Dynorphin is a specific endogenous ligand of the kappa opioid receptor.

In the guinea pig ileum myenteric plexus--longitudinal muscle preparation, dynorphin-(1--13) and the prototypical kappa agonist ethylketocyclazocine had equally poor sensitivity to naloxone antagonism and showed selective cross protection in receptor inactivation experiments with the alkylating antagonist beta-chlornaltrexamine. In binding assays with membranes from guinea pig brain, ethylketocyclazocine and dynorphin-(1--13) amide were more potent in displacing tritium-labeled ethylketocyclazocine than in displacing typical mu and delta opioid receptor ligands. In the two preparations studied, the dynorphin receptor appears to be the same as the kappa opioid receptor.

Redefining the structure-activity relationships of 2,6-methano-3-benzazocines. Part 7: syntheses and opioid receptor properties of cyclic variants of cyclazocine.

A series of 7,8- and 8,9-fused triazole and imidazole analogues of cyclazocine have been made and characterized in opioid receptor binding and [(35)S]GTPgammaS assays. Target compounds were designed to explore the SAR surrounding our lead molecule for this study, namely the 8,9-fused pyrrolo analogue 2 of cyclazocine. Compared to 2, many of the new compounds in this study displayed very high affinity for opioid receptors.

Redefining the structure-activity relationships of 2,6-methano-3-benzazocines. 5. Opioid receptor binding properties of N-((4'-phenyl)-phenethyl) analogues of 8-CAC.

A series of aryl-containing N-monosubstituted analogues of the lead compound 8-[N-((4'-phenyl)-phenethyl)]-carboxamidocyclazocine were synthesized and evaluated to probe a putative hydrophobic binding pocket of opioid receptors. Very high binding affinity to the mu opioid receptor was achieved though the N-(2-(4'-methoxybiphenyl-4-yl)ethyl) analogue of 8-CAC. High binding affinity to mu and very high binding affinity to kappa opioid receptors was observed for the N-(3-bromophenethyl) analogue of 8-CAC. High binding affinity to all three opioid receptors were observed for the N-(2-naphthylethyl) analogue of 8-CAC.

Opiate receptor subtypes in the rat hypothalamus and neurointermediate lobe.

The potent opiate radioligands [3H]etorphine, [3H]ethylketocyclazocine (EKC), and [3H]naloxone, bound specifically and saturably to a single class of membrane-binding sites in rat neurointermediate lobe (NIL), with Kd values of 3.7, 24, and 51 nM, respectively. In the hypothalamus (Ht), [3H]etorphine bound to specific and saturable sites with a Kd of 2.9 nM. Binding-inhibition studies with [3H]etorphine and unlabeled etorphine-HCl as well as [3H]EKC and unlabeled EKC, revealed high and low affinity binding sites in rat Ht and NIL as well as in the neural lobe of the bovine pituitary gland. [3H]naloxone also bound specifically to two classes of sites in Ht membranes, but to only a single class of low affinity sites in NIL membranes. Specific binding represented 80-90% of total [3H]etorphine binding, about 75% of total [3H]EKC binding, and 45-55% of total [3H]naloxone binding at 22 C in NIL and Ht, respectively. Relative binding potencies derived from Ki values for binding-inhibition studies of [3H]etorphine with opioid peptides and opiates were: NIL, etorphine-HCl greater than dynorphin A greater than naloxone-HCl greater than dynorphin-(1-9) greater than beta-endorphin much greater than alpha-neoendorphin approximately (Leu5)enkephalin approximately DAGO (Tyr-D-Ala-Gly-NMe-Phe-Gly-ol); Ht, etorphine HCl greater than naloxone-HCl greater than beta-endorphin greater than dynorphin A much greater than DAGO greater than morphiceptin much greater than (Leu5)enkephalin. Specific [3H]etorphine binding was also demonstrable after preincubation of NIL membranes with DAGO and (Leu5)enkephalin and after preincubation of Ht membranes with morphiceptin and (Leu5)enkephalin; such binding could be displaced by nonradioactive dynorphin A. In addition, [3H]etorphine binding to bovine neural lobe was displaceable by naloxone-HCl, with an ED50 of 43 nM. Specific ligands for sigma-opiate receptors, such as (+)SKF 10,047 (N-allylnorcyclazocine), phencyclidine (PCP), and (-)cyclazocine, displaced specifically bound [3H]etorphine and [3H]EKC from NIL membranes only at high (micromolar) concentrations. However, specific [3H]PCP sites were of higher affinity in NIL and Ht membranes, with similar Kd values of 102 and 190 nM respectively, and different concentrations (0.15 and 1.32 pmol/mg protein, respectively). These data have revealed several differences in the opiate-binding properties of rat Ht and NIL membranes.(ABSTRACT TRUNCATED AT 400 WORDS)

Functional opioid receptor sites in human placentas.

We characterized the presence of opioid peptide receptor sites in plasma membranes and cells from human midterm and term placentas. Incubations with [3H]ethylketo-cyclazocine (EKC) at increasing doses revealed the presence of high affinity, low capacity, opioid peptide receptor-specific binding of the kappa-type. Scatchard analysis of the binding data showed, in the plasma membranes, linear plots at both stages of pregnancy with similar mean equilibrium association constants of 1.31 +/- 0.29 (+/- SE) X 10(9) mol/L-1 (n = 4) at midterm and 0.52 +/- 0.63 X 10(9) mol/L-1 at term (n = 4). In placental cells (n = 4) from term gestations, the binding plots were curvilinear; the first component had a Ka of 5.51 +/- 0.50 X 10(9) mol/L-1, and the second component had a Ka of 1.33 +/- 0.81 X 10(8) mol/L-1 (P less than 0.01). When standardized per mg tissue protein, the number of binding sites in plasma membranes increased from 13.8 +/- 9.8 fmol at midterm to 50.0 +/- 18.6 fmol at term (P less than 0.05). For term placental cells, the concentration of binding sites was 81.2 +/- 36.0 fmol for the high affinity sites and 713 +/- 390 fmol for the lower affinity sites. Specificity for the kappa-type of OPR was found based on the inability of mu- or delta-opioid peptides, as well as LHRH and TRH, to compete for [3H]EKC binding. Term placental cells incubated with various doses of opioid peptides had a 50% increase in placental lactogen production. The increase was significantly higher than controls only with kappa-agonists (P less than 0.05), maximal with 10(-9) mol/L EKC, and completely inhibited by 5 X 10(-6) mol/L naloxone. These results expand on previous data demonstrating the presence of opioid peptide receptor in placental plasma membranes and suggest a role for opioid peptides in regulating secretion of placental lactogen by placental cells.

Separation of kappa-opioid receptor subtype from frog brain.

Complete separation of the [3H]ethylketocyclazocine [( 3H]EKC) specific binding (kappa subtype) from tritiated Tyr-D-Ala2-Me-Phe4-Gly-ol5 enkephalin (DAGO) and Tyr-D-Ala2-L-Leu5-enkephalin (DALA) binding (mu-and delta-subtypes, respectively) was achieved by Sepharose-6B chromatography and sucrose density gradient centrifugation of digitonin solubilized frog brain membranes. The apparent sedimentation coefficient (s20.w) for the kappa receptor-detergent complex was 13.1 S and the corresponding Stokes radius 64 A. The isolated fractions exhibited high affinity for EKC and bremazocine, whereas mu- and delta-specific ligands were unable to compete for the [3H]EKC binding sites, indicating that the kappa subtype represents a separate molecular to compete for the [3H]EKC binding sites, indicating that the kappa subtype represents a separate molecular entity from the mu and delta receptor sites.

Binding characteristics of kappa opioids in rat brain. A comparison of in vitro binding paradigms.

Putative kappa binding was investigated in homogenates of the brain of the rat using [3H]ethylketocylazocine and [3H]diprenorphine under conditions where mu and delta sites were blocked. Under blocked conditions, the binding of [3H]ethylketocyclazocine labelled a single site, as defined by kinetic and equilibrium analysis, which showed high affinity for dynorphin 1-17, U50,488H, and benzomorphan compounds. However, blocked binding of [3H]diprenorphine showed a biphasic dissociation curve, and did not show high affinity for the specific kappa agonists dynorphin 1-17 or U50,488H. It is proposed that blocked [3H]ethylketocyclazocine is the more appropriate paradigm to study putative kappa binding, while blocked [3H]diprenorphine may label additional non-mu/non-delta sites.

Opioid ligand binding sites in the spinal cord of the guinea-pig.

The properties of opioid binding sites in membranes from the spinal cord of the guinea-pig were analyzed in experiments employing radiolabeled opioid ligands, selective or partially selective for mu, delta and kappa-type binding sites. Incubation was conducted at 37 degrees C in a quasi-physiological modified Krebs medium, containing sodium and magnesium. The types of binding sites were discriminated on the basis of their affinities for [3H-D-Ala2-MePhe4-Gly5-ol]enkephalin ([3H]DAGO), [3H-D-Ala2-D-Leu5]enkephalin, and [3H]ethylketocyclazocine and the relative potencies of the displacing ligands, DAGO, [D-Ser2-Leu5]enkephalyl-Thr and trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)- cyclohexyl]benzeneacetamide methanesulfonate hydrate (U50488H), which are selective for mu, delta and kappa type binding sites respectively. In membranes from whole spinal cord, kappa type sites comprised about 60%, mu about 30% and delta about 10% of the total of mu, delta and kappa binding sites. Binding sites of the mu type were also found in the lumbo-sacral region of guinea-pig spinal cord, in contrast to earlier reports of their absence from this tissue. Morphine showed a better than 500-fold selectivity for mu over kappa sites in spinal cord, while nalbuphine and (-)1-cyclopentyl-5-(1,2,3,4,5,6-hexahydro-8-hydroxy-3,6,11- trimethyl-2,6-methano-3-benzazocin-11-yl)3-pentanone methanesulfonate (WIN 44441-3) showed about a 10-fold selectivity for mu sites. The drug U50488H had about a 150-fold greater affinity for kappa than mu-type binding sites.

Interaction between [3H]ethylketocyclazocine and [3H]etorphine and opioid receptors in membranes from rat brain. A kinetic analysis.

Scatchard analysis of the binding to opioid receptors of [3H]ethylketocyclazocine ([3H]EKC) and [3H]etorphine at equilibrium yielded biphasic plots and computer fitting of the data resulted in a minimal model of two independent saturable binding sites. The KD values for the high- and low-affinity sites were 0.58 and 38 nM for [3H]EKC, and 0.13 and 22 nM for [3H]etorphine. The corresponding density of binding sites was 157 and 418 fmol/mg protein for [3H]EKC, and 220 and 289 fmol/mg protein for [3H]etorphine. The KD values calculated from the association and dissociation rate constants corresponded to those observed at equilibrium. In the course of equilibrium binding, various opioids competed with [3H]EKC and [3H]etorphine preferentially at the high-affinity opioid receptor sites. No difference between the competition patterns of putative mu and kappa ligands was observed. The kinetics of association and dissociation of [3H]EKC and [3H]etorphine revealed that the apparently homogeneous high-affinity binding site observed at equilibrium consisted of two components characterized by their fast and slow equilibrium times, respectively. While none of the mu and kappa opiates investigated altered the rate of dissociation of [3H]EKC or [3H]etorphine, in the presence of sodium ions the rapidly dissociating binding component of [3H]etorphine became refractory to inhibition by mu but not kappa agonists. The results underline the advantages of evaluating both equilibrium binding and the kinetics of ligand-receptor interactions.

High affinity [3H]ethylketazocine binding: evidence for specific kappa receptors.

A study has been made of the inactivation of mu(mu) ([3H]-dihydromorphine), delta (delta) ([3H](D-ala2-D-leu5)enkephalin) and kappa (kappa) ([3H]ethylketazocine) opiate receptor binding sites by N-ethylmaleimide (NEM) and it was observed that in contrast to mu and delta sites, the kappa sites of rat brain membrane preparations were resistant to low concentrations of N-ethylmaleimide. Furthermore, this kappa site was selectively protected, from inactivation with high concentrations of N-ethylmaleimide, by the kappa agonists ethylketazocine and (-)-alpha-(1R,5R,9R)-5,9-dimethyl-2-(L-tetrahydrofurfuryl)-2'-hydroxy-6,7-benzo morphan (MR-2034) but not by morphine or (D-ala2-D-leu5)-enkephalin. These studies suggest that a unique kappa receptor is present in the rat CNS.

Inhibition of [3H][3-MeHis2]thyrotropin releasing hormone recognition sites in the rat brain by tifluadom, a kappa opiate receptor agonist.

The effect of tifluadom (TIF), a postulated kappa-opiate agonist with a benzodiazepine (Bz) structure, on the binding of [3H][3-MeHis2]thyrotropin releasing hormone [( 3H]MeTRH) to receptors for thyrotropin releasing hormone (TRH) in membranes from rat brain was determined. Tifluadom inhibited the binding of [3H]MeTRH with an IC50 value of 1.88 microM. When the binding was carried out in the presence of an IC20 concentration of tifluadom, the Bmax value of [3H]MeTRH was decreased by 20% but no change in the Kd value was noted, indicating that the inhibition was apparently noncompetitive. Ro 15-1788 a benzodiazepine antagonist, as well as bicuculline, a gamma-aminobutyric acid (GABA) antagonist did not antagonize the effect of tifluadom on the binding of [3H]MeTRH suggesting that the benzodiazepine receptors are not involved in the action of tifluadom. Since tifluadom is suggested to be a kappa-opioid agonist, the effect of other kappa-opiate ligands were also tested for their ability to affect TRH receptors. The drugs used were ethylketocyclazocine, dynorphin(1-13) and 5-bromo-6-(2-imidazoline-2-ylamino)quinoxaline (U-50,488H). The order of potency of these compounds to inhibit the binding of [3H]MeTRH to membrane from the rat brain was in the following order: tifluadom greater than U-50,488H greater than dynorphin-(1-13) greater than ethylketocyclazocine. It is concluded that tifluadom inhibits the binding of [3H]MeTRH to receptors in brain in a noncompetitive manner. The effect does not appear to be mediated through benzodiazepine receptors but possibly through kappa-opiate receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in multiple opioid receptors in regions of the brain in rats treated chronically with thyroxine.

The effect of the administration of thyroxine (T4) (1 mg/kg, s.c.), to male Sprague-Dawley rats on alternate days for 18 days, on the binding of opioid ligands, [3H]Tyr-D-Ala-Gly-MePhe-Gly-ol (DAGO, mu receptors), [3H]Tyr-D-Ser-Gly-Phe-Leu-Thr (DSTLE, delta receptors) and [3H]ethylketocyclazocine (EKC, kappa receptors) to membranes of regions of the brain was determined. The chronic administration of thyroxine to rats decreased their rate of gain of body weight, increased colonic temperature and increased systolic blood pressure and heart rate, in comparison to vehicle-injected rats. The administration of thyroxine also increased the serum concentration of triiodothyronine (total T3) and thyroxine, when compared to vehicle-injected rats. The binding of [3H]DAGO to membranes of the striatum of rats treated with thyroxine was greater than in vehicle-treated rats; however, the binding to membranes of pons and medulla, amygdala, hypothalamus, midbrain and cortex in the two treatment groups did not differ. The increased binding of [3H]DAGO in rats treated with thyroxine was due to an increase in the Bmax value. The binding of [3H]DSTLE in the midbrain, hypothalamus, pons and medulla and striatum of rat treated with thyroxine and vehicle-injected rats did not differ but it was significantly less in the amygdala of rats injected with thyroxine than in vehicle-injected rats. The decreased binding in the amygdala was due to changes in the Kd value of [3H]DSTLE.(ABSTRACT TRUNCATED AT 250 WORDS)

Binaltorphimine-related bivalent ligands and their kappa opioid receptor antagonist selectivity.

In an effort to develop selective antagonists for kappa opioid receptors, bivalent ligands that contain opioid antagonist pharmacophores derived from naltrexone or other morphinans were synthesized and tested on the guinea pig ileum (GPI) and mouse vas deferens (MVD) preparations. The minimum requirements for kappa selectivity are at least one free phenolic OH group and one N-cyclopropyl or N-ally substituent. Several compounds (3, 8, 10) with kappa selectivity as good as or better than norbinaltorphimine (nor-BNI, 2) were discovered. The structure-activity relationship revealed that the pyrrole ring functions strictly as a spacer and does not contribute to kappa selectivity. The pharmacologic data suggest that only one antagonist pharmacophore may be required for kappa selectivity and that the other morphinan portion of the molecule confers selectivity by interacting with a unique subsite proximal to the antagonist pharmacophore recognition locus.

Synthesis and sigma binding properties of 1'- and 3'-halo- and 1',3'-dihalo-N-normetazocine analogues.

The synthesis and sigma 1 and sigma 2 binding properties of several 1'- and 3'-halo- and 1',3'-dihalo-substituted analogues of (+)-N-benzyl- and (+)- and (-)-N-dimethylallyl-N-normetazocine are presented. Structure-activity relationship analyses of the binding data showed that halogen substitution at the 1'-position of these N-substituted N-normetazocine analogues had little effect on sigma 1 binding affinity, whereas 3'-halo substitution as well as 1',3'-dihalo substitution resulted in a reduction of affinity. sigma 2 affinity was increased by the presence of a 3'-bromo substituent in this series of (+)-N-substituted N-normetazocines.

Selective protection and functionalization of morphine: synthesis and opioid receptor binding properties of 3-amino-3-desoxymorphine derivatives.

As part of an effort to identify novel opioid receptor interactive agents, we recently prepared a series of 8-(substituted)amino analogues of cyclazocine. We found the chiral 8-phenylamino (NHC(6)H(5)) cyclazocine derivative to have subnanomolar affinity for kappa opioid receptors and a 2-fold lower affinity for mu, opioid receptors. To determine if the benefits of (substituted)amino groups could be extended to the morphine core structure, we have made five novel 3-amino-3-desoxymorphine derivatives of general structure 5 where RR'N = H(2)N, CH(3)NH, (CH(3))(2)N, C(6)H(5)NH, and C(6)H(5)CH(2)NH. Relative to morphine, these derivatives had 38-273-fold, 11-41-fold, and 10-141-fold lower affinity for mu, delta, and kappa opioid receptors, respectively. Target compounds were made via Pd-catalyzed amination of a morphine 3-trifluoromethylsulfonate substrate where the 6-OH group was protected with a tert-butyldiphenylsilyl group. To make 6-tert-butyldiphenylsilyloxymorphine selectively, a new high-yield method was developed whereby morphine was bis-silylated using normal conditions followed by selective removal of the 3-tert-butyldiphenylsilyl group with catalytic tetrabutylammonium fluoride.

Non-peptide ligands for opioid receptors. Design of kappa-specific agonists.

A series of phenyl carboxyl esters 5a-d derived from N-(cyclopropylmethyl)normetazocine was synthesized and evaluated for its selectivity at mu, kappa, and delta opioid receptors. Compound 5a, although 43 times less potent than the reference compound U50488, was specific for kappa receptors, having no detectable affinity for either mu or delta receptors. Greater binding affinity was seen with the diastereoisomer having the 1'R,2'S stereochemistry in the cyclopropyl ring of the nitrogen substituent, which was only 12 times less active than U50488. Antinociceptive activity in the mouse tail flick was only slightly lower than that of U50488 (ED50 = 7.66 vs 4.52 mg/kg). Naloxone fully prevented antinociception induced by (1'R,2'S)-5a at the doses of 2.0 mg/kg. Compound (1'R,2'S)-5a is one of the most kappa-selective non-peptide compounds reported to date. The implications of these results in terms of requirements for kappa ligands are discussed.

(+)-cis-N-(para-, meta-, and ortho-substituted benzyl)-N-normetazocines: synthesis and binding affinity at the [3H]-(+)-pentazocine-labeled (sigma 1) site and quantitative structure-affinity relationship studies.

sigma 1 receptor ligands have potential pharmacological significance as antipsychotic drugs, as tools in the study of drug-induced motor function disorders, and as radiopharmaceutical imaging agents for the noninvasive imaging of malignant tumors in human subjects. A series of substituted N-benzyl-N-normetazocines were synthesized and their binding affinity at the sigma 1 receptor evaluated in order to examine the details of the structure--affinity relationships (SAR) of a previously determined high-affinity lead compound, (+)-cis-N-benzyl-N-normetazocine (Ki = 0.67 nM). Variation in the benzyl substituents of these compounds produced a 1590-fold range in affinity at the sigma 1 receptor from the unsubstituted benzyl analog to the lowest affinity p-tert-butylbenzyl analog (Ki = 1066 nM). The nanomolar binding affinity for the sigma 1 receptor of (+)-cis-N-(4-fluorobenzyl)-N-normetzocine suggests that this analog may be a useful PET imaging agent.

Synthesis and receptor binding properties of fluoro- and iodo-substituted high affinity sigma receptor ligands: identification of potential PET and SPECT sigma receptor imaging agents.

Unlabeled fluoro- and iodo-substituted ligands exhibiting very high affinity and selectivity for sigma receptors were synthesized based on three different structural classes of sigma receptor ligands. These compounds were evaluated for sigma receptor affinity and specificity in order to assess their potential as PET/SPECT imaging agents. Thus, (+)- and (-)-N-(5-fluoro-1-pentyl)normetazocines [(+)- and (-)-4] based on the (+)-benzomorphan class of sigma ligands were synthesized via N-alkylation of optically pure (+)- and (-)-normetazocine with 5-[(methylsulfonyl)oxy]-1-pentyl fluoride (11). (+)- and (-)-4 displaced [3H](+)-3-PPP with Ki values of 0.29 and 73.6 nM and [3H](+)-pentazocine with Ki values of 10.5 and 38.9 nM, respectively. The second class of PET/SPECT ligands was based upon the N-(arylethyl)-N-alkyl-2-(1-pyrrolidinyl)ethylamine class of sigma ligands; N-[2-(3,4-dichlorophenyl)-1-ethyl]-N-(3-fluoro-1-propyl)-2-(1- pyrrolidinyl)ethylamine (5) was obtained via N-alkylation of N-[2-(3,4-dichlorophenyl)-1-ethyl]-2-(1-pyrrolidinyl)ethylamine (14) with 3-fluoropropyl p-toluenesulfonate. 5 exhibited Ki values of 4.22 and 5.07 nM for displacement of [3H](+)-3-PPP and [3H](+)-pentazocine, respectively, comparable with the parent N-propyl compound. Attempts to synthesize N-[2-(3,4-dichlorophenyl)-1-ethyl]-N-[3- [(methylsulfonyl)oxy]-1-propyl]-2-(1-pyrrolidinyl)ethylamine (26), a precursor to 5 that could conceivably be converted to [18F]-5 by treatment with 18F-, proved unsuccessful. The sequence of regioselective nitration, catalytic hydrogenation, and diazotization followed by NaI quench of N-[2-(3,4-dichlorophenyl)-1-ethyl]-N-methyl-2-(1- pyrrolidinyl)ethylamine (2) afforded the iodinated ethylenediamine N-[2-(2-iodo-4,5-dichlorophenyl)-1-ethyl]-N-methyl-2-(1- pyrrolidinyl)ethylamine (8), a potential SPECT ligand for sigma receptors. This compound showed an affinity of 0.54 nM ([3H](+)-3-PPP) comparable with the parent compound 2 (Ki = 0.34 nM, [3H](+)-3-PPP). Ligand 8 exhibited a similar potency against [3H](+)-pentazocine. The third class of high-affinity sigma receptor ligands was rationalized based on rearrangement of the bonds in ethylenediamine 2 to give 1-[2-(3,4-dichlorophenyl)-1-ethyl]-4-(1-propyl)piperazine (3). This compound exhibited very high affinity (Ki = 0.31 nM, [3H](+)-3-PPP) and selectivity for sigma receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

Antipodal alpha-N-(methyl through decyl)-N-normetazocines (5,9 alpha-dimethyl-2'-hydroxy-6,7-benzomorphans): in vitro and in vivo properties.

The enantiomeric (-)- and (+)-N-(methyl through decyl) normetazocines (5,9 alpha-dimethyl-2'-hydroxy-6,7-benzomorphans) were synthesized and their in vitro and in vivo activities determined. Increasingly bulky enantiomeric N-alkyl homologs were prepared until their interaction with the sigma 1 receptor decreased and their insolubility became a hindrance to their evaluation in vivo and/or in vitro. The (-)-methyl, -pentyl, -hexyl, and -heptyl homologs were essentially as potent as, or more potent than, morphine in the tail-flick, phenylquinone, and hot-plate assays for antinociceptive activity; the (-)-propyl homolog had narcotic antagonist activity between that of nalorphine and naloxone in the tail-flick vs morphine assay, and it also displayed antagonist properties in the single-dose suppression assay in the rhesus monkey. The antinociceptively potent (-)-heptyl homolog did not substitute for morphine in monkeys but did show morphine-like properties in a primary physical-dependence study in continuously infused rats. All five potent compounds showed high affinity for the mu opioid receptor from both rat and monkey preparations and the kappa opioid receptor (< 0.05 microM), and all except the (-)-methyl homolog interacted reasonably well at the delta receptor (K(i) < 0.1 microM). The (-)-propyl compound was equipotent (K(i) 1.5-2.0 nM) at mu and kappa receptors. The pattern of interaction of the (-)-enantiomeric homologs with mu receptors from rat and monkey preparations was similar, but not identical. The enantioselectivity of the homologs for mu receptors was greater in the rat than in the monkey preparation for all but the N-H and butyl compounds, and the enantioselectivity of the lower homologs (methyl through butyl) for the mu (monkey) receptor was greater than for the kappa or delta receptors. However, bulkier homologs (hexyl through decyl) displayed higher enantioselectivity at kappa or delta receptors than at the mu (monkey) receptor. The (+)-butyl through (+)-octyl homologs were essentially equipotent with, or more potent than, (+)-pentazocine at the sigma receptor. Only the (+)-H and (+)-methyl homologs had high affinity (< 0.05 microM) at PCP binding sites.

Characterization of the kappa-subtype of the opiate receptor in the guinea-pig brain.

1. In homogenates of guinea-pig brain, the characteristics of the binding of [3H]-ethylketazocine, an agonist for the putative kappa-receptor, were determined by estimation of the affinity and capacity of binding, by competitive inhibition for the binding site by unlabelled ligands and by selective protection of the binding site from alkylation by phenoxybenzamine. 2. At 25 degrees C the maximum number of binding sites for [3H]-ethylketazocine was about 14 pmol/g fresh brain, of which about 50% were high affinity sites. 3. In competition experiments, the high affinity binding of [3H]-ethylketazocine to the kappa-binding site was readily displaced by several kappa-agonists but not by the selective mu-ligand, D-Ala2, MePhe4, Gly-ol5-enkephalin or the selective delta ligand, D-Ala2, D-Leu5-enkephalin. In contrast, the kappa-agonists tested so far exhibit a high degree of cross-reactivity with the mu-binding site but somewhat less with the sigma-binding site. Similar specificities were observed in protection experiments. 4. The approximate proportions of the three subtypes of opiate receptor in the guinea-pig brain are 25% mu-binding sites, 45% delta-binding sites and 30% kappa-binding sites. 5. The endogenous opioids, Met-enkephalin, Leu-enkephalin and porcine beta-endorphin have only a low affinity for the kappa-binding site.