The (-)-N-2-cyanoethyl (3), -allyl (8), and -but-3-ynyl (13) analogues had good affinity (<10 nM) for delta-opioid receptors. Two homologues in the (+)-(1S,5S,9S)-normetazocine series, N-pent-4-enyl (24) and N-hex-5-enyl (25), were high-affinity and selective sigma(1)-ligands (K(i) = 2 nM, sigma(2)/sigma(1) = 1250, and 1 nM, sigma(2)/sigma(1) = 750, respectively); in contrast, N-allylnormetazocine (22) had relatively poor affinity at sigma(1), and its sigma(1)/sigma(2) ratio was <100.

Synthesis and biological evaluation of 14-alkoxymorphinans. 11. 3-Hydroxycyprodime and analogues: opioid antagonist profile in comparison to cyprodime.

A series of 3-hydroxy-substituted analogues (3-7) of the mu selective opioid antagonist cyprodime has been synthesized in order to evaluate the role of a hydroxy group at C-3 concerning mu opioid antagonist selectivity. Compounds 3-7 were tested in bioassays (electrical stimulated mouse vas deferens preparation and myenteric-plexus longitudinal muscle preparation of the guinea pig ileum) and opioid receptor binding assays. Antagonism of mu receptor-mediated responses induced by the mu selective agonist DAMGO afforded equilibrium dissociation constants in the mouse vas deferens preparation (Ke values) for compounds 3-7 which agreed closely with their affinities as determined by opioid receptor binding assays (Ki values). At kappa and delta receptors differences were apparent. Although the compounds had high affinity for both kappa and delta receptors in opioid receptor binding, they were very poor at antagonizing agonist responses mediated by kappa and particularly delta agonists in the mouse vas deferens preparation. None of the compounds tested showed agonist potency in the mouse vas deferens preparation or the myenteric-plexus longitudinal muscle preparation of the guinea pig ileum.

3-Deoxyclocinnamox: the first high-affinity, nonpeptide mu-opioid antagonist lacking a phenolic hydroxyl group.

The C(3)-substituent in morphinan opioids is of critical importance; the 3-OH group is usually associated with very much higher affinity for mu-receptors than H or -OMe. However in this series of 14beta-cinnamoylamino derivatives the codeinones (e.g. methoclocinnamox, MC-CAM) had unexpectedly high mu-opioid receptor affinity, similar to that of the morphinone (clocinnamox, C-CAM). The current report relates to the synthesis and in vitro evaluation of deoxyclocinnamox (DOC-CAM) which acted as a high-affinity opioid antagonist similar to C-CAM but with greater mu selectivity. Thus it appears that the C(3)-substituent does not play a major role in the binding of the 14beta-cinnamoyl series and that the cinnamoyl group itself may in fact be the dominant binding feature.

3-Alkyl ethers of clocinnamox: delayed long-term mu-antagonists with variable mu efficacy.

In recent years there has been considerable interest in the relationship between clocinnamox (C-CAM) and its methyl ether methoclocinnamox (MC-CAM). While C-CAM appears to be an insurmountable mu-antagonist, MC-CAM has been shown to be a potent partial agonist at mu-opioid receptors. To further investigate this relationship we prepared other ethers of C-CAM and evaluated these in opioid receptor binding assays and in vivo in mouse antinociceptive assays and in morphine-dependent monkeys. In opioid binding assays, the ethers were generally mu-selective with affinity equivalent to that of C-CAM itself. Although they displayed little or no efficacy in vitro, some of the ethers showed substantial agonist activity in the in vivo antinociceptive tests. Two of the ethers, the propargyl ether 7 and the cyclopropylmethyl ether 5, were chosen for more detailed analysis in vivo. 7 was shown to have significant mu-agonist character and was able to substitute for morphine in morphine-dependent monkeys. Interestingly, when this agonist effect abated, 7 displayed long-lasting mu-antagonism. In contrast, 5 displayed little agonist activity in vivo and was characterized as a potent, long-acting mu antagonist. Although further work is needed to determine whether metabolism is a crucial factor in determining the pharmacological profile of these ethers, it is clear that 3-O-alkylation is a useful means of varying the mu efficacy displayed by this class of acyl-substituted 14-aminomorphinones. MC-CAM itself has generated considerable interest as a potential pharmacotherapy for opiate abuse. These analogues with differing mu efficacy but retaining the long-lasting mu-antagonist effects provide further opportunities for the development of treatment drugs.

Evidence for lack of modulation of mu-opioid agonist action by delta-opioid agonists in the mouse vas deferens and guinea-pig ileum.

1. There is evidence from in vivo studies for an interaction of mu- and delta-opioid ligands. In the present work this concept has been investigated using the mouse vas deferens and guinea-pig ileum myenteric plexus-longitudinal preparations. 2. In field stimulated vasa deferentia of the mouse, co-administration of sub-effective concentrations of the delta-opioid agonist [D-Pen2,D-Pen5]enkephalin (DPDPE) and [Met5]- or [Leu5]enkephalin had no effect on the dose-response curves of the mu-agonists [D-Ala2,MePhe4, Gly-ol5]enkephalin (DAMGO) and morphine. Similarly, the delta-opioid agonists did not alter the potency of morphine and DAMGO when added at different times prior to the mu-opioid agonists, or when EC50 concentrations of delta-opioid ligands were co-administered. Compounds with preferred activity for the putative delta 1-(DPDPE) or delta 2-([D-Ala2,Glu4]deltorphin II (Delt II)) opioid receptors were ineffective in this respect. 3. The guinea-pig ileum contains delta-opioid receptors. No function of these receptors in mediating blockage of field-stimulated contractions was observed with ligands having affinity for the putative delta 1 or delta 2 subtypes nor were the agonists able to modulate responses to mu-opioid ligands in this tissue. 4. The results demonstrate the modulation of mu-opioid agonists by delta-opioid agonists does not occur in the isolated peripheral tissues examined. Thus the findings do not support the concept of a functional coupling of opioid receptors, though the results may be explained by differences between opioid systems in the brain and peripheral tissues examined.

Alkylation with beta-funaltrexamine suggests differences between mu-opioid receptor systems in guinea-pig brain and myenteric-plexus.

1. The effects of pre-incubation with beta-funaltrexamine (beta-FNA) on the binding of [3H]-[D-Ala2, MePhe4, Gly-ol5]enkephalin ([3H]-DAMGO) to homogenates of guinea-pig brain and myenteric-plexus longitudinal muscle have been studied. 2. beta-FNA pretreatment of brain homogenates in Tris-HCl buffer reduced the amount of [3H]-DAMGO binding. This was principally due to a reduction in the maximal number of binding sites measurable. However, approximately 30% of sites labelled by 1 nM [3H]-DAMGO were insensitive to 1 microM beta-FNA. Similar findings were obtained when the alkylation was performed in brain homogenates prepared in Krebs solution buffered with HEPES. 3. beta-FNA pretreatment of whole myenteric-plexus longitudinal muscle strips caused an increase in the IC50 values of mu-agonists, but not of kappa-agonists. However, the binding of [3H]-DAMGO to homogenates of myenteric-plexus longitudinal muscle was not altered by pre-incubation with beta-FNA in Tris-HCl buffer. On the other hand when the pretreatment was carried out in whole tissue in Krebs solution, or in homogenates in the presence of NaCl and Gpp(NH)p, a marked reduction in [3H]-DAMGO binding was observed. 4. These results suggest that a low affinity form of the mu-opioid receptor is the physiologically relevant site for beta-FNA alkylation in the myenteric-plexus and that differences exist between mu-receptor systems in guinea-pig myenteric plexus and brain.

Evidence that the agonist action of dynorphin A(1-8) in the guinea-pig myenteric-plexus may be mediated partly through conversion to [Leu5]enkephalin.

1. The agonist action of the opioid peptide dynorphin A(1-8) on the myenteric plexus-longitudinal muscle of the guinea-pig ileum has been characterized. 2. The endogenous opioid peptide dynorphin A(1-8) was rapidly degraded by slices of myenteric plexus-longitudinal muscle of the guinea-pig ileum. 3. A product of the degradation was the delta-receptor preferring [Leu5]enkephalin. Levels of [Leu5]enkephalin were markedly increased in the presence of the peptidase inhibitors bestatin, thiorphan and captopril. 4. In the myenteric plexus dynorphin A(1-8) acted as a kappa-receptor agonist. In the presence of bestatin, thiorphan and captopril a mu-receptor agonist effect was observed. This mu-agonist action was lost in the presence of N-[1-(RS)-carboxy-2-phenylethyl]Ala-Ala-Phe-p-aminobenzoate, an inhibitor of the endopeptidase enzyme EC 3.4.24.15. 5. The results suggest that formation of [Leu5]enkephalin from dynorphin A(1-8) may be an important conversion process. The enzyme responsible may be the Zn2(+)-metalloendopeptidase, EC 3.4.24.15.

Tolerance to mu-opioid agonists in human neuroblastoma SH-SY5Y cells as determined by changes in guanosine-5'-O-(3-[35S]-thio)triphosphate binding.

1. The agonist action of morphine on membranes prepared from human neuroblastoma SH-SY5Y cells was measured by an increase in the binding of the GTP analogue [35S]-GTPgammaS. Morphine increased the binding of [35S]-GTPgammaS to SH-SY5Y cell membranes by 30 fmol mg(-1) protein with an EC50 value of 76 +/- 10 nM. 2. Incubation of SH-SY5Y cells with 10 microM morphine for 48 h caused a tolerance to morphine manifested by a 2.5 fold shift to the right in the EC50 value with a 31 +/- 6% decrease in the maximum stimulation of [35S]-GTPgammaS binding. The response caused by the partial agonist pentazocine was reduced to a greater extent. 3. Chronic treatment of the cells with the more efficacious mu-ligand [D-Ala2, MePhe4, Gly-ol5]enkephalin (DAMGO, 10 microM) for 48 h afforded a greater effect than treatment with morphine. The maximal agonist effect of morphine was reduced to 58.9 +/- 6% of that seen in control cells while the maximal effect of DAMGO was reduced to 62.8 +/- 4%. There was a complete loss of agonist activity for pentazocine. 4. The development of tolerance was complete within 24 h and was blocked by naloxone and by the nonselective protein kinase inhibitor H7, but not by the putative beta-adrenoceptor kinase (beta-ARK) inhibitor suramin. 5. The observed tolerance effect was accompanied by a down-regulation of mu-opioid receptors determined by a decrease in the maximal binding capacity for the opioid antagonist [3H]-diprenorphine of 66 +/- 4%, but with no change in binding affinity. Binding of the agonist [3H]-DAMGO was similarly reduced. 6. The modulation of [35S]-GTPgammaS binding in SH-SY5Y cell membranes by opioids provides a simple method for the study of opioid tolerance at a site early in the signal transduction cascade.

Constitutive activity of the delta-opioid receptor expressed in C6 glioma cells: identification of non-peptide delta-inverse agonists.

1. G-protein coupled receptors can exhibit constitutive activity resulting in the formation of active ternary complexes in the absence of an agonist. In this study we have investigated constitutive activity in C6 glioma cells expressing either the cloned delta-(OP1) receptor (C6delta), or the cloned mu-(OP3) opioid receptor (C6mu). 2. Constitutive activity was measured in the absence of Na+ ions to provide an increased signal. The degree of constitutive activity was defined as the level of [35S]-GTPgammaS binding that could be inhibited by pre-treatment with pertussis toxin (PTX). In C6delta cells the level of basal [35S]-GTPgammaS binding was reduced by 51.9+/-6.1 fmols mg-1 protein, whereas in C6mu; and C6 wild-type cells treatment with PTX reduced basal [35S]-GTPgammaS binding by only 10.0+/-3.5 and 8.6+/-3.1 fmols mg-1 protein respectively. 3. The delta-antagonists N, N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH (ICI 174,864), 7-benzylidenenaltrexone (BNTX) and naltriben (NTB), in addition to clocinnamox (C-CAM), acted as delta-opioid receptor inverse agonists. Naloxone, buprenorphine, and naltrindole were neutral antagonists. Furthermore, naltrindole blocked the reduction in [35S]-GTPgammaS binding caused by the inverse agonists. The inverse agonists did not inhibit basal [35S]-GTPgammaS binding in C6mu; or C6 wild-type cell membranes. 4. Competition binding assays in C6delta cell membranes revealed a leftward shift in the displacement curve of [3H]-naltrindole by ICI 174,864 and C-CAM in the presence of NaCl and the GTP analogue, GppNHp. There was no change in the displacement curve for BNTX or NTB under these conditions. 5. These data confirm the presence of constitutive activity associated with the delta-opioid receptor and identify three novel, non-peptide, delta-opioid inverse agonists.