Opioid activity of C8813, a novel and potent opioid analgesic

Compound trans-4-(p-bromophenyl)-4-(dimethylamino)-1-(2-thiophen-2-yl-ethyl)-cyclohexanol (C8813), structurally unrelated to morphine, is a novel analgesic. The present study examined the antinociception, opioid receptor selectivity and in vitro activity of C8813. The antinociceptive activity was evaluated using mouse hot plate and acetic acid writhing tests. In mouse hot plate test, the antinociceptive ED(50) of C8813 was 11.5 microg/kg, being 591 times and 3.4 times more potent than morphine and fentanyl respectively. In mouse writhing test, the antinociceptive ED(50) of C8813 was 16.9 microg/kg, being 55 times and 2.3 times more active than morphine and fentanyl respectively. In the opioid receptor binding assay, C8813 showed high affinity for mu-opioid receptor (K(i) = 1.37 nM) and delta-opioid receptor (K(i) = 3.24 nM) but almost no affinity for kappa-opioid receptor (at 1 microM). In the bioassay, the inhibitory effect of C8813 in the guinea-pig ileum (GPI) was 16.5 times more potent than in the mouse vas deferens (MVD). The inhibitory effects of C8813 in the GPI and MVD could be antagonized by mu-opioid receptor antagonist naloxone and delta-opioid receptor antagonist ICI174,864 respectively. However, the inhibitory effect of C8813 in the rabbit vas deferens was very weak. These results indicated that C8813 was a potent analgesic and a high affinity agonist for the mu- and delta-opioid receptors.     

Activation of G-proteins in the mouse pons/medulla by beta-endorphin is mediated by the stimulation of mu- and putative epsilon-receptors

The activation of mu-, delta- and kappa1-opioid receptors by their respective agonists increases the binding of the non-hydrolyzable GTP analog guanosine-5'-(gamma-thio)-triphosphate (GTPgammaS) to G proteins. Beta-endorphin is an endogenous opioid peptide which binds nonselectively to mu-, delta- and putative epsilon-opioid receptors. The present experiment was designed to determine which opioid receptors are involved in the stimulation of [35S]GTPgammaS binding induced by beta-endorphin in the mouse pons/medulla. The mouse pons/medulla membranes were incubated in an assay buffer containing 50 pM [35S]GTPgammaS, 30 microM GDP and various concentrations of beta-endorphin. Beta-endorphin (0.1 nM-10 microM) increased [35S]GTPgammaS binding in a concentration-dependent manner, and 10 microM beta-endorphin produced a maximal stimulation of approximately 260% over baseline. This stimulation of [35S]GTPgammaS binding by beta-endorphin was partially attenuated by the mu-opioid receptor antagonist beta-funaltrexamine (beta-FNA), but not by the delta-opioid receptor antagonist naltrindole (NTI) or the kappa1-opioid receptor antagonist nor-binaltorphimine (nor-BNI). Beta-endorphin stimulated [35S]GTPgammaS binding by about 80% in the presence of 10 microM beta-FNA, 30 nM NTI and 100 nM nor-BNI. The same concentrations of these antagonists completely blocked the stimulation of [35S]GTPgammaS binding induced by 10 microM [D-Ala2,NHPhe4,Gly-ol]enkephalin, [D-Pen(2,5)]enkephalin and U50,488H, respectively. Moreover, the residual stimulation of [35S]GTPgammaS binding induced by beta-endorphin in the presence of the three opioid receptor antagonists was significantly attenuated by 100 nM of the putative epsilon-opioid receptor partial agonist beta-endorphin (1-27). These results indicate that the stimulation of [35S]GTPgammaS binding induced by beta-endorphin is mediated by the stimulation of both mu- and putative epsilon-opioid receptors in the mouse pons/medulla.     

Drug design and synthesis of epsilon opioid receptor agonist: 17-(cyclopropylmethyl)-4,5alpha-epoxy-3,6beta-dihydroxy-6,14-endoethenomorphinan-7alpha-(N-methyl-N-phenethyl)carboxamide (TAN-821) inducing antinociception mediated by putative epsilon opioid receptor

Here we report the new drug design and synthesis of a series of 6,14-endoethenomorphinan-7-carboxamide derivatives as a putative epsilon opioid receptor agonist. One of these compounds, 17-(cyclopropylmethyl)-4,5alpha-epoxy-3,6beta-dihydroxy-6,14-endoethenomorphinan-7alpha-(N-methyl-N-phenethyl)carboxamide (TAN-821), showed agonistic activity for a putative epsilon opioid receptor (IC(50) = 71.71nM) in the rat vas deferens (RVD) preparations. TAN-821 stimulated the binding of the nonhydrolyzable guanosine 5'-triphosphate analog, guanosine 5'-(gamma-thio)-triphosphate (GTPgammaS), to the mouse pons/medulla membrane via the activation of putative epsilon opioid receptor. Moreover, TAN-821 given intracerebroventricularly (i.c.v.) produced a marked antinociception in the tail-flick test (ED(50) = 1.73 microg) and the hot-plate test (ED(50) = 2.05 microg) in a dose-dependent manner. The antinociception induced by TAN-821 administered i.c.v. was blocked by the i.c.v.-pretreatment with a putative epsilon opioid receptor partial agonist beta-endorphin [1-27], but not a mu opioid receptor antagonist beta-FNA, a delta opioid receptor antagonist NTI, or a kappa opioid receptor antagonist nor-BNI. The present results suggest that TAN-821 may be a useful tool for the investigation on the pharmacological properties of the putative epsilon opioid receptor.     

The K(+)-evoked release of [3H]acetylcholine from slices of rat globus pallidus: modulation by delta-opioid receptors.

Previous investigations have shown that the activation of delta-opioid receptors depresses the release of acetylcholine (ACh) in the rat caudate putamen. This finding raised the possibility that the release of ACh is similarly modulated in the globus pallidus, a region containing a distinct population of cholinergic neurons and enriched in enkephalinergic nerve terminals. In the present study the pallidal release of ACh was characterized and the effects of delta-opioid receptor activation on this release were examined. The results show that this release is stimulated by high K+ in a concentration- and Ca(2+)-dependent manner. D-Pen2,L-Pen5-enkephalin (0.1-10 microM), a selective delta-opioid receptor agonist, produced a dose-related inhibition of the 25 mM K(+)-evoked tritium release. The maximal inhibitory effect, representing a 34% decrease in the K(+)-induced tritium release, was observed at a concentration of 1 microM. This opioid effect was attenuated by the selective delta-opioid receptor antagonist, ICI 174864 (1 microM). These findings support the role of a delta-opioid receptor in the modulation of ACh release in the rat globus pallidus.     

Relationship Between Opioid-Receptor Occupancy and Stimulation of Low-Km GTPase in Brain Membranes

Treatment of rat brain membranes with the irreversible opioid ligand cis-3-methylfentanylisothiocyanate (Superfit) was used to reduce gradually the number of available binding sites for the delta-selective agonist [3H][D-Ser2,Leu5]enkephalin-Thr6 ([ 3H]DSLET). Subsequently, the correlation between ligand binding and low-Km GTPase was investigated. Alkylation with 10 microM and 25 microM Superfit inactivated 66% and 71% of high-affinity (KD, 1 nM) binding sites without decreasing the affinity of the remaining sites and the stimulation of low-Km GTPase by DSLET. Following exposure of the membranes to 50 microM and 75 microM Superfit, ligand binding was confined to the low-affinity (KD, 20 nM) sites. In these membranes, the delta-agonists DSLET and [D-Pen2,D-Pen5]enkephalin still stimulated low-Km GTPase, and these effects were blocked by ICI 174864 (N,N-diallyl-Tyr-AIB-AIB-Phe-Leu-OH; AIB, alpha-aminoisobutyric acid), a delta-selective antagonist. A similar relationship between low-affinity ligand binding and GTPase stimulation was observed following alkylation of the delta-opioid receptor with the non-selective irreversible antagonist beta-chlornaltrexamine in the presence of protective concentrations of DSLET. The results reveal spare receptor sites in the coupling of the delta-opioid receptor to low-Km GTPase in brain and identify low-affinity ligand binding as a functional component in the process.     

δ antagonist and κ agonist activity of naltriben: Evidence for differential κ interaction with the δ1 and δ2 opioid receptor subtypes

The selective δ₂ receptor antagonist Naltriben (NTB) has played an important role in the identification of subtypes of the δ opioid receptor, termed δ₁ and δ₂, and their role in antinociception. However, the majority of these studies have been conducted in the mouse. The present study determined the opioid receptor selectivity of subcutaneously (s.c.) administered NTB in the rat. Five minute pretreatment with 1 mg/kg s.c. NTB antagonized the increase in TFL produced by i.t. administration of equieffective doses of the δ₂ receptor agonist [D-Ala², Glu⁴]deltorphin (DELT) or the δ₁ receptor agonist [D-Pen², D-Pen⁵]enkephalin (DPDPE), but did not antagonize the μ receptor agonist [D-Ala², MePhe⁴, Gly-ol⁵]enkephalin (DAMGO). These data confirm previous reports that NTB is a selective δ opioid receptor antagonist. However, this dose of NTB antagonized DELT and DPDPE to an equivalent extent, suggesting that its selectivity for the δ₂ receptor is not maintained after s.c. administration in the rat. A lower dose of NTB (0.56 mg/kg s.c.) was ineffective. When the dose of NTB was increased to 3 mg/kg s.c. the antagonism of DELT and of DPDPE was unexpectedly lost. Pretreatment with the κ receptor antagonist nor-binaltorphimine (nor-BNI) partially restored the antagonism of DELT, but not DPDPE by this dose of NTB and did not modify the antagonism of DAMGO by NTB. These data suggest that high doses of NTB have κ receptor agonist-like activity and support the proposal that κ opioid agonists diminish the actions of δ receptor antagonists. They also suggest that nor-BNI-sensitive κ opioid receptors interact with δ₂, but not δ₁ opioid receptors in the spinal cord.     

Agonist and antagonist activities of ligands derived from naltrexone and oxymorphone.

The pharmacological profile of naltrindole (NTI) and three of its analogues, N-methyl-NTI (N-Me-NTI), oxymorphindole (OMI) and naltriben (NTB) were studied in antinociceptive assays. The compounds were found to have agonist activities that appear to be mediated mainly by kappa opioid receptors because norbinaltorphimine (nor-BNI), the selective kappa opioid receptor antagonist inhibited their effects significantly. All of the compounds, behaved as antagonists at doses that were lower than those that produced agonist effects and they possessed a profile that was very selective for inhibiting the antinociceptive activities of delta opioid receptor agonists. Differential antagonism by NTB of the activities of DSLET and DPDPE was demonstrated.     

Pharmacological effects of naltriben as a ligand for opioid mu and kappa receptors in rat cerebral cortex

Naltriben (NTB) has been used to differentiate the subtypes of delta opioid receptors, delta1 and delta2. However, there is considerable evidence suggesting that NTB may act on other types of opioid receptors too. We examined the effects of NTB on the specific binding of radiolabeled ligands for opioid mu and kappa2 receptors, and the effects on the release of [3H]norepinephrine ([3H]NE) in rat cerebral cortex slices. NTB displaced the specific binding of [3H]DAMGO with Ki value of 19.79 +/- 1.12 nM in rat cortex membranes. Specific binding of [3H]diprenorphine ([3H]DIP) was inhibited by NTB with Ki value of 82.75 +/- 6.32 nM in the presence of DAMGO and DPDPE. High K+ (15 mM)-stimulated release of [3H]NE was attenuated by DAMGO in rat cerebral cortex slices. NTB (30 nM) shifted the dose-response curve of DAMGO to the right and attenuated the maximal effect. In the meantime, NTB inhibited high K+-stimulated [3H]NE release at concentrations above 100 nM. The inhibitory effect of NTB was not attenuated by CTAP (10 nM) and naloxone (3 nM) but by higher concentration of naloxone (30 nM), nor-BNI (300 nM) and bremazocine (3 nM). These results indicate that NTB, depending on the dosage, could acts not only as an antagonist at delta but also as a noncompetitive antagonist for mu receptors, and as an agonist for kappa2 receptors in rat cerebral cortex.     

Synthesis and biological evaluation of N-substituted quinolinimides, as potential ligands for in vivo imaging studies of delta-opioid receptors

We report here the syntheses of N-substituted quinolinimide derivatives displaying sufficient affinity and high selectivity for delta-opioid receptors. Among 9-subsituted derivatives, one showed much higher selectivity for the delta receptor in binding assays than the delta antagonist methylnaltrindole (6: Ki = 42 nM; micro/delta and kappa/delta > 238 on rat brain membranes) and antagonist properties. This compound was labeled with carbon-11 (t1/2 = 20.4 min) as a potential radioligand for the noninvasive assessment of delta opioid receptors in vivo with positron emission tomography (PET). A high yielding radiosynthesis of [11C]-6, based on the [11C]methyl introduction on the pyridine moiety by a Stille reaction, was described (radiochemical yield = 60 +/- 10%, specific activities = 0.8 to 1.5 Ci/micromol). The in vivo pharmacological profile in rats indicated that the radiotracer crossed the blood-brain barrier but was not stable and underwent rapid degradation in both plasma and brain. No specific binding was consequently revealed.     

Opioid Control of the In Vitro Release of Cholecystokinin-Like Material from the Rat Substantia Nigra

Possible interactions between Met-enkephalin and cholecystokinin (CCK)-containing neurons in the rat substantia nigra were investigated by looking for the effects of various opioid receptor ligands and inhibitors of enkephalin-degrading enzymes on the K(+)-evoked overflow of CCK-like material (CCKLM) from substantia nigra slices. The delta-opioid agonists D-Pen2, D-Pen5-enkephalin (50 microM) and Tyr-D-Thr-Gly-Phe-Leu-Thr (DTLET; 3 microM) enhanced, whereas the mu-opioid agonists Tyr-D-Ala-Gly-MePhe-Gly-ol (DAGO; 10 microM) and MePhe3, D-Pro4-morphiceptin (PL 017; 10 microM) decreased, the K(+)-evoked release of CCKLM. By contrast, the kappa-opioid agonist U-50488 H (5 microM) was inactive. The stimulatory effect of DTLET could be prevented by the delta antagonist ICI-154129 (50 microM), but not by the mu antagonist naloxone (1 microM). Conversely, the latter drug, but not ICI-154129, prevented the inhibitory effect of DAGO and PL 017. A significant increase in CCKLM overflow was observed upon tissue superfusion with the peptidase inhibitors kelatorphan or bestatin plus thiorphan. This effect probably resulted from the stimulation of delta-opioid receptors by endogenous enkephalins protected from degradation, because it could be prevented by ICI-154129 (50 microM). Furthermore the peptidase inhibitors did not enhance CCKLM release further when delta-opioid receptors were stimulated directly by DTLET (3 microM). These data indicate that opioids acting on delta and mu receptors may exert an opposite influence, i.e., excitatory and inhibitory, respectively, on CCK-containing neurons in the rat substantia nigra.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis, biology, NMR and conformation studies of the topographically constrained delta-opioid selective peptide analogs of [beta-iPrPhe(3)]deltorphin I.

Replacement of Phe3 in the endogenous delta-opioid selective peptide deltorphin I with four optically pure stereoisomers of the topographically constrained, highly hydrophobic novel amino acid beta-isopropylphenylalanine (beta-iPrPhe) produced four pharmacologically different deltorphin I peptidomimetics. Radiolabeled ligand-binding assays and in vitro biological evaluation indicate that the stereoconfiguration of the iPrPhe residue plays a crucial role in determining the binding affinity, bioactivity and selectivity of [beta-iPrPhe3]deltorphin I analogs: a (2S,3R) configuration of the iPrPhe3 residue in [beta-iPrPhe3]deltorphin I provided the most desirable biological properties with binding affinity (IC50 = 2 nM), bioassay potency (IC50 = 1.23 nM in MVD assay) and exceptional selectivity for the delta-opioid receptor over the mu-opioid receptor (30 000). Further conformational studies based on two-dimensional NMR and computer-assisted molecular modeling suggested a model for the possible bioactive conformation in which the Tyr1 and (2S,3R)-beta-iPrPhe3 residues adopt trans side-chain conformations, and the linear peptide backbone favors a distorted beta-turn conformation.     

Pharmacological and functional biochemical properties of D-Ala2-D-Nle5-enkephalin-Arg-Phe

Tyr-D-Ala-Gly-Phe-D-Nle-Arg-Phe (DADN) a synthetic analogue of the endogenous Met-enkephalin-Arg-Phe (Tyr-Gly-Gly-Phe-Met-Arg-Phe; MERF), was investigated in radioligand binding assays, [(35)S]GTPgammaS stimulation experiments as well as in in vivo algesiometric tests. Binding properties of [(3)H]DADN were measured in crude membrane fractions of rat spinal cord tissues and in homogenates of Chinese hamster ovary (CHO) cells selectively expressing delta-, kappa-or micro-opioid receptors. The highest affinity for [(3)H]DADN binding was observed in membranes from CHO cells transfected with micro-opioid receptors confirming the micro-selectivity of the peptide. Unlabeled DADN was also investigated in functional biochemical experiments by measuring opioid receptor-mediated G-protein activation in rat brain membrane fractions. The peptide stimulated the activity of the regulatory G-proteins in a concentration dependent manner, and the stimulation was efficiently inhibited in the presence of micro-receptor specific antagonist ligands further supporting the selectivity profile of DADN. Intrathecally administered DADN produced a dose-related, naloxone-reversible antinociception in rat hot water tail-flick tests. Among the selective opioid antagonists tested, the delta-selective naltrindole (NTI) and the kappa-specific norbinaltorphimine (norBNI) showed only slight blocking effects compared with naloxone. The results obtained in the in vitro agonist-stimulated [(35)S]GTPgammaS binding assays are in good agreement with the opioid agonist effect seen in the in vivo pain test.     

(N-stearyl, norleucine17) VIP hybrid inhibits the growth of pancreatic cancer cell lines

The effects vasoactive intestinal peptide (VIP) antagonists were investigated on pancreatic cancer cell lines. (N-Stearyl, Norleucine17) VIP hybrid ((SN)VIPhyb) inhibited 125I-VIP binding to human Capan-2 cells with an IC50 value of 0.01 microM whereas VIP hybrid had an IC50 value of 0.2 microM. By RT-PCR and Northern blot, VPAC1 receptor mRNA was detected in CAPAN-2 cells. One microM (SN)VIPhyb and 10 microM VIPhyb inhibited the ability of 30 nM VIP to elevate cyclic AMP and increase c-fos mRNA. (SN)VIPhyb, 1 microM inhibited the clonal growth of CAPAN-2 cells in vitro. In vivo, (SN)VIPhyb (10 microg/day s.c.) inhibited CAPAN-2 xenograft growth in nude mice. These results indicate that (SN)VIPhyb is a pancreatic cancer VPAC receptor antagonist.     

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.     

Arylacetamide kappa opioid receptor agonists with reduced cytochrome P450 2D6 inhibitory activity

Some kappa opioid receptor agonists of the arylacetamide class, for example, ICI 199441 (1), were found to strongly inhibit the activity of cytochrome P450 2D6 (CYP2D6) (1: CYP2D6 IC50=26 nM). Certain analogs bearing a substituted sulfonylamino group, for example, 13, were discovered to have significantly reduced CYP2D6 inhibitory activity (13: CYP2D6 IC50>10 microM) while displaying high affinity toward the cloned human kappa opioid receptor, good kappa/delta and kappa/mu selectivity, and potent in vitro and in vivo agonist activity.     

Morphine-induced in vivo release of spinal cholecystokinin is mediated by δ-opioid receptors – effect of peripheral axotomy

Morphine and other opioid agonists induce spinal in vivo release of cholecystokinin (CCK), a neuropeptide with anti-opioid properties. However, so far the opioid receptor subtype responsible for this effect has not been determined. In the present in vivo microdialysis study, the morphine-induced release of cholecystokinin-like immunoreactivity (CCK-LI) in the dorsal horn was completely blocked by the delta-opioid antagonist naltrindole (10 microM in the perfusion fluid). Neither the mu-opioid receptor antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr amide (CTOP; 10 microM in the perfusion fluid), nor the kappa-opioid receptor antagonist nor-binaltorphimine (nor-BNI); 10 microM in the perfusion fluid) had any significant effect in this respect. In addition, systemic administration of the delta-opioid receptor agonist BW373U86 (1 mg/kg, s.c.) and spinal administration of the delta(2)-opioid receptor agonist, Tyr-D-Ala-Phe-Glu-Val-Val-Gly amide ([D-Ala(2)] deltorphin II) (1 microM in the perfusion fluid) induced a significant increase of the CCK-LI level. The effect of BW373U86 on spinal CCK-LI release was completely blocked by spinal administration of naltrindole. The mu-opioid receptor agonist [D-ala(2)-N-Me-Phe(4)-Gly(5)-ol]-enkephalin (DAMGO) (1 microM in the perfusion fluid or 1 mg/kg, s.c.) failed to alter the CCK-LI level. Peripheral nerve lesions have previously been shown to down-regulate mu- and delta-opioid receptors in the dorsal horn, to increase the gene-expression of CCK and CCK-receptor mRNA in dorsal root ganglion neurons and to alter the potassium-induced spinal CCK-LI release. After complete sciatic nerve transection, administration of the two selective delta-opioid receptor agonists induced a significant release of CCK-LI, which was comparable to controls. In contrast, neither systemic nor spinal administration of morphine and DAMGO altered the spinal CCK-LI release in axotomized animals. The present data indicate that the delta-opioid receptor mediates morphine-induced CCK-LI release in the spinal cord.     

Discrimination of two angiotensin II receptor subtypes with a selective agonist analogue of angiotensin II, p-aminophenylalanine6 angiotensin II

Angiotensin II receptor binding sites in rat liver and PC12 cells differ in their affinities for a nonpeptidic antagonist, DuP 753, and p-aminophenylalanine6 angiotensin II. In liver, which primarily contains the sulfhydryl reducing agent-inhibited type of angiotensin II receptor, which we refer to as the AII alpha subtype, DuP 753 displays an IC50 of 55 nM, while p-aminophenylalanine6 angiotensin II displays an IC50 of 8-9 microM. In PC12 cells, which primarily contain the angiotensin II receptor type whose binding affinity is enhanced by sulfhydryl reducing agents (AII beta), DuP 753 displays an IC50 in excess of 100 microM, while p-aminophenylalanine6 angiotensin II displays an IC50 of 12 nM. p-Aminophenylalanine6 angiotensin II binding affinity in liver is decreased in the presence of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) suggesting that this analogue is an agonist.     

Spinal δ2 but not δ1 opioid receptors are involved in intracereboventricular β-endorphin-induced antinociception in the mouse

The antinociception induced by β-endorphin given intracerebroventricularly (i.c.v.) has been previously demonstrated to be mediated by the release of Met-enkephalin and subsequent stimulation of δ receptors in the spinal cord for antinociception. The present study was designed to determine what type of opioid receptor, δ1 or δ2, in the spinal cord is involved in i.c.v. β-endorphin-induced antinociception. Antinociception was assessed by the tail-flick test in male ICR mice. NTB (0.2–20 nmol) and NTI0 (0.22–2.2 nmol),selective δ2 receptor antagonists, given intrathecally (i.t.) dose-dependently attenuated i.c.v. β-endorphin-induced inhibition of the tail-flick response. On the other hand, BNTX (0.02–2.2 nmol), a selective δ1 receptor antagonist, given i.t., did not block i.c.v. β-endorphin-induced antinociception. The tail-flick inhibition induced by DAMGO, a μ receptor agonist, or U50,488H, a к receptor agonist, was not blocked by i.t. BNTX, NTB or NTI. It is concluded that δ2 but not δ1 receptors in the spinal cord are involved in i.c.v. β-endorphin-induced antinociception.     

Opioid binding site in EL-4 thymoma cell line

Using EL-4 thymoma cell-line we found a binding site similar to the k opioid receptor of the nervous system. The Scatchard analysis of the binding of [3H] bremazocine indicated a single site with a KD = 60 +/- 17 nM and Bmax = 2.7 +/- 0.8 pmols/10(6) cells (51 pmols/mg total cell proteins). To characterize this binding site, competition studies were performed using selective compounds for the various opioid receptors. The k agonist U-50,488H was the most potent displacer of [3H] bremazocine with an IC50 value = 0.57 microM. The two stereoisomers levorphanol and dextrorphan showed the same affinity for this site (IC50 = 2.9 microM and 1.9 microM respectively). While morphine, [D-Pen2, D-Pen5] enkephalin and beta-endorphin failed to displace, except at very high concentrations, codeine demonstrated a IC50 = 60 microM, that was similar to naloxone (IC50 = 69 microM).     

Affinity identification of delta-opioid receptors using latex nanoparticles

Three types of latex nanoparticles carrying naltrindole (NTI) derivatives were synthesized as probes for the affinity isolation of their binding proteins including the delta-opioid receptor. The effect of the attachment of NTI to different positions on the linker was investigated. Only latex nanoparticles in which the NTI derivative was linked through the phenol group were useful for isolating the recombinant delta-opioid receptor solubilized from CHO cell membrane. These latex nanoparticles could be a useful tool for investigations of the pharmacological activity of NTI.