Association of enkephalin catabolism inhibitors and CCKB antagonists: A potential use in the management of pain and opioid addiction

The overlapping distribution of opioid and cholecystokinin (CCK) peptides and their receptors (μ and δ opioid receptors; CCK-A and CCK-B receptors) in the central nervous system have led to a large number of studies aimed at clarifying the functional relationships between these two neuropeptides. Most of the pharmacological studies devoted to the role of CCK and enkephalins have been focused on the control of pain. Recently the existence of regulatory mechanisms between both systems have been proposed, and the physiological antagonism between CCK and endogenous opioid systems has been definitely demonstrated by coadministration of CCK-B selective antagonists with RB 101, a systemically active inhibitor, which fully protects enkephalins from their degradation. Several studies have also been done to investigate the functional relationships between both systems in development of opioid side-effects and in behavioral responses. This article will review the experimental pharmacology of association of enkephalin-degrading enzyme inhibitors and CCK-B antagonists to demonstrate the interest of these molecules in the management of both pain and opioid addiction. Special issue dedicated to Dr. Eric J. Simon.     

Effects of opioids on the immune system

Both therapeutic and chronic uses of opioids compromise the optimal functioning of the immune system. Overwhelming evidence suggests that opioid use affects both innate immunity and adaptive immunity. Chronic administration of opioids decreases the proliferative capacity of macrophage progenitor cells and lymphocytes. Additionally, the differentiated function of immune cells is significantly affected by opioids. These effects are mediated by either a direct action of opioids on the target cells or by indirect centrally mediated pathways. Molecular biological and biochemical characterization suggest that immune cells differentially express classical opioid receptors. Interestingly, these studies also reveal the presence of a novel class of opioid receptors in immune cells. We believe that this low affinity morphine binding site mediates the antiproliferative effects of morphine. Special issue dedicated to Dr. Eric J. Simon.     

Modulatory effects of Gs-coupled excitatory opioid receptor functions on opioid analgesia,tolerance, and dependence

Electrophysiologic studies of opioid effects on nociceptive types of dorsal root ganglion (DRG) neurons in organotypic cultures have shown that morphine and mostμ, δ, and κ opioid agonists can elicit bimodal excitatory as well as inhibitory modulation of the action potential duration (APD) of these cells. Excitatory opioid effects have been shown to be mediated by opioid receptors that are coupled via Gs to cyclic AMP-dependent ionic conductances that prolong the APD, whereas inhibitory opioid effects are mediated by opioid receptors coupled via Gi/Go to ionic conductuances that shorten the APD. Selective blockade of excitatory opioid receptor functions by low (ca. pM) concentrations of naloxone, naltrexone, etorphine and other specific agents markedly increases the inhibitory potency of morphine or other bimodally acting agonists and attenuates development of tolerance/dependence. These in vitro studies have been confirmed by tail-flick assays showin that acute co-treatment of mice with morphine plus ultra-low-dose naltrexone or etorphine remarkably enhances the antinociceptive potency of morphine whereas chronic co-treatment attenuates development of tolerance and naloxone-precipitated withdrawal-jumping symptoms. Special issue dedicated to Dr. Eric J. Simon.     

Stimulatory effects of opioids on transmitter release and possible cellular mechanisms: Overview and original results

Opiates and opioid peptides carry out their regulatory effects mainly by inhibiting neuronal activity. At the cellular level, opioids block voltage-dependent calcium channels, activate potassium channels and inhibit adenylate cyclase, thus reducing neurotransmitter release. An increasing body of evidence indicates an additional opposite, stimulatory activity of opioids. The present review summarizes the potentiating effects of opioids on transmitter release and the possible cellular events underlying this potentiation: elevation of cytosolic calcium level (by either activating Ca²⁺ influx or mobilizing intracellular stores), blockage of K⁺ channels and stimulation of adenylate cyclase. Biochemical, pharmacological and molecular biology studies suggest several molecular mechanisms of the bimodal activity of opioids, including the coupling of opioid receptors to various GTP-binding proteins, the involvement of different subunits of these proteins, and the activation of several intracellular signal transduction pathways. Among the many experimental preparations used to study the bimodal opioid activity, the SK-N-SH neuroblastoma cell line is presented here as a suitable model for studying the complete chain of events leading from binding to receptors down to regulation of transmitter release, and for elucidating the molecular mechanism involved in the stimulatory effects of opioid agonists. Special issue dedicated to Dr. Eric J. Simon.     

Application of the message-address concept to the docking of naltrexone and selective naltrexone-derived opioid antagonists into opioid receptor models

A binding site model for the opioid family of G-protein coupled receptors (GPCRs) is proposed based on the message-address concept of ligand recognition. Using ligand docking studies of the universal opioid antagonist, naltrexone, the structural basis for ‘message’ recognition is explored across all three receptor types, μ, δ, and κ. The binding mode proposed and basis for selectivity are also rationalized using the naltrexone-derived ligands, naltrindole (NTI) and norbinaltorphimine (nor BNI). These ligands are docked to the receptor according to the common naltrexone core or message. The resulting orientation places key ‘address’ elements in close proximity to amino acid residues critical to selectivity among receptor types. Selectivity is explained by sequence differences in the μ, δ, and κ receptors at these recognition points. Support for the model is derived from site directed mutagenesis studies and ligand binding data for the opioid receptors and other related GPCRs. Special issue dedicated to Dr. Eric J. Simon     

New perspectives on cocaine addiction: recent findings from animal research

Research with laboratory animals has provided several insights into the nature of cocaine abuse and addiction. First, the nature of drug addiction has been reevaluated and the emphasis has shifted from physical dependence to compulsive drug-taking behavior. Second, animal studies suggest that cocaine is at least as addictive as heroin and possibly even more addictive. Third, cocaine is potentially more dangerous than heroin as evidenced by the higher fatality rate seen in laboratory animals given unlimited access to these drugs. Fourth, the neural basis of cocaine reinforcement has been identified and involves an enhancement of dopaminergic neurotransmission in the ventral tegmental dopamine system. Other addictive drugs (e.g., opiates) may also derive at least part of their reinforcing impact by pharmacologically activating this reward system. Fifth, although the biological consequences of repeated cocaine self-administration on central nervous system functioning are poorly understood, preliminary findings suggest that intravenous cocaine self-administration may decrease neural functioning in this brain reward system. This has important clinical implications because diminished functioning of an important brain reward system may significantly contribute to relapse into cocaine addiction. These and other findings from experimentation with laboratory animals suggest new considerations for the etiology and treatment of drug addiction.     

Laminar distribution of the multiple opioid receptors in the human cerebral cortex

Quantitative autoradiographic assessment of cerebral cortical laminar distribution of μ, δ and κ opioid receptors was carried out in coronal sections of five post-mortem human brains obtained at autopsy. The cortical areas studied were: cingulate, frontal, insular, parietal, parahippocampal, temporal, occipitotemporal, occipital and striate area. In general, the laminar patterns of distribution for the three types of receptors are distinctive. Peak levels of δ opioid binding are in laminae I, II, and IIa. μ-Receptors are located in lamina III followed by I and II in cingulate, frontal, insular and parietal cortices and lamina IV in temporal and occiptotemporal cortices. κ-Receptors are found concentrated in laminae V and VI. The patterns of opioid binding in cortical laminae showed remarkable consistency in all five brains examined. In contrast to other cortical areas, the parahippocampal gyrus, at the level of the amygdaloid formation, demonstrated peak κ receptor density in laminae I, II and III. μ-Opioid binding was undetectable in the lateral occipital cortex and in the striate area. Special issue dedicated to Dr. Eric J. Simon.     

Genetic association analyses of PDYN polymorphisms with heroin and cocaine addiction

Genetic factors are believed to account for 30-50% of the risk for cocaine and heroin addiction. Dynorphin peptides, derived from the prodynorphin (PDYN) precursor, bind to opioid receptors, preferentially the kappa-opioid receptor, and may mediate the aversive effects of drugs of abuse. Dynorphin peptides produce place aversion in animals and produce dysphoria in humans. Cocaine and heroin have both been shown to increase expression of PDYN in brain regions relevant for drug reward and use. Polymorphisms in PDYN are therefore hypothesized to increase risk for addiction to drugs of abuse. In this study, 3 polymorphisms in PDYN (rs1022563, rs910080 and rs1997794) were genotyped in opioid-addicted [248 African Americans (AAs) and 1040 European Americans (EAs)], cocaine-addicted (1248 AAs and 336 EAs) and control individuals (674 AAs and 656 EAs). Sex-specific analyses were also performed as a previous study identified PDYN polymorphisms to be more significantly associated with female opioid addicts. We found rs1022563 to be significantly associated with opioid addiction in EAs [P = 0.03, odds ratio (OR) = 1.31; false discovery rate (FDR) corrected q-value]; however, when we performed female-specific association analyses, the OR increased from 1.31 to 1.51. Increased ORs were observed for rs910080 and rs199774 in female opioid addicts also in EAs. No statistically significant associations were observed with cocaine or opioid addiction in AAs. These data show that polymorphisms in PDYN are associated with opioid addiction in EAs and provide further evidence that these risk variants may be more relevant in females.     

Sensitivity of opioid receptor binding to N-substituted maleimides and methanethiosulfonate derivatives

A series of N-substituted maleimides was shown to effectively inactivate bremazocine binding to δ opioid receptors. Apparent second order rate constants for inactivation increased with increasing size of the N-substituent: N-methyl < N-ethyl < N-butyl < N-phenylmaleimide. It is suggested that the positive chain length effect is attributed to nonpolar interactions with the receptor in the vicinity of the reactive group. Binding to μ and δ opioid receptors was equally sensitive to inactivation by (2-aminoethyl)methanethiosulfonate; the [2-(trimethylammonium)ethyl] and (2-sulfonatoethyl) derivatives were less active. Site-directed mutagenesis of the μ opioid receptor indicated that Cys¹⁵⁹, Cys¹⁹⁰, Cys²³⁵, Cys²⁹², or Cys³²¹, residing in transmembrane domain 3, 4, 5, 6, and 7, respectively, werenot the site of modification. Special issue dedicated to Dr. Eric J. Simon.     

Cyclazocine revisited

Recently synthesized compounds which have long-term mu antagonist activity and short-term kappa agonist effects prevent self-administration of cocaine and morphine in rats. Cyclazocine, a compound synthesized in 1962 and studied in animals and man in the 1960's and in the early 1970's is a mu antagonist in rats and man and is a potent kappa agonist in both species. It also prevents self-administration of cocaine and morphine in rats. Although it produces unpleasant side effects in man, subjects become tolerant to these side effects but not to the antagonistic actions of the drug after prolonged administration. Deceased Special issue dedicated to Dr. Eric J. Simon.     

Opioid receptor interactions: local and nonlocal,symmetric and asymmetric,physical and functional

The pharmacological effects of opioid drugs and endogenous opioid peptides are mediated by several kinds of receptors, the major ones being mu, delta and kappa. Though classically it has been thought that a particular effect mediated by a drug or other ligand results from its interaction with a single type of receptor, accumulating evidence demonstrates that interactions between receptors play a major role in opioid actions. These interactions may be either local, involving receptors within the same tissue, or nonlocal, between receptors located in different tissues. Nonlocal interactions always involve intercellular mechanisms, whereas local interactions may involve either intercellular or intracellular interactions, the latter including physical association of receptors. Both local and nonlocal interactions, moreover, may be either symmetric, with ligand interaction at one receptor affecting interaction at the other, or asymmetric; and either potentiating or inhibitory. In this article we discuss major examples of these kinds of interactions, and their role in the acute and chronic effects of opioids. Knowledge of these interactions may have important implications for the design of opioids with more specific actions, and for eliminating the addictive liability of these drugs.     

The genetics of the opioid system and specific drug addictions

Addiction to drugs is a chronic, relapsing brain disease that has major medical, social, and economic complications. It has been established that genetic factors contribute to the vulnerability to develop drug addiction and to the effectiveness of its treatment. Identification of these factors may increase our understanding of the disorders, help in the development of new treatments and advance personalized medicine. In this review, we will describe the genetics of the major genes of the opioid system (opioid receptors and their endogenous ligands) in connection to addiction to opioids, cocaine, alcohol and methamphetamines. Particular emphasis is given to association and functional studies of specific variants. We will provide information on the sample populations and the size of each study, as well as a list of the variants implicated in association with addiction-related phenotypes, and with the effectiveness of pharmacotherapy for addiction.     

Aminopeptidase activity in the postmortem brain of human heroin addicts

Several studies have reported that the chronic administration of opioids induces changes in the biosynthesis of endogenous opioid peptides or their precursors in specific brain regions of the adult central nervous system. However, little is known about the catabolic regulation of opioid peptides and its contribution to neuroadaptative changes underlying drug addiction. In the present study, we have analyzed the activity of two enkephalin-degrading enzymes (puromycin-sensitive aminopeptidase or PSA and aminopeptidase N or APN) and two functionally different, soluble aminopeptidases (aminopeptidase B and aspartyl-aminopeptidase) in postmortem samples of prefrontal cortex and caudate nucleus of eight human heroin addict brains and eight matched-controls. Enzyme activities were fluorimetrically measured using beta-naphthylamide derivatives. An increase in the activity of soluble PSA in the prefrontal cortex of heroin abusers was observed (heroin addict group: 51,452+/-3892 UAP/mg protein versus control group: 42,003+/-2597 UAP/mg protein; P<0.05), while the activity of the other peptidases in both brain regions remained unaltered. This result agrees with previous findings in morphine-tolerant rats, and indicates that soluble PSA may be involved in neurobiological processes which underlie heroin addiction.     

[35S]GTPγS binding: A tool to evaluate functional activity of a cloned opioid receptor transiently expressed in COS cells

In this paper we propose a powerful procedure to measure functional activation of the mouse δ-opioid receptor transiently expressed in mammalian cells. Receptor stimulation was assessed using a population of electroporated COS cells, transfected at a 50% efficiency. Under those conditions, agonist-promoted activation of the receptor was measured by [³⁵S]GTPγS binding. Both BW373U86, an alkaloid compound, and DADLE, a peptide agonist, elicited increase of specific [³⁵S]GTPγS binding representing 300% of basal level. Maximal activation was compared to that obtained for the cloned receptor stably expressed in CHO cells. Agonist efficacy was similar in both expressions systems, demonstrating the high sensitivity of the proposed method applied to transient expression. Finally dose-response curves were found highly reproducible across transfection experiments, opening the possibility for a direct comparison of distinct recombinant receptor preparations. This method represents a powerfull tool for the study of opioid signal transduction at the receptor level. It may also be extended to investigate signalling properties of other Gi/Go coupled receptors. Special issue dedicated to Dr. Eric J. Simon.     

Sigma1 binding in a human neuroblastoma cell line

Behaviorally, sigma₁ agents modulate opioid analgesia. To examine possible mechanisms responsible for these interactions, we have identified a cell line containing both sigma₁ and opioid receptors. [³H](+)-pentazocine binding in BE(2)-C human neuroblastoma cells is high affinity (K_D 3.4±0.7 nM) and high density (B_max 2.98±0.14 pmol/mg protein). Competition studies reveal a selectivity profile similar to that of sigma₁ sites in guinea pig brain. (+)-Pentazocine has no effect upon either basal or forskolin-stimulated cyclase in the BE(2)-C cells, but cAMP accumulation is inhibited by the morphine, DPDPE and naloxone benzoylhydrazone. (+)-Pentazocine at concentrations as high as 10 μM does not affect this opioid effect, implying that sigma₁/opioid interactions are not mediated at the level of the cell. This suggests that their behavioral interactions result from interacting neural circuits. Although (+)-pentazocine is without effect in the cyclase system, it does block carbachol-stimulated phosphoinositol turnover (IC₅₀ 6.5±1.14 μM). The specificity of the effect is confirmed by the ability of haloperidol (1 μM) to shift the IC₅₀ value of (+)-pentazocine 2-fold to the right. Special issue dedicated to Dr. Eric J. Simon.     

Feeding,drug abuse,and the sensitization of reward by metabolic need

The incentive-motivating effects of external stimuli are dependent, in part, upon the internal need state of the organism. The increased rewarding efficacy of food as a function of energy deficit, for example, has obvious adaptive value. The enhancement of food reward extends, however, to drugs of abuse and electrical brain stimulation, probably due to a shared neural substrate. Research reviewed in this paper uses lateral hypothalamic electrical stimulation to probe the sensitivity of the brain reward system and investigate mechanisms through which metabolic need, induced by chronic food restriction and streptozotocin-induced diabetes, sensitizes this system. Results indicate that sensitivity to rewarding brain stimulation varies inversely with declining body weight. The effect is not mimicked by pharmacological glucoprivation or lipoprivation in ad libitum fed animals; sensitization appears to depend on persistent metabolic need or adipose depletion. While the literature suggests elevated plasma corticosterone as a peripheral trigger of reward sensitization, sensitization was not reversed by meal-induced or pharmacological suppression of plasma corticosterone. Centrally, reward sensitization is mediated by opioid receptors, since the effect is reversed by intracerebroventricular (i.c.v.) infusion of naltrexone, TCTAP (μ antagonist) and nor-binaltorphimine (κ antagonist). The fact that these same treatments, as well as i.c.v. infusion of dynorphin A antiserum, block the feeding response to lateral hypothalamic stimulation suggests that feeding and reward sensitization are mediated by a common opioid mechanism. Using in vitro autoradiography, radioimmunoassays and a solution hybridization mRNA assay, brain regional μ and κ opioid receptor binding, levels of prodynorphin-derived peptides, and prodynorphin mRNA, respectively, were measured in food-restricted and diabetic rats. Changes that could plausibly be involved in reward sensitization are discussed, with emphasis on the increased dynorphin A_1–8 and prodynorphin mRNA levels in lateral hypothalamic neurons that innervate the pontine parabrachial nucleus, where μ binding decreased and κ binding increased. Finally, the possible linkage between metabolic need and activation of a brain opioid mechanism is discussed, as is evidence supporting the relevance of these findings to drug abuse. Special issue dedicated to Dr. Eric J. Simon.     

Opioidergic modulation of cocaine conditioned place preferences.

Recent studies have begun to assess the utility of opioid agonists and antagonists for the treatment of cocaine addiction. The present studies assess the effects of naltrexone or methadone on cocaine's reinforcing properties using the conditioned place preference (CPP) test. The results indicate that a 56 mg/kg dose of naltrexone, given 4 hr prior to conditioning, attenuates cocaine's CPP. In contrast, methadone (8 mg/kg), given 1 hr prior to conditioning, enhanced cocaine's reinforcing properties. These results support the suggestion that opioid antagonists may have clinical utility in treating cocaine addiction. The results with methadone lead to a possible explanation for the higher rates of cocaine use in methadone-treated heroin addicts.     

A review of the effects of dopaminergic agents on humans,animals, and drug-seeking behavior,and its implications for medication development

Medication development for cocaine abuse has focused on potential mechanisms of action related to the abuse of cocaine. The hypothesis that mesolimbic dopamine (DA) is the key neurochemical mediator of cocaine’s addictive and reinforcing effects is well supported by a wide variety of data from animal studies. On the other hand, medications that increase DA or block its actions in humans can produce effects that appear incompatible with this hypothesis. This article reviews these incompatibilities between animal and human data with a focus on the DAergic actions of drugs, including DA reuptake inhibitors, direct DA agonists, DA increasers, and DA antagonists. Possible reasons for these discrepancies are discussed, and the potential role of high-affinity DA uptake inhibitors, such as GBR12909, for pharmacotherapeutic application to treat cocaine abuse is discussed. Since progress in developing pharmacotherapies for treating cocaine addiction in humans is likely to come from understanding its mechanisms of action, it is clear that further research on the effects of cocaine in humans and animals will be critical to the medication development effort. A shortened version of this paper was presented at the Satellite Meeting of the International Society for Neurochemistry on “Cellular and Molecular Mechanisms of Drugs of Abuse: Cocaine and Methamphetamine” held on August 19–20, 1993 in Nice, France.     

Cocaine Modulates the Expression of Opioid Receptors and miR-let-7d in Zebrafish Embryos

Prenatal exposure to cocaine, in mammals, has been shown to interfere with the expression of opioid receptors, which can have repercussions in its activity. Likewise, microRNAs, such as let-7, have been shown to regulate the expression of opioid receptors and hence their functions in mammals and in vitro experiments. In light of this, using the zebrafish embryos as a model our aim here was to evaluate the actions of cocaine in the expression of opioid receptors and let-7d miRNA during embryogenesis. In order to determine the effects produced by cocaine on the opioid receptors (zfmor, zfdor1 and zfdor2) and let-7d miRNA (dre-let-7d) and its precursors (dre-let-7d-1 and dre-let-7d-2), embryos were exposed to 1.5 µM cocaine hydrochloride (HCl). Our results revealed that cocaine upregulated dre-let-7d and its precursors, and also increased the expression of zfmor, zfdor1 and zfdor2 during early developmental stages and decreased them in late embryonic stages. The changes observed in the expression of opioid receptors might occur through dre-let-7d, since DNA sequences and the morpholinos of opioid receptors microinjections altered the expression of dre-let-7d and its precursors. Likewise, opioid receptors and dre-let-7d showed similar distributions in the central nervous system (CNS) and at the periphery, pointing to a possible interrelationship between them.In conclusion, the silencing and overexpression of opioid receptors altered the expression of dre-let-7d, which points to the notion that cocaine via dre-let-7 can modulate the expression of opioid receptors. Our study provides new insights into the actions of cocaine during zebrafish embryogenesis, indicating a role of miRNAs, let-7d, in development and its relationship with gene expression of opioid receptors, related to pain and addiction process.