Redefining the structure-activity relationships of 2,6-methano-3-benzazocines. Part 8. High affinity ligands for opioid receptors in the picomolar Ki range: oxygenated N-(2-[1,1'-biphenyl]-4-ylethyl) analogues of 8-CAC.

N-[2-(4'-methoxy[1,1'-biphenyl]-4-yl)ethyl]-8-CAC (1) is a high affinity (K(i)=0.084 nM) ligand for the µ opioid receptor and served as the lead compound for this study. Analogues of 1 were made in hopes of identifying an SAR within a series of oxygenated (distal) phenyl derivatives. A number of new analogues were made having single-digit pM affinity for the µ receptor. The most potent was the 3',4'-methylenedioxy analogue 18 (K(i)=1.6 pM).

Drug discrimination training with progressively lowered doses.

Rats were trained to discriminate drug from no-drug conditions in a two-lever operant task. Moderately high dosages were used initially. Whenever the discrimination was learned, training was continued with progressively reduced dosages. Eventually the rats discriminated extremely low doses of phenobarbital, chlordiazepoxide, cyclazocine, and fentanyl.

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. Part 6: Opioid receptor binding properties of cyclic variants of 8-carboxamidocyclazocine.

A series of 7,8- and 8,9-fused pyrimidinone, aminopyrimidine and pyridone derivatives of 8-carboxamidocyclazocine (8-CAC) have been prepared and evaluated in opioid receptor binding assays. Targets were designed to corroborate a pharmacophore hypothesis regarding the bioactive conformation of the carboxamide of 8-CAC. In addition to the results from this study strongly supporting this pharmacophore hypothesis, a number of novel compounds with high affinity to opioid receptors have been identified.

Redefining the structure-activity relationships of 2,6-methano-3-benzazocines. 4. Opioid receptor binding properties of 8-[N-(4'-phenyl)-phenethyl)carboxamido] analogues of cyclazocine and ethylketocycalzocine.

The synthesis and evaluation of a series of aryl-containing N-monosubstituted analogues of the lead compound 8-carboxamidocyclazocine were performed to probe a putative hydrophobic binding pocket of opioid receptors. High binding affinity to mu, kappa, and delta opioid receptors was observed for the 8-[N-(4'-phenyl)-phenethyl)carboxamido] analogue.

Binding of opioids to human MCF-7 breast cancer cells and their effects on growth.

The well characterized human breast cancer cell line, MCF-7, has been shown to possess membrane receptors for various opioid ligands, and these compounds have been shown to modulate the growth of the cells in culture. Using specific radioligands for the receptor types, we were able to demonstrate that the MCF-7 cells possess multiple opioid receptor types. Relatively high-affinity-binding sites are present for the mu- and kappa-specific ligands, while lower affinity sites are present for the delta-agonist. Opioid ligands specific for the different receptor types significantly inhibited the growth of the MCF-7 cells in a dose-dependent manner when grown in the presence of 10% fetal bovine serum. This inhibitory effect was reversed by the simultaneous administration of the opioid receptor antagonist, naloxone. However, the opioid effect appears to be restricted to the hormonally responsive fraction of the MCF-7 cell growth. Cells grown in the presence of charcoal-stripped fetal bovine serum are refractory to the effects of the opioids unless the media is supplemented with estradiol. The data presented here suggest an important regulatory role for opioids in the growth and development of human breast cancers.

Intracranial self-stimulation thresholds: a model for the hedonic effects of drugs of abuse.

We present the thesis that many drugs of abuse are used for their hedonic effects and that a relevant animal model for the study of these effects is the action of these drugs on the pathways that support rewarding intracranial self-stimulation. A relationship between abuse potential of a drug and its ability to lower the threshold for rewarding brain stimulation in the rat was found. Of all the compounds we have studied, morphine and cocaine were the drugs that caused the maximum lowering of the rewarding threshold. Phencyclidine hydrochloride and the mixed agonist-antagonist pentazocine also lowered the threshold to a lesser degree, while the mixed agonist-antagonists cyclazocine and nalorphine hydrochloride had inconsistent effects. Naloxone hydrochloride, at the doses tested, had no effect on the threshold. Further, there is no evidence that tolerance develops to the threshold-lowering effect of morphine, suggesting that continued use of narcotics by the physically dependent individual is not simply due to an effort to avoid the pain of withdrawal.

Direct action of opiates on bromocriptine-inhibited prolactin release by human prolactinoma cells in primary culture.

The present study was undertaken in order to examine the existence of opioid binding sites on cell membranes of human PRL-secreting tumors. Determination of opioid binding sites using different opiate ligands revealed one class of high affinity (KD, 1.3 nM) binding sites. Pharmacological characterization revealed kappa-1 selectivity (high affinity ethylketocyclazocine (EKC) binding, insensitive to 5 microM (D-Ala2, D-Leu5]enkephalin). Subsequently EKC was added to hPRL-secreting tumor cells in primary culture, alone or in combination with the dopaminergic agonist bromocriptine, and PRL release was measured. Opiates had no direct effect on PRL release by prolactinoma cells. When cells were preincubated with bromocriptine [6.6 +/- 4.8 (SD) X 10(-11) M], EKC (10(-11) to 10(-9) M) antagonized, in a dose-dependent manner, the dopaminergic inhibition of PRL release. The opiate effect was reversed by the opiate antagonist diprenorphine (10(-7) M). Cross-competition studies indicated that this effect was not due to the interaction of opiates with the dopaminergic receptor. In conclusion, opioid binding sites are found on prolactinoma cells. The binding of kappa-1 type opioid ligands modulates the inhibitory effect of dopamine upon PRL release.

Kappa opiate receptor agonists: effects on behavior and on brain function and structure in rhesus monkeys.

In the present study several drugs that are predominantly agonists of kappa receptors were tested in rhesus monkeys prepared with deep and surface brain electrodes. The administration of two benzomorphan derivatives and of ethylketocyclazocine induced acute behavioral effects resembling catatonia concomitant with generalized spike and slow-wave electroencephalographic activity at widespread brain sites, that lasted for about 1 hr. After administration of one to three doses of these agonists (the benzomorphan derivatives to five monkeys and the ethylketocyclazocine to two monkeys), chronic recording changes developed, characterized by continuous high-amplitude spiking activity focal at the anterior septal region and periaqueductal gray of the mesencephalon. They increased in intensity with the passage of time, the monkeys having been followed as long as 5 months without further drug administration. Light microscopy and electron microscopy showed no structural abnormalities in the monkey brains at the sites of altered recordings, although occasional dendritic atrophy was noted at all cortical and subcortical brain sites examined. Chronic recording changes did not develop in a monkey that received U-50,488H on eight occasions. And none of the electrode-implanted monkeys that served as controls (having received no kappa agonists) developed recording changes. The sites affected by the active kappa agonists were those at which abnormal activity has been correlated with psychotic behavior. The ability of the kappa agonists to induce a lasting physiologic change without corresponding structural change at those focal sites implicated in schizophrenia may prove a useful probe in further investigations into the cause of schizophrenia and its ultimate treatment.

Effects of ketazocine, ethylketazocine and phenazocine on schedule-controlled behavior: antagonism by naloxone.

Dose-effect curves were determined for phenazocine (0.64-2.5 mg/kg), ketazocine (1.25-80 mg/kg) and ethylketazocine (1.25-80 mg/kg) in pigeons responding under a multiple fixed-ratio 30-response, fixed-interval 5-min schedule of grain presentation. All three opioid agonists decreased responding with the larger doses. The effects of phenazocine were completely antagonized by small doses of naloxone (0.01-1 mg/kg), whereas the effects of ethylketazocine required larger doses of naloxone (1-10 mg/kg) to be completely antagonized. The behavioral effects of ketazocine were partially attenuated by naloxone, but were not antagonized completely even by a 10 mg/kg dose of naloxone. These data from the pigeon are consistent with previous interpretations that the effects of phenazocine are mediated by actions at a mu opioid receptor, whereas the effects of ketazocine and ethylketazocine are mediated by actions at a kappa opioid receptor.

U50,488: a kappa-selective agent with poor affinity for mu1 opiate binding sites.

Although a number of potent kappa ligands have been reported, virtually all also label mu receptors with very high affinity. In contrast, we found that U50,488 is highly selective for kappa sites (Ki 12 nM) when compared to both morphine-selective (mu2) or delta receptors (both Ki values greater than 500 nM) confirming earlier reports. However, these reports did not examine interactions with mu1 receptors. In marked distinction to all other kappa-active agents examined which typically compete mu1 binding with Ki values under 1 nM, U50,488 competed mu1 binding quite poorly (Ki 370 nM). Thus, U50,488 is a highly selective kappa agent with very poor affinity for both subtypes of mu and delta receptors.

The epileptogenic spectrum of opiate agonists.

The present authors gave mu, delta, kappa, epsilon and sigma opiate receptor agonists intracerebroventricularly to rats both singly and in combination while monitoring the electroencephalogram from cortical and depth electrodes. Dose-response curves were plotted with naloxone against the changes produced by each agonist, and the effect of a number of anticonvulsant drugs on agonist-induced seizures was ascertained. Each opiate agonist produced a different seizure pattern with a different naloxone dose-response curve and anticonvulsant profile. The order of convulsive potency was epsilon greater than delta greater than mu greater than sigma much greater than kappa. Petit mal-like seizure activity was unique to the delta agonist, leucine-enkephalin, while only the mu agonist, morphine produced generalized convulsive seizures. These experiments raise the possibility that opiate systems in the brain may be involved in the pathogenesis of a wide spectrum of seizure disorders.

Classification of opioid and 5-hydroxytryptamine receptors by means of discriminative drug effects.

Behavioural studies can help to validate, modify and refine schemes for classifying receptors that are developed from electrophysiological and biochemical experiments. Drug discrimination constitutes one family of behavioural techniques that is being extensively used for studying subtypes of receptors, mainly because the methods often have remarkably high pharmacological specificity but can be applied to agents from a diverse range of classes. This article reviews briefly studies on agents acting through opioid and 5-hydroxytryptamine systems, where the results of the behavioural studies are very largely consistent with findings from other approaches. Many drugs used in such work have limited selectivity for putative subtypes of receptor, but little is known about how such compound pharmacological stimuli are processed in drug discrimination experiments. The characteristics of the discriminative stimuli produced by a mixture of drugs are discussed with respect to implications for effects of single drugs with multiple actions. Based on these initial experiments on discrimination of a mixture of nicotine and midazolam, it appears that the components of a compound pharmacological stimulus may be perceived and processed independently.

Evaluation of the direct actions of drugs with a serotonergic link in spinal analgesia on the release of [3H]serotonin from spinal cord synaptosomes.

Morphine, ketamine, ethylketocyclazocine and quipazine, drugs with an apparent local spinal serotonergic action, which contributes to their analgesic effects, were tested for their ability to alter the release of [3H]serotonin ([3H]5-HT) from a synaptosomal preparation from the spinal cord of the rat. Related compounds including [D-Ala2, D-Leu5]enkephalin (DADLE), n-allylnormetazocine and phencyclidine were also examined. None of the drugs was found to be capable of inducing a direct release of [3H]5-HT or of facilitating potassium-induced release of 5-HT. However, quipazine inhibited the depressant action of exogenous 5-HT on overflow of 3H (mediated through the 5-HT autoreceptor), an action that should facilitate serotonergic neurotransmission. In contrast to the other drugs, DADLE was found to depress K+ stimulated release of 5-HT. The results suggests that the serotonergic mechanism involved in the antinociceptive action of some of these drugs (i.e. ketamine, morphine and ethyl-ketocyclazocine) is not related to direct presynaptic interactions to promote release of 5-HT. On the other hand, a small population of serotonergic nerves critical for analgesia may be involved and are not detected using tissue from the whole spinal cord, However, it seems equally plausible that these drugs may produce their antinociceptive action through interactions with other neurotransmitter systems that in turn interface with the serotonergic nerves, perhaps through interneurons or collateral connections.

Effects of tifluadom on food consumption compared with chlordiazepoxide and kappa agonists in the rat.

Tifluadom (0.625-10.0 mg kg-1) was administered to non-deprived male rats which had been accustomed to eating a highly palatable diet in a 30 min test period. This compound, an opioid benzodiazepine, produced a significant increase in consumption of food when administered by the subcutaneous route, but not after intraperitoneal injection. Both chlordiazepoxide (1.25-20.0 mg kg-1) and the selective kappa opiate receptor agonist U-50,488 (0.3125-2.5 mg kg-1) also produced significant hyperphagic effects in the same feeding situation. In contrast, the two kappa opiate receptor agonists, ethylketocyclazocine (0.1-3.0 mg kg-1) and bremazocine (0.078-1.25 mg kg-1) brought about a dose-related suppression of food intake. Hence, the effects of kappa opiate receptor agonists in the feeding situation described here were not uniform. Furthermore, tifluadom could be likened either to a benzodiazepine or to a selective kappa receptor agonist. The hyperphagia induced by tifluadom was antagonized by naloxone, suggesting that the effect was mediated by an action at opiate receptors. It was not antagonized however by Ro15-1788 (10.0 and 20.0 mg kg-1), a selective benzodiazepine receptor antagonist, ruling out possible mediation by benzodiazepine receptors. The benzodiazepine receptor antagonist, CGS 8216, exhibited intrinsic activity when administered alone, and significantly reduced food consumption in tifluadom-treated and control animals.

Differential neuropharmacological effects of mu, kappa and sigma opioid agonists on cortical EEG power spectra in the rat. Stereospecificity and naloxone antagonism.

The study was designed to determine and compare the acute effects of the enantiomers of mu, kappa and sigma opioid agonists on the cortical EEG with the spectral analysis technique. The relative ability of naloxone to antagonize such effects was also assessed. Adult female Sprague-Dawley rats were implanted with chronic cortical EEG and temporalis muscle EMG recording electrodes, and with permanent indwelling external jugular cannulae. (-)-Methadone(mu agonist) produced increases in spectral power over the zero to 10Hz range, while (-)-ketocyclazocine (kappa agonist) produced increases in the 5-8 Hz band as a predominant peak. The(+) enantiomers of methadone and ketocyclazocine were inactive. The drug (+)-SK-10,047 (sigma agonist), produced a predominant spectral peak in the 7-9 HZ band that was associated with behavior that suggested psychotomimetic effects. The effects of morphine (mu agonist) on EEG and EEG power spectra were more sensitive to antagonism by naloxone than those produced by ketocyclazocine. The effects of (+/-)-SKF-10,047 and (+)-SKF-10,047 were not antagonized by 10 mg/kg of naloxone, while the effects of (-)-SKF-10,047 were partially antagonized by 10 mg/kg of naloxone. These findings further delineate the specificity of the differential effects of mu, kappa and sigma opioid agonists on the EEG and EEg power spectra in the rat.

Differential tolerance and cross-tolerance to repeated daily injections of mu and kappa opioid agonists in the rat.

This study was designed to compare the development of tolerance to the effects of morphine and ethylketocyclazocine ( EKC ) on EEG, EEG power spectra and behavior and to assess any cross-tolerance. Adult female Sprague-Dawley rats were implanted with chronic cortical EEG and temporalis muscle EMG recording electrodes and with permanent cannulae in the external jugular vein. In non-tolerant rats, 10 mg/kg (i.v.) injections of morphine and of ethylketocyclazocine produced biphasic EEG and behavioral profiles lasting for 3 and 2 hr , respectively. In both cases, a stuporous phase, associated with high-voltage cortical EEG bursts, was followed by a hyperactive phase, associated with low-voltage desynchronized EEG. However, power spectra derived from epochs of EEG bursting produced by morphine and ethylketocyclazocine were qualitatively different. One group of rats was then given a series of automatic, intravenous injections of morphine, while a second group received ethylketocyclazocine. Following chronic treatment, the duration of the biphasic EEG and behavioral profiles induced by morphine and ethylketocyclazocine were both significantly reduced. In both cases, the intensity of EEG bursting was also reduced as reflected by significant quantitative reductions in EEG power spectral densities. In assessments of cross-tolerance, ethylketocyclazocine tolerant rats were found to be cross-tolerant to the effects of morphine. However, no cross-tolerance to the effects of ethylketocyclazocine in morphine-tolerant rats was observed. These data contribute to a further understanding of the relative heterogeneity of mu and kappa receptor populations and to the differential pharmacodynamics of morphine and ethylketocyclazocine.

Modulation of mu, delta and kappa opioid receptors in rat brain by metal ions and histidine.

The effect of zinc (Zn2+) and several other trace elements was studied on the binding of the opioid receptor agonists [3H] DAGO [( ([Tyr-D-Ala-Gly-Methyl-Phe-Glyol]-enkephalin)a, [3H] DSTLE ([Tyr-D-Ser-Gly-Phe-Leu-Thr]-enkephalin) and [3H] EKC (ethylketocyclazocine), which are specific for the mu, delta and kappa opioid receptors, respectively, in the cerebral cortex of the rat. Physiological concentrations of zinc were inhibitory to mu receptor binding, whereas the delta and kappa receptors were relatively insensitive to this inhibition. Scatchard analysis, using these opioid agonists, revealed curvilinear plots; concentrations of zinc equal to or less than the IC50 (the concentration of cation which caused 50% inhibition of the binding of opioid ligand to its receptor), increased the KD (the dissociation constant) of all three subtypes of receptor, with no effect on the Bmax (the maximum number of binding sites) and abolished the high affinity sites of the delta and kappa receptors. Copper, cadmium and mercury also inhibited the binding of these ligands to their receptors. Histidine was most effective in preventing the inhibitory effects of zinc and copper, whereas it was less effective on cadmium and without any effect on the inhibition caused by mercury. Magnesium and manganese were stimulatory to opioid receptor binding, whereas cobalt and nickel had dual (stimulatory and inhibitory) effects. Non-inhibitory concentrations of zinc significantly decreased the stimulatory effects of magnesium and manganese on the mu and delta receptors, suggesting that part of the effect of zinc was through prevention of the actions of stimulatory cations.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of ketamine and (+)cyclazocine on 4-aminopyridine and "magnesium free" epileptogenic activity in hippocampal slices of rats.

The effects of ketamine and (+)cyclazocine on three in vitro models of epilepsy: the "Mg2+ free", the 4-aminopyridine (4-AP) and, for comparison, the penicillin model were studied. These data indicate that the two compounds had an inhibitory effect in hippocampal slices of rats, bathed in "Mg2+ free" solution at a concentration that did not influence the basal field potential. They also had an inhibitory effect on the penicillin model, but at concentrations ten times greater than those effective against "Mg2+ free" model. On the other hand, (+)cyclazocine was equally active against epileptogenic activity produced by 4-AP and "Mg2+ free" solution, while ketamine failed to produce an effect on epileptiform activity induced by 4-AP.

Binding of [3H]clonidine to membranes from cow neurohypophysis and cerebral cortex of the rat.

The binding of [3H]clonidine to membranes prepared from rat cerebral cortex and cow neurohypophysis was compared. Both tissues showed a high affinity site and a low affinity site when examined using a variety of techniques. The binding data, together with displacement studies using a variety of alpha 2-adrenoceptor agonists and antagonists, indicated that the binding activity of clonidine in the two tissues was very similar, however when the putative sigma opiate compound SKF 10,047 was examined it was found to have only a weak activity in displacing clonidine on the cortical membranes but a much stronger activity on the neurohypophysial membranes, other opiates showed little activity on binding of clonidine in either tissue. It is proposed that the binding of clonidine to neurohypophysial membranes may explain in part the various reports of suppression by clonidine of release of hormones from the neurohypophysis and that the effects of SKF 10,047 may be similarly associated with the reports of diuresis induced by phencyclidine.