A chimeric opioid peptide with mixed mu agonist/delta antagonist properties.

There is evidence to indicate that opioid compounds with mixed mu agonist/delta antagonist properties are analgesics with low propensity to produce tolerance and physical dependence. A chimeric peptide containing the potent and selective mu agonist H-Dmt-D-Arg-Phe-Lys-NH2 ([Dmt1]DALDA) (Dmt=2',6'-dimethyltyrosine) and the potent and selective delta antagonist H-Tyr-TicPsi[CH2-NH]Cha-Phe-OH (TICP[Psi]) (Cha=cyclohexylalanine), connected 'tail-to-tail' via a short linker, was synthesized using a combination of solid-phase and solution techniques. The resulting peptide, H-Dmt-->D-Arg-->Phe-->Lys-NH-CH2-CH2-NH-Phe<--Cha[NH-CH2]PsiTic<--Tyr-H, showed the expected mu agonist/delta antagonist profile in the guinea-pig ileum and mouse vas deferens assays. Its mu and delta receptor binding affinities were in the low nanomolar range, as determined in rat brain membrane binding assays.     

Aladan3]TIPP: a fluorescent delta-opioid antagonist with high delta-receptor binding affinity and delta selectivity

Fluorescent analogues of the potent and highly selective delta-opioid antagonist TIPP (H-Tyr-Tic-Phe-Phe-OH) and TIP (H-Tyr-Tic-Phe-OH) containing the exceptionally environmentally sensitive fluorescent amino acid beta-(6'-dimethylamino-2'-naphthoyl)alanine (Aladan [Ald]) in place of Phe3 were synthesized. The Ald3- and D-Ald3 analogues of TIPP and TIP all retained delta-opioid antagonist properties. The most potent analogue, [Ald3]TIPP, showed a K(e) value of 2.03 nM in the mouse vas deferens assay and five times higher delta vs. mu selectivity (K(i)mu/K(i)delta = 7930) than the TIPP parent peptide in the opioid receptor binding assays. Theoretical conformational analyses of [Ald3]TIPP and [Ald3]TIP using molecular mechanics calculations resulted in a number of low-energy conformers, including some showing various patterns of aromatic ring stacking and others with the Ald side chain and a carbonyl group (fluorescence quencher) in close proximity. These ensembles of low-energy conformers are in agreement with the results of steady-state fluorescence experiments (fluorescence emission maxima and quantum yields) and fluorescence decay measurements (fluorescence lifetime components), which indicated that the fluorophore was either engaged in intramolecular hydrophobic interactions or in proximity of a fluorescence quencher (e.g., a carbonyl group). These fluorescent TIP(P) delta-opioid antagonists represent valuable pharmacological tools for various applications, including studies on membrane interactions, binding to receptors, cellular uptake and intracellular distribution, and tissue distribution.     

Subtleties of structure-agonist versus antagonist relationships of opioid peptides and peptidomimetics

The development of novel delta opioid antagonists and delta opioid agonists structurally derived from the prototype delta antagonist TIPP (H-Tyr-Tic-Phe-Phe-OH), is reviewed. Both delta antagonists and delta agonists with extraordinary potency and unprecedented delta receptor selectivity were discovered. Some of them are already widely used as pharmacological tools and are also of interest as potential therapeutic agents for use in analgesia. The results of the performed structure-activity studies revealed that the delta antagonist versus delta agonist behavior of this class of compounds depended on very subtle structural differences in diverse locations of the molecule. These observations can be best explained with a receptor model involving a number of different inactive and active receptor conformations.     

Reduced peptide bond cyclic somatostatin based opioid octapeptides. Synthesis, conformational properties and pharmacological characterization.

The conformational and pharmacological properties that result from peptide bond reduction as well as the use of secondary amino acids in a series of cyclic peptides related to the mu opioid receptor selective antagonist D-Phe1-Cys2-Tyr3-D-Trp4-Orn5-Thr6-Pen7+ ++-Thr8-NH2 (IV), have been investigated. Peptide analogues that contain [CH2NH] and [CH2N] pseudo-peptide bonds (in primary and secondary amino acids, respectively) were synthesized on a solid support. Substitution of Tyr3 in IV by the cyclic, secondary amino acid 1,2,3,4-tetrahydroisoquinoline carboxylate (Tic) and of D-Trp4 with D-1,2,3,4-tetrahydro-beta-carboline(D-Tca4), gave peptides 4 and 1, respectively. Both analogues displayed reduced affinities for mu opioid receptors. Conformational analysis based on extensive NMR investigations demonstrated that the backbone conformations of 1 and 4 are similar to those of the potent and selective analogue D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH2 (I), while the conformational properties of the side chains of Tic3 (4) and D-Tca4 (1) resulted in topographical properties that were not well recognized by the mu opioid receptor. Peptide bond modifications were made including (Tyr3-psi[CH2NH]-D-Trp4), 3; (Tyr3-psi[CH2N]-D-Tca4), 2; and (Cys2-psi[CH2N]-Tic3), 6. These analogues showed decreases in their mu opioid receptor affinities relative to the parent compounds IV, 1, and 4, respectively. 1H NMR based conformational analysis in conjunction with receptor binding data led to the conclusion that the reduced peptide bonds in 2, 3, 5, and 6 do not contribute to the process of discrimination between mu and delta opioid receptors, and in spite of their different dynamic behaviors (relative to 1 and 4), they are still capable of attaining similar receptor bound conformations, possibly due to their increased flexibility.     

Conversion of delta-, kappa- and mu-receptor selective opioid peptide agonists into delta-, kappa- and mu-selective antagonists

2',6'-Dimethyl substitution of the Tyr(1) residue of opioid agonist peptides and deletion of the positively charged N-terminal amino group or its replacement with a methyl group has recently been shown to represent a general structural modification to convert opioid peptide agonists into antagonists. This conversion requires the syntheses of opioid peptide analogues containing either 3-(2,6-dimethyl-4-hydroxyphenyl)propanoic acid (Dhp) or (2S)-2-methyl-3-(2,6-dimethyl-4-hydroxyphenyl)propanoic acid [(2S)-Mdp] in place of Tyr(1). Using this approach, delta-, kappa- and mu-selective opioid peptide agonist peptides were successfully converted into corresponding delta-, kappa- and mu-selective antagonists, whereby receptor selectivity was often maintained or even improved. Thus, two (2S)-Mdp(1)-analogues of the delta-selective cyclic enkephalin analogue H-Tyr-c[D-Pen-Gly-Phe(pF)-Pen]-Phe-OH turned out to be potent and selective delta antagonists. Most successful was the development of kappa antagonists derived from dynorphin A (Dyn A), including the highly potent and selective kappa-antagonist [(2S)-Mdp(1)]Dyn A(1-11)-NH(2) (dynantin) and the enzymatically stable octapeptide analogue [(2S)-Mdp(1),MeArg(7),D-Leu(8)]Dyn A(1-8)-NH(2). The (2S)-Mdp(1)-analogues of dynorphin B and alpha-neoendorphin also were kappa antagonists and may be useful as pharmacological tools in studies of kappa receptor subtypes. Finally, the Dhp(1)-analogues of the mu-selective cyclic enkephalin analogue H-Tyr-c[N(epsilon ),N(beta)-carbonyl-D-Lys(2),Dap(5)]enkephalinamide and of endomorphin-2 were moderately potent mu opioid antagonists.     

Structure-activity relationships of arodyn, a novel acetylated kappa opioid receptor antagonist.

We previously reported that the novel dynorphin A (Dyn A, Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys-Trp-Asp-Asn-Gln) analog arodyn (Ac[Phe(1,2,3),Arg(4),d-Ala(8)]Dyn A-(1-11)NH(2), Bennett, M.A., Murray, T.F. & Aldrich, J.V. (2002) J. Med. Chem. vol. 45, pp. 5617-5619) is a kappa opioid receptor-selective peptide [K(i)(kappa) = 10 nm, K(i) ratio (kappa/mu/delta) = 1/174/583] which exhibits antagonist activity at kappa opioid receptors. In this study, a series of arodyn analogs was prepared and evaluated to explore the structure-activity relationships (SAR) of this peptide; this included an alanine scan of the entire arodyn sequence, sequential isomeric d-amino acid substitution in the N-terminal 'message' sequence, NMePhe substitution individually in positions 1-3, and modifications in position 1. The results for the Ala-substituted derivatives indicated that Arg(6) and Arg(7) are the most important residues for arodyn's nanomolar binding affinity for kappa opioid receptors. Ala substitution of the other basic residues (Arg(4), Arg(9) and Lys(11)) resulted in lower decreases in affinity for kappa opioid receptors (three- to fivefold compared with arodyn). Of particular interest, while [Ala(10)]arodyn exhibits similar kappa opioid receptor binding as arodyn, it displays higher kappa vs. mu opioid receptor selectivity [K(i) ratio (kappa/mu) = 1/350] than arodyn because of a twofold loss in affinity at mu opioid receptors. Surprisingly, the Tyr(1) analog exhibits a sevenfold decrease in kappa opioid receptor affinity, indicating that arodyn displays significantly different SAR than Dyn A; [Tyr(1)]arodyn also unexpectedly exhibits inverse agonist activity in the adenylyl cyclase assay using Chinese hamster ovary cells stably expressing kappa opioid receptors. Substitution of NMePhe in position 1 gave [NMePhe(1)]arodyn which exhibits high affinity [K(i)(kappa) = 4.56 nm] and exceptional selectivity for kappa opioid receptors [K(i) ratio (kappa/mu/delta) = 1/1100/>2170]. This peptide exhibits antagonistic activity in the adenylyl cyclase assay, reversing the agonism of 10 nm Dyn A-(1-13)NH(2). Thus [NMePhe(1)]arodyn is a highly kappa opioid receptor-selective antagonist that could be a useful pharmacological tool to study kappa opioid receptor-mediated activities.     

Reciprocal opioid-opioid interactions between the ventral tegmental area and nucleus accumbens regions in mediating mu agonist-induced feeding in rats

Feeding elicited by the mu-selective agonist, [D-Ala2, M-Phe4, Gly-ol5]-encephalin administered into the nucleus accumbens is blocked by accumbal pre-treatment with mu, delta1, delta2 and kappa, but not mu1 opioid antagonists. Correspondingly, mu-agonist-induced feeding elicited from the ventral tegmental area is blocked by ventral tegmental area pre-treatment with mu and kappa, but not delta opioid antagonists. A bi-directional opioid-opioid feeding interaction has been firmly established such that mu-agonist-induced feeding elicited from the ventral tegmental area is blocked by accumbal naltrexone, and that accumbal mu-agonist-induced feeding is blocked by naltrexone pre-treatment in the ventral tegmental area. To determine which opioid receptor subtypes mediate the regional bi-directional opioid-opioid feeding interactions between these two sites, the present study examined the dose-dependent ability of either general (naltrexone), mu (beta-funaltrexamine), kappa (nor-binaltorphamine) or delta (naltrindole) opioid antagonists administered into one site to block mu-agonist-induced feeding elicited from the other site. General, mu and kappa, but not delta opioid receptor antagonist pre-treatment in the ventral tegmental area dose-dependently reduced mu-agonist-induced feeding elicited from the nucleus accumbens. General, mu and delta, and to a lesser degree kappa, opioid receptor antagonist pre-treatment in the nucleus accumbens dose-dependently reduced mu-agonist-induced feeding elicited from the ventral tegmental area. Thus, multiple, but different opioid receptor subtypes are involved in mediating opioid-opioid feeding interactions between the nucleus accumbens and ventral tegmental area regions.     

High-affinity carbamate analogues of morphinan at opioid receptors

A series of carbamate analogues were synthesized from levorphanol (1a), cyclorphan (2a) or butorphan (3a) and evaluated in vitro for their binding affinity at mu, delta, and kappa opioid receptors. Functional activities of these compounds were measured in the [(35)S]GTPgammaS binding assay. Phenyl carbamate derivatives 2d and 3d showed the highest binding affinity for kappa receptor (K(i)=0.046 and 0.051 nM) and for mu receptor (K(i)=0.11 and 0.12 nM). Compound 1c showed the highest mu selectivity. The preliminary assay for agonist and antagonist properties of these ligands in stimulating [(35)S]GTPgammaS binding mediated by the kappa opioid receptor illustrated that all of these ligands were kappa agonists. At the mu receptor, compounds 1b, 1c, 2b, and 3b were agonists, while compounds 2c-e and 3c-e were mu agonists/antagonists.     

Characterization of [125I]AR-M100613, a high-affinity radioligand for delta opioid receptors

AR-M100613 ([I]-Dmt-c[-D-Orn-2-Nal-D-Pro-D-Ala-]) is the iodinated analog of a cyclic casomorphin previously shown to be a potent antagonist at the delta opioid receptor. Specific [125I]AR-M100613 binding to rat whole brain membranes was saturable, reversible, and best fit to a one-site model (Kd = 0.080 +/- 0.008 nM, Bmax = 45.2 +/- 4.4 fmol/mg protein). [125I]AR-M100613 binding was displaced with high affinity by the delta opioid receptor ligands SNC-80, Deltorphin II and DPDPE but not the mu or kappa-selective receptor ligands DAMGO and U69593. Residual non-selective binding of [125I]AR-M 100613 to mu opioid receptors is blocked by the addition of CTOP to the assay buffer. [35S]GTPgammaS binding assays indicate that AR-M100613 is a potent, selective, and reversible antagonist for delta opioid receptors in rat brain membranes. The high-affinity, high specific activity, low nonspecific binding and antagonist profile of [125I]AR-M100613 favor its use as a radiochemical probe for delta opioid receptors.     

Co-expression of mu and delta opioid receptors as receptor-G protein fusions enhances both mu and delta signalling via distinct mechanisms

Mu and delta opioid receptors (MORs and DORs) were co-expressed as fusion proteins between a receptor and a pertussis insensitive mutant Gα_i/o protein in human embryonic kidney 293 cells. Signalling efficiency was then monitored following inactivation of endogenous Gα_i/o proteins by pertussis toxin. Co-expression resulted in increased delta opioid signalling which was insensitive to the mu specific antagonist d -Phe-Cys-Tyr- d -Trp-Arg-Thr-Pen-Thr-NH₂. Under these conditions, mu opioid signalling was also increased and insensitive to the delta specific antagonist Tic-deltorphin. In this latter case, however, no G protein activation was observed in the presence of the delta specific inverse agonist N , N (CH3)₂-Dmt-Tic-NH₂. When a MOR fused to a non-functional Gα subunit was co-expressed with the DOR-Gα protein fusion, delta opioid signalling was not affected whereas mu opioid signalling was restored. Altogether our results suggest that increased delta opioid signalling is due to enhanced DOR coupling to its tethered Gα subunit. On the other hand, our data indicate that increased mu opioid signalling requires an active conformation of the DOR and also results in activation of the Gα subunit fused the DOR.     

Pharmacophore elements of the TIPP class of delta opioid receptor antagonists

Summary A series of tri-and tetrapeptides sharing the amino-terminal dipeptide unit Tyr-Tic, found in the high-affinity delta opioid receptor antagonist Tyr-Tic-Phe-Phe (TIPP), was prepared and evaluated in receptor binding assays to explore the role(s) of the phenylalanine residues in positions 3 and 4. It was found that aromaticity of residues 3 and 4 is not required for high affinity, a lipophilic side chain in either location being sufficient, as evidenced by the high delta receptor binding affinities observed for the tetrapeptide Tyr-Tic-Ala-Leu and the tripeptide Tyr-Tic-Leu. These results support the suggestion of Temussi et al. [Biochem. Biophys. Res. Commun., 198 (1994) 933] that the aromatic side chain of the Tic residue corresponds to the aromatic side chain found in residues 3 or 4 in other delta-selective peptide series.     

Benzylideneoxymorphone: A new lead for development of bifunctional mu/delta opioid receptor ligands

Opioid analgesic tolerance remains a considerable drawback to chronic pain management. The finding that concomitant administration of delta opioid receptor (DOR) antagonists attenuates the development of tolerance to mu opioid receptor (MOR) agonists has led to interest in producing bifunctional MOR agonist/DOR antagonist ligands. Herein, we present 7-benzylideneoxymorphone (6, UMB 246) displaying MOR partial agonist/DOR antagonist activity, representing a new lead for designing bifunctional MOR/DOR ligands.     

Novel TIPP (H-Tyr-Tic-Phe-Phe-OH) analogues displaying a wide range of efficacies at the δ opioid receptor. Discovery of two highly potent and selective δ opioid agonists

Analogues of the δ opioid antagonist peptide TIPP (H-Tyr-Tic-Phe-Phe-OH; Tic=1,2,3,4-tetrahydroisoquinoline3-carboxylic acid) containing various 4'-[N-(alkyl or aralkyl)carboxamido]phenylalanine analogues in place of Tyr(1) were synthesized. The compounds showed subnanomolar or low nanomolar δ opioid receptor binding affinity and various efficacy at the δ receptor (antagonism, partial agonism, full agonism) in the [(35)S]GTPγS binding assay. Two analogues, [1-Ncp(1)]TIPP (1-Ncp=4'-[N-(2-(naphthalene-1-yl)ethyl)carboxamido]phenylalanine) and [2-Ncp(1)]TIPP (2-Ncp=4'-[N-(2-(naphthalene-2-yl)ethyl)carboxamido]phenylalanine), were identified as potent and selective δ opioid agonists.     

Modifications of the cyclic mu receptor selective tetrapeptide Tyr-c[D-Cys-Phe-D-Pen]NH2 (Et): effects on opioid receptor binding and activation.

The previously described cyclic mu opioid receptor-selective tetrapeptide Tyr-c[D-Cys-Phe-D-Pen]NH2 (Et) (JOM-6) was modified at residues 1 and 3 by substitution with various natural and synthetic amino acids, and/or by alteration of the cyclic system. Effects on mu and delta opioid receptor binding affinities, and on potencies and efficacies as measured by the [35S]-GTPgammaS assay, were evaluated. Affinities at mu and delta receptors were not influenced dramatically by substitution of Tyr1 with conformationally restricted phenolic amino acids. In the [35S]-GTPgammaS assay, all of the peptides tested exhibited a maximal response comparable with that of fentanyl at the mu opioid receptor, and all showed high potency, in the range 0.4-9nM. However, potency changes did not always correlate with affinity, suggesting that the conformation required for binding and the conformation required for activation of the opioid receptors are different. At the delta opioid receptor, none of the peptides were able to produce a response equivalent to that of the full delta agonist BW 373,U86 and only one had an EC50 value of less than 100nM. Lastly, we have identified a peptide, D-Hat-c[D-Cys-Phe-D-Pen]NH2 (Et), with high potency and > 1,000-fold functional selectivity for the mu over delta opioid receptor as measured by the [35S]-GTPgammaS assay.     

Cyclic enkephalin analogs containing various para-substituted phenylalanine derivatives in place of Tyr1 are potent opioid agonists.

The cyclic enkephalin analog H-Tyr-c[D-Cys-Gly-Phe(pNO(2))-D-Cys]NH(2) is a highly potent opioid agonist with IC(50)s of 35 pm and 19 pm in the guinea-pig ileum (GPI) and mouse vas deferens (MVD) assays, respectively. The Phe(1)-analog of this peptide showed 370-fold and 6790-fold lower agonist potency in the GPI and MVD assays, respectively, indicating the importance of the Tyr(1) hydroxyl-group in the interaction with mu and delta opioid receptors. In the present study, the effect of various substituents (-NH(2), -NO(2), -CN, -CH(3), -COOH, -COCH(3), -CONH(2)) introduced in the para-position of the Phe(1)-residue of H-Phe-c[D-Cys-Gly-Phe(pNO(2))-D-Cys]NH(2) on the in vitro opioid activity profile was examined. Most analogs showed enhanced mu and delta agonist potencies in the two bioassays, except for the Phe(pCOOH)(1)-analog, which was weakly active, probably as a consequence of the negative charge. The most potent compounds were the Phe(pCOH(3))(1)- and the Phe(pCONH(2))(1)-analogs. The latter compound showed subnanomolar mu and delta agonist potencies and represents the most potent enkephalin analog lacking the Tyr(1) hydroxyl-group reported to date. Taken together, these results indicate that various substituents introduced in the para-position of Phe(1) enhance opioid activity via hydrogen bonding or hydrophobic interactions with the receptor. Comparison with existing structure-activity relationship on phenolic hydroxyl replacements in morphinans indicates that these nonpeptide opiates and some of the cyclic enkephalin analogs described here may have different modes of binding to the receptor.     

A human neuroblastoma cell line expresses mu and delta opioid receptor sites

A series of neuroblastoma cell lines were screened for the presence of opioid receptor sites with the tracers [3H]diprenorphine (mu, delta, kappa ligand) and [3H]naloxone (mu-selective ligand). One human neuroblastoma cell line, SK-N-SH, displayed avid binding for both tracers. Binding experiments with multiple tracers revealed the presence of both mu and delta sites. These sites were stereospecific, saturable, and proteinaceous in character. Saturation binding experiments provided an estimate of 50,000 mu and 10,000 delta sites/cell. NaCl (100 mM) and guanine nucleotide, guanylyl imidodiphosphate (50 microM), reduced opioid agonist but not antagonist binding to these sites. Etorphine at 1 nM inhibited prostaglandin E1-stimulated cyclic AMP production by approximately 20%, which was reversible by naloxone. The opioid-binding sites on SK-N-SH cells closely resemble the previously reported mu and delta sites in human and rodent brain. Therefore, the SK-N-SH neuroblastoma cell line represents a useful tool to study the molecular functions of opioid receptors.     

Reduced peptide bond pseudopeptide analogues of substance P. A new class of substance P receptor antagonists with enhanced specificity

Each of the last 6 peptide bonds in the COOH terminus of [Leu11]substance P [( Leu11]SP) and [Nle11]spantide were replaced with [CH2NH], and each analogue was tested for SP agonist or antagonist activity by determining its ability to interact with SP receptors on dispersed acini from guinea pig pancreas. Each of the 6 spantide and 5 of the 6 SP analogues had no agonist activity, whereas [psi 9-10]SP was an agonist. For the spantide pseudopeptides, the psi 10-11 analogue (Ki,2.8 microM) was equipotent as an antagonist to spantide itself, whereas the psi 9-10, psi 8-9, psi 7-8, and psi 6-7 analogues were 2.5, 7, 5, and 3 times less potent. For the SP pseudopeptides, the psi 10-11 analogue was the most potent antagonist (Ki, 6.2 microM), whereas the psi 8-9, psi 7-8, and psi 6-7 analogues were 7-, 36-, and 39-fold less potent. There was a close correlation between the ability of each pseudopeptide to inhibit binding of 125I-Bolton-Hunter-SP and to affect amylase secretion. [psi 10-11]SP inhibited SP-stimulated amylase release in a competitive manner, and its inhibitory ability was specific for the SP receptor. Despite [psi 10-11]SP, spantide, and [psi 10-11]spantide having similar affinities for the SP receptor (Ki, 2-6 microM), for inhibition of binding of 125I-[Tyr4]bombesin, the analogues differed with [psi 10-11]SP having a 50-fold lower affinity than for the SP receptor, whereas [psi 10-11]spantide had a 4-fold lower affinity and spantide a 1.5-fold lower affinity for the SP receptor. These results demonstrate that SP pseudopeptides represent a new class of SP receptor antagonists and, in contrast to the currently described SP receptor antagonists, are more specific for SP receptors.     

Expression of delta, kappa and mu human opioid receptors in Escherichia coli and reconstitution of the high-affinity state for agonist with heterotrimeric G proteins.

Human opioid receptors of the delta, mu and kappa subtypes were successfully expressed in Escherichia coli as fusions to the C-terminus of the periplasmic maltose-binding protein, MBP. Expression levels of correctly folded receptor molecules were comparable for the three subtypes and reached an average of 30 receptors.cell-1 or 0.5 pmol.mg-1 membrane protein. Binding of [3H]diprenorphine to intact cells or membrane preparations was saturatable, with a dissociation constant, KD, of 2.5 nM, 0.66 nM and 0.75 nM for human delta, mu and kappa opioid receptors (hDOR, hMOR and hKOR, respectively). Recombinant receptors of the three subtypes retained selectivity and nanomolar affinity for their specific antagonists. Agonist affinities were decreased by one to three orders of magnitude as compared to values measured for receptors expressed in mammalian cells. The effect of sodium on agonist binding to E. coli-expressed receptors was investigated. Receptor high-affinity state for agonists was reconstituted in the presence of heterotrimeric G proteins. We also report affinity values of endomorphins 1 and 2 for mu opioid receptors expressed both in E. coli and in COS cells. Our results confirm that opioid receptors can be expressed in a functional form in bacteria and point out the advantages of E. coli as an expression system for pharmacological studies.     

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.     

Opioid receptors in human neuroblastoma SH-SY5Y cells: evidence for distinct morphine (mu) and enkephalin (delta) binding sites

Human neuroblastoma SH-SY5Y cells exhibited a heterogeneous population of mu and delta types of opioid binding sites. These specific binding sites displayed the characteristic saturability, stereospecificity and reversibility, expected of a receptor. Scatchard analysis of [3H]-D-Ala2-D-Leu5-enkephalin (DADLE) in the presence of 10(-5) M D-Pro4-morphiceptin (to block the mu receptors) and the competitive displacement by various highly selective ligands yielded the binding parameters of delta sites which closely resemble those of the delta receptors in brain and mouse neuroblastoma clones. Similarly, the high affinity binding of [3H]-dihydromorphine, together with the higher potency of morphine analogues to displace [3H]-naloxone binding established the presence of mu sites. Guanine nucleotides and NaCl significantly inhibited the association and increased the dissociation of [3H]-DADLE binding. The observed heterogeneity of opioid receptors in cultured SH-SY5Y cells would serve as an excellent model for the biochemical and pharmacological characterization of brain opiate receptors.