Opioid and non-opioid effects of novel butyrophenone analogues.

Haloperidol, haloperidol propionate, and a haloperidol analogue N-3-(p-fluorobenzoyl) propyl-4-phenyl-4-propionyl-oxypiperidine (NIH 10495) were evaluated in several in vitro and in vivo tests of opioid effects. Haloperidol bound to opioid receptors with very low affinity and had no opioid agonist effects in the other test systems. Haloperidol propionate was 10 times less potent than NIH 10495 in the binding assay and in the smooth-muscle assay. Both of these haloperidol analogues decreased the rate and volume of respiration in air and in 5% CO2 with NIH 10495 being approximately 50 times more potent than haloperidol propionate. The NIH 10495, but not the haloperidol propionate, attenuated naltrexone-like discriminative stimulus effects in morphine-dependent withdrawn rhesus monkeys. Intravenously delivered NIH 10495 maintained higher rates of responding than did haloperidol propionate when evaluated for reinforcing effects. These drugs appear to have novel spectra of action that suggest possible value for this synthetic approach to the development of clinically useful analgesics and to the development of novel neuroleptics.

Coupling of multiple opioid receptors to GTPase following selective receptor alkylation in brain membranes.

Opioid agonists of the mu, kappa and delta types stimulated low-Km guanosine triphosphatase (GTPase) in membranes, from the brain of the rat by up to 34%, with potencies the rank order of which corresponded to the respective binding affinities to opioid receptor. In general, kappa ligands stimulated GTPase to a lesser degree than mu or delta opiates. The coupling of a given type of opioid receptor to GTPase was resolved by direct or protective alkylation of the other receptors. Treatment of the membranes with beta-funaltrexamine abolished the stimulation of GTPase by sufentanil and levorphanol (mu), but not by bremazocine (kappa) or DSLET (delta). On the other hand, prior incubation with Superfit, an alkylating agent with selectivity for the delta opioid receptor, specifically eliminated the effect of DSLET. Partial alkylation by increasing concentrations of Superfit gradually reduced the extent of stimulation of GTPase by DSLET. The successive treatment of membranes with Superfit and beta-funaltrexamine blocked the actions of DSLET, sufentanil and levorphanol, but had no effect on the stimulation of the GTPase by bremazocine. Selective coupling of an opioid receptor to GTPase was also obtained after incubation of membranes with beta-chlornaltrexamine in the presence of protective concentrations of mu, kappa or delta opioid ligands. Alkylation resolved the coupling of the non-selective opiate etorphine: the sum of stimulation of GTPase in the receptor-selective membranes equalled maximal stimulation of enzyme in untreated membranes. Naloxone blocked the stimulation of GTPase by mu, kappa or delta agonists, but ICI-174,864 specifically inhibited the effect of DSLET.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

Synthesis and characterization of 7-nitrobenzo-2-oxa-1,3-diazole (NBD)-labeled fluorescent opioids.

Alkylation of sarcosine with 4-chloro-nitrobenzo-2-oxa-1,3-diazole (NBD-chloride) furnished a fluorescent tag that was coupled with a tetrahydrothebaine derivative and beta-naltrexamine, respectively, to yield the fluorescent opioids 7 alpha-(1R)-1-hydroxy-1-methyl-3-(4-hydroxyphenyl)-propyl]-6,14- endoethenotetrahydrothebaine NBD-sarcosinate (ASM-5-10) and N-cyclopropylmethyl-3-hydroxy-14 beta-hydroxy-6 beta-(NBD sarcosinyl)-amino-epoxymorphinan (ASM-5-67). The fluorescence intensity of the novel opioids allowed their detection at subnanomolar concentrations, and was dependent on the polarity of the solvent. Maximum quantum yield was obtained in ethyl acetate and ethanol, and minimal fluorescence in heptane and water. Compounds ASM-5-10 and ASM-5-67 displaced the opioid receptor binding of [3H]Tyr-D-Ala-Gly-(Me)Phe-Gly-ol in monkey brain membranes with IC50 values of 8.4 and 1.5 nM, respectively. Whereas ASM-5-67 bound to mu, delta, and kappa receptors with comparable affinities, ASM-5-10 was mu-selective, with selectivity indices (ratio of respective IC50 values) of 0.04 for both mu/delta and mu/kappa. The sodium response ratio in binding revealed a pronounced agonist property of ASM-5-10. Both opioids were lipophilic, with octanol-water partition coefficients (log Papp) of 2.8 (ASM-5-10) and 1.0 (ASM-5-67). ASM-5-10 exhibited particularly strong membrane retention that was not reversible by four washes. Their favorable characteristics in fluorescence, receptor binding, and membrane interaction make these newly developed ligands useful molecular probes to study opioid receptor mechanisms.

Synthesis and characterization of 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY)-labeled fluorescent ligands for the mu opioid receptor.

A series of opioid ligands utilizing the 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) fluorophores 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene++ +-3-propionic acid or 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza- s-indacene-3-propionic acid were synthesized and characterized for their ability to act as a suitable fluorescent label for the mu opioid receptor. All compounds displaced the mu opioid receptor binding of [3H]Tyr-D-Ala-Gly-(Me)Phe-Gly-ol in monkey brain membranes with high affinity. The binding of fluorescent ligands to delta and kappa receptors was highly variable. 5,7-Dimethyl-BODIPY naltrexamine, "6-BNX," displayed subnanomolar affinities for the mu and kappa opioid receptors (Ki 0.07 and 0.43 nM, respectively) and nanomolar affinity at the delta (Ki 1.4 nM) receptor. Using fluorescence spectroscopy, the binding of 6-BNX in membranes from C6 glioma cells transfected with the cloned mu opioid receptor was investigated. In these membranes containing a high receptor density (10-80 pmol/mg protein), 6-BNX labeling was saturable, mu opioid specific, stereoselective (as determined with the isomers dextrorphan and levorphanol), and more than 90% specific. The results describe a series of newly developed fluorescent ligands for the mu opioid receptor and the use of one of these ligands as a label for the cloned mu receptor. These ligands provide a new approach for studying the structural and biophysical nature of opioid receptors.

Selectivity of ligand binding to opioid receptors in brain membranes from the rat, monkey and guinea pig.

Conditions for the equilibrium binding to opioid receptor of [3H]sufentanil (mu selective), [3H][D-Pen2,D-Pen5]enkephalin (delta selective), and [3H]U69,593 (kappa selective) were established in membranes from rat brain cerebrum, monkey cortex, or guinea pig cerebellum. The selectivity index of various opioid alkaloids and peptides in binding to the mu, delta, or kappa opioid receptors was expressed as the ratio of their EC50 values in displacing two selective radiolabeled ligands: [3H]sufentanil/[3H](D-Pen2,D-Pen5)enkephalin (selectivity: mu/delta), [3H]sufentanil/[3H]U69,593 (selectivity: mu/kappa), or [3H][D-Pen2,D-Pen5]enkephalin/[3H]U69,593 (selectivity: delta/kappa). High resolution in binding selectivity was observed: in rat brain the mu/delta selectivity for Tyr-D-Ala-Gly-(Me)Phe-Gly-ol and sufentanil were 0.02 and 0.03, whereas for [D-Pen2,D-Pen5]enkephalin and ICI 174,864 they were 1,200 and 998. Compared to mu opiates, the specific binding of delta and kappa agonists was less sensitive to sodium. The results describe a routinely applicable methodological approach for the assessment of selective ligand binding to the mu, delta and kappa opioid receptors in rodent and monkey brain membranes.

Activation of G protein by opioid receptors: role of receptor number and G-protein concentration.

The collision-coupling model for receptor-G-protein interaction predicts that the rate of G-protein activation is dependent on receptor density, but not G-protein levels. C6 cells expressing mu- or delta-opioid receptors, or SH-SY5Y cells, were treated with beta-funaltrexamine (mu) or naltrindole-5'-isothiocyanate (delta) to decrease receptor number. The time course of full or partial agonist-stimulated ¿35SGTPgammaS binding did not vary in C6 cell membranes containing <1-25 pmol/mg mu-opioid receptor, or 1. 4-4.3 pmol/mg delta-opioid receptor, or in SHSY5Y cells containing 0. 16-0.39 pmol/mg receptor. The association of ¿35SGTPgammaS binding was faster in membranes from C6mu cells than from C6delta cells. A 10-fold reduction in functional G-protein, following pertussis toxin treatment, lowered the maximal level of ¿35SGTPgammaS binding but not the association rate. These data indicate a compartmentalization of opioid receptors and G protein at the cell membrane.

Intraocular pressure effects of carbonic anhydrase inhibitors in primary open-angle glaucoma.

We tested the effect on intraocular pressure of three commonly used oral carbonic anhydrase inhibitor preparations in a controlled, randomized, comparative study on patients with primary open-angle glaucoma. Preparations tested included acetazolamide tablets, acetazolamide Sequels, and methazolamide tablets. The effect of the three carbonic anhydrase inhibitors was assessed by using a statistical modeling approach as well as by evaluating the average maximum reduction in intraocular pressure for each preparation. Dosage and time effects were also determined. As expected, each drug preparation was more effective in reducing intraocular pressure when administered to a patient who had already been treated with the carbonic anhydrase inhibitor preparation. The amount of intraocular pressure lowering was directly related to dose for both acetazolamide preparations. Of particular interest was the finding that maximal rapid reduction of intraocular pressure was obtained with a 500-mg dosage of acetazolamide tablets.

Zipeprol: preclinical assessment of abuse potential.

Zipeprol was evaluated in a number of in vitro and in vivo assays predictive of stimulant, depressant, or opioid abuse potential. Zipeprol had affinity for mu and kappa opioid binding sites as well as sigma binding sites. However, it failed to exert opioid-like agonist actions in rodents, and did not attenuate withdrawal signs in morphine- or pentobarbital-dependent rats. Zipeprol did not substitute for either amphetamine or pentobarbital in drug discrimination assays in rhesus monkeys. On the other hand, it suppressed morphine withdrawal signs in rhesus monkeys in two assays, and it acted as a quadazocine-sensitive reinforcer in monkeys trained to self-inject alfentanil. Zipeprol also acted as a reinforcer in monkeys trained to self-inject methohexital. In a dose range of 10-18 mg/kg, zipeprol induced convulsions in monkeys. Zipeprol appears to have abuse potential and a novel spectrum of action involving both opioid and non-opioid effects.

Lipophilicity of opioids determined by a novel micromethod.

The lipophilicity of various mu-selective opioids was determined by measuring their distribution between n-octanol and Tris.HCl buffer, pH 7.4, by a procedure requiring submicromolar concentrations (submilligram amounts) of the compounds. After partitioning at 25 degrees C, the concentrations of opioids in the aqueous phase was quantified by their displacement of bound [3H]Tyr-Ala-Gly-(Me)Phe-Gly-ol (DAMGO) from opioid receptor in brain membranes. The obtained distribution coefficients (log Papp) agreed well with respective values determined previously with other, less sensitive or more cumbersome, methods of quantitation. The procedure is precise and versatile, and offers the routine assessment of lipophilicity as part of the in vitro characterization of opioids frequently available in limited quantities. In principle, the method is applicable to any compound whose binding to its receptor is quantifiable.

An artifactual component of drug-protein binding generated in vitro.

In the course of investigating the binding of imipramine to soluble cellular fractions from brain and leukocytes using equilibrium dialysis, an artifactual binding component was produced. On Scatchard and double-reciprocal plots of the data, the component appeared as a homogenous population of sites displaying a binding affinity of 170 nM. The obtained pattern of biphasic interaction bore a marked resemblance to reported Scatchard plots representing the interaction of drugs with bovine serum albumin, and depicting two components of widely differing binding affinity and capacity. The artifact occurred when solutions were transferred after dialysis and before quantitation to intermediate containers, and resulted from binding of 3H-imipramine to the walls of these containers. The latter interaction decreased the concentration of radiolabeled drug in the dialysate but not in the dialyzed solution, and thus mimicked increased imipramine binding to the biological material under study. The effect was particularly pronounced at low drug concentrations, and was particularly pronounced at low drug concentrations, and was prevented by the presence of either proteinaceous material, or of an excess of another basic compound such as methadone. The concentration dependence of the phenomenon led to its appearance as a discrete binding component. The artifact was eliminated either by applying an appropriate correction factor, or by transferring the dialyzed solutions directly into scintillation vials for counting.

Receptor-related interactions of opiates with PGE-induced adenylate cyclase in brain.

The inhibition by opiates of the PGE2-induced formation of cAMP in slices from rat brain striatum was investigated. A maximal, 3.5-fold increase over the basal level of cAMP was obtained with an EC50 for PGE2 of 3 microM. Opiate agonists of both mu and kappa type were inhibitory. The IC50 values for morphine, levorphanol and ethylketocyclazocine (EKC) were 110 nM, 80 nM and 25 nM, respectively. These values were similar to the potencies of the compounds in displacing stereospecifically bound 3H-etorphine in rat brain membranes. As evidenced by the inactivity of dextrorphan, the inhibition of PGE2-dependent cAMP formation was stereospecific. Also ineffective were the opiate antagonists naloxone, naltrexone and MR 2266. These compounds did, however, reverse the inhibition by agonists, displaying thereby selectivity toward the putative mu and kappa opiates. Thus, the inhibition by morphine was antagonized to a greater degree by naloxone than by MR 2266, and the action of EKC was blocked more effectively by MR 2266 relative to naloxone.

Evidence for coupling of the kappa opioid receptor to brain GTPase.

In membranes from guinea pig cerebellum, a tissue which predominantly contains kappa opioid receptors, exogenous and endogenous kappa-selective opioid agonists stimulated low-km GTPase activity by 11-20% with concentrations for half-maximal stimulation of 3-23 microM. Opioid ligands of the mu and delta type had no effect on GTPase in these membranes. Similar stimulation of GTPase by kappa opiates was obtained in rat and monkey brain membranes pretreated with beta-funaltrexamine (beta-FNA) and cis-(+/-)-3-methylfentanyl isothiocyanate (superfit) to alkylate the mu and delta receptors, respectively. The stimulation of brain GTPase by kappa opiates in both types of membranes was inhibited by naloxone with IC50's of 0.35 microM and 0.40 microM. The results demonstrate the coupling of the kappa opioid receptor to high affinity GTPase, the Ni regulatory protein of the adenylate cyclase complex.

Cyclic, disulfide- and dithioether-containing opioid tetrapeptides: development of a ligand with high delta opioid receptor selectivity and affinity.

Tetrapeptides of primary sequence Tyr-X-Phe-YNH2, where X is D-Cys or D-Pen (penicillamine) and where Y is D-Pen or L-Pen, were prepared and were cyclized via the side chain sulfurs of residues 2 and 4 to disulfide or dithioether-containing analogs. These peptides are related to previously reported penicillamine-containing pentapeptide enkephalin analogs but lack the central glycine residue of the latter and were designed to assess the effect of decreased ring size on opioid activity. Binding affinities of the tetrapeptides were determined to both mu and delta opioid receptors. Binding affinity and selectivity in the tetrapeptide series were observed to be highly dependent on primary sequence. For example, L-Pen4 analogs displayed low affinity and were nonselective, while the corresponding D-Pen4 diastereomers were of variable affinity and higher selectivity. Among the latter compounds were examples of potent analogs in which selectivity shifted from delta selective to mu selective as the ring size was increased. The relatively high binding affinity and delta receptor selectivity observed with one of the carboxamide terminal disulfide analogs led to the synthesis of the corresponding carboxylic acid terminal, Tyr-D-Cys-Phe-D-PenOH. This analog displayed delta receptor binding selectivity similar to that of the standard delta ligand, [D-Pen2,D-Pen5]enkephalin (DPDPE), and was found to have a 3.5-fold higher binding affinity than DPDPE. All the tetrapeptides were further evaluated in the isolated mouse vas deferens (mvd) assay and all displayed opioid agonist activity. In general, tetrapeptide potencies in the mouse vas deferens correlated well with binding affinities but were somewhat lower. Receptor selectivity in the mvd, assessed by examining the effect of opioid antagonists on the tetrapeptide concentration-effect curves, was similar to that determined in the binding studies.

Stereospecific binding of 3H-phencyclidine in brain membranes.

Phencyclidine (PCP) displaceable binding of 3H-PCP to glass-fiber filters was eliminated and total binding markedly reduced by initial treatment of the discs with 0.05% polyethyleneimine. Assessed with treated filters, unlabeled PCP displaced 3H-PCP in both rat and pigeon brain membranes with an EC50 of 1 microM. Of similar high inhibitory potency were dextrorphan, levorphanol, SKF 10047 and ketamine, while morphine, naloxone and etorphine had EC50 values higher then 1 mM. Using the dissociative anesthetic dexoxadrol and its inactive isomer levoxadrol as displacing agents, stereospecific binding of 3H-PCP was obtained in rat and pigeon brain membranes. The markedly higher potency of dexoxadrol, relative to levoxadrol, in displacing bound 3H-PCP is compatible with behavioral data for these enantiomers. However, they were equipotent in displacing 3H-PCP bound to glass-fiber filters in the absence of tissue. Heat denaturation, but not freezing, abolished stereospecific binding of 3H-PCP, which was also absent in rat liver membranes. The stereospecific binding component in brain displayed biphasic saturability at 60-70 nM and 300-400 nM, respectively.

Receptor binding, antagonist, and withdrawal precipitating properties of opiate antagonists.

A number of opiate antagonists and the dextro isomers of some of these drugs were studied for antagonism of acute opiate effects on ilea isolated from opiate-naive guinea pigs, precipitation of a withdrawal contraction of ilea isolated from morphine-dependent guinea pigs, precipitation of withdrawal in morphine-dependent rhesus monkeys and stereospecific displacement of 3H-etorphine binding to rat-brain membranes. With the exception of d-naloxone, all of the compounds displaced 3H-etorphine. With the exception of d-naloxone, nalorphine, and quaternary nalorphine, all of the antagonists caused a contraction of ilea isolated from morphine-dependent guinea pigs. Moreover, the IC 50 values of the compounds for displacing 3H-etorphine binding were well correlated with both their Ke values for antagonism in the ileum (r = 0.95) and with their EC 50 values for precipitating a contraction in this preparation (r = 0.92). Generally, the concentration of antagonist necessary to precipitate half maximal contracture was 30-fold greater than the Ke value of the antagonist. Most of the opiate antagonists also precipitated withdrawal when administered to morphine-dependent rhesus monkeys and their in vivo potencies were well correlated with their in vitro potencies in ileum (with Ke: r = 0.95; with EC 50: r = 0.99) and in displacing 3H-etorphine (r = 0.95). The quaternary derivative of naltrexone, however, was an effective opiate antagonist only in vitro, and was ineffective in precipitating withdrawal in morphine-dependent rhesus monkeys. These results suggest that the receptor sites labeled by 3H-etorphine are the same as those involved in antagonism of acute opiate actions and in precipitation of withdrawal.

Resolution in the receptor binding of putative mu and kappa opiates.

Conditions for the stereospecific binding of 3H-ethylketocyclazocine and 3H-etorphine were established in membranes from rat and pigeon brain. In displacing the specific binding of the radiolabeled ligands, putative mu and kappa opiates displayed different sensitivity toward sodium. In membranes from both species, the ratio of the sodium responses exhibited by a given drug in displacing 3H-ethylketocyclazocine and 3H-etorphine, respectively, ("double sodium ratio" = DSR) distinguished between mu and kappa opiates. Compounds characterized on the basis of their pharmacological effects as kappa opiates had DSR values between 0.3 and 2.2, regardless of their nature as agonists or antagonists. In contrast, the DSR for mu opiates ranged from 3.4 to 11. In rat brain membranes, UM 1382 (U-50,488, a compound with pronounced kappa activity) exhibited a DSR of 0.3, while the corresponding value for morphine was 7.4. Dynorphin-(1-13) had a DSR of 1. Within each of the two groups, the simple sodium ratio continued to serve as an index for the agonist or antagonist property of the tested opiates.