KOR Antagonists Research Articles

Design, synthesis, and pharmacological evaluation of JDTic analogs to examine the significance of replacement of the 3-hydroxyphenyl group with pyridine or thiophene bioisosteres

The potent and selective KOR antagonist JDTic was derived from the N-substituted trans-3,4-dimethyl-4-(3-hydroxyphenyl)piperidine class of pure opioid antagonists. In previous studies we reported that compounds that did not have a hydroxyl on the 3-hydroxyphenyl group and did not have methyl groups at the 3- and 4-position of the piperidine ring were still potent and selective KOR antagonists. In this study we report JDTic analogs 2, 3a–b, 4a–b, and 5, where the 3-hydroxyphenyl ring has been replaced by a 2-, 3-, or 4-pyridyl or 3-thienyl group and do not have the 3-methyl or 3,4-dimethyl groups, remain potent and selective KOR antagonists. Of these, (3R)-7-hydroxy-N-(1S)-2-methyl-[4-methyl-4-pyridine-3-yl-carboxamide (3b) had the best overall binding potency and selectivity in a [35S]GTPγS functional assay, with a Ke=0.18nM at the KOR and 273- and 16,700-fold selectivity for the KOR relative to the MOR and DOR, respectively. Calculated physiochemical properties for 3b suggest that it will cross the blood–brain barrier.

Species differences in the effects of the κ-opioid receptor antagonist zyklophin.

We have shown that dysregulation of the dynorphin/kappa-opioid receptor (DYN/KOR) system contributes to escalated alcohol self-administration in alcohol dependence and that KOR antagonists with extended durations of action selectively reduce escalated alcohol consumption in alcohol-dependent animals. As KOR antagonism has gained widespread attention as a potential therapeutic target to treat alcoholism and multiple neuropsychiatric disorders, we tested the effect of zyklophin (a short-acting KOR antagonist) on escalated alcohol self-administration in rats made alcohol-dependent using intermittent alcohol vapor exposure. Following dependence induction, zyklophin was infused centrally prior to alcohol self-administration sessions and locomotor activity tests during acute withdrawal. Zyklophin did not impact alcohol self-administration or locomotor activity in either exposure condition. To investigate the neurobiological basis of this atypical effect for a KOR antagonist, we utilized a κ-, μ-, and δ-opioid receptor agonist-stimulated GTPyS coupling assay to examine the opioid receptor specificity of zyklophin in the rat brain and mouse brain. In rats, zyklophin did not affect U50488-, DAMGO-, or DADLE-stimulated GTPyS coupling, whereas the prototypical KOR antagonist nor-binaltorphimine (norBNI) attenuated U50488-induced stimulation in the rat brain tissue at concentrations that did not impact μ- and δ-receptor function. To reconcile the discrepancy between the present rat data and published mouse data, comparable GTPyS assays were conducted using mouse brain tissue; zyklophin effects were consistent with KOR antagonism in mice. Moreover, at higher concentrations, zyklophin exhibited agonist properties in rat and mouse brains. These results identify species differences in zyklophin efficacy that, given the rising interest in the development of short-duration KOR antagonists, should provide valuable information for therapeutic development efforts.

Effects of the kappa opioid receptor antagonist nor-binaltorphimine (nor-BNI) on cocaine versus food choice and extended-access cocaine intake in rhesus monkeys.

The dynorphin/kappa opioid receptor (KOR) system has been implicated as one potential neurobiological modulator of the abuse-related effects of cocaine and as a potential target for medications development. This study determined effects of the KOR antagonist nor-binaltorphimine (nor-BNI) on cocaine self-administration under a novel procedure that featured two daily components: (1) a 2-hour 'choice' component (9:00-11:00 am) when monkeys could choose between food pellets and cocaine injections (0-0.1 mg/kg per injection, intravenous) and (2) a 20-hour 'extended-access' component (noon to 8:00 am) when cocaine (0.1 mg/kg per injection) was available under a fixed-ratio schedule to promote high daily cocaine intakes. Rhesus monkeys (n = 4) were given 14 days of exposure to the choice + extended-access procedure then treated with nor-BNI (3.2 or 10.0 mg/kg, intramuscular), and cocaine choice and extended-access cocaine intake were evaluated for an additional 14 days. Consistent with previous studies, cocaine maintained both a dose-dependent increase in cocaine choice during choice components and a high level of cocaine intake during extended-access components. Neither 3.2 nor 10 mg/kg nor-BNI significantly altered cocaine choice or extended-access cocaine intake. In two additional monkeys, nor-BNI also had no effect on cocaine choice or extended-access cocaine intake when it was administered at the beginning of exposure to the extended-access components. Overall, these results do not support a major role for the dynorphin/KOR system in modulating cocaine self-administration under these conditions in non-human primates nor do they support the clinical utility of KOR antagonists as a pharmacotherapeutic strategy for cocaine addiction.

Design, synthesis, and biological evaluation of (3R)-1,2,3,4-tetrahydro-7-hydroxy-N-[(1S)-1-[[(3R,4R)-4-(3-hydroxyphenyl)-3,4-dimethyl-1-piperidinyl]methyl]-2-methylpropyl]-3-isoquinolinecarboxamide (JDTic) analogues: in vitro pharmacology and ADME profile.

JDTic analogues 4–15 which have the hydroxyl groups replaced with other groups were synthesized and their in vitro efficacy at the μ, δ, and κ opioid receptors determined and compared to JDTic using [35S]GTPγS assays. Compounds 4, 5, 6, 13, 14, and 15 had Ke = 0.024, 0.01, 0.039, 0.02, 0.11, and 0.041 nM compared to the Ke = 0.02 nM for JDTic at the κ receptor and were highly selective for the κ receptor relative to the μ and δ opioid receptors. Unexpectedly, replacement of the 3-hydroxyl substituent of the 4-(3-hydroxyphenyl) group of JDTic with a H, F, or Cl substituent leads to potent and selective KOR antagonists. In vitro studies to determine various ADME properties combined with calculated TPSA, clogP, and logBB values suggests that the potent and selective κ opioid receptors 4, 5, 13, and 14 deserve consideration for further development toward potential drugs for CNS disorders.

Characterization of the kappa opioid receptor‐mediated G protein signaling in mouse striatum using a [35S]GTPγS binding assay (659.6)

The kappa opioid receptor/dynorphin system is an endogenous system involve in modulating stress responses. Disruption of the ability to respond appropriately to stress can lead to elevated risks of developing mood disorders including depression, anxiety, and drug addiction. The dynorphin peptides are products cleaved from prodynorphin of varying amino acid length and are considered the cognate high affinity ligands for the KORs. However, they also promiscuously bind to and activate other opioid receptors in the brain. Here we seek to determine what component of dynorphin signaling can be attributed specifically to KOR by investigating the pharmacological profiles of dynorphin peptides in wild‐type (WT), KOR knockout (KOR‐KO), and mu opioid receptor knockout (MOR‐KO) mice using [35S]GTPγS binding assay in striatal membrane preparations. We find that dynorphin peptides are efficacious in stimulating [35S]GTPγS binding in WT striatum in a manner that resists concentration‐induced plateau. While KOR activation contributes significantly to dynorphin‐induced [35S]GTPγS binding in striatum, our studies identify a significant contribution of MOR‐induced coupling as well. Moreover, we find that KOR displays a constitutive activity in striatum that can be inhibited by antagonist pretreatment; the constitutive activity is not present in KOR‐KO striatum. Recently developed, functionally selective small molecule KOR agonists as well as new selective KOR antagonists were also tested in striata. Our studies in characterization of dynorphin’s effects at the KOR in brain will be essential for developing ex vivo assays that assess the selective contributions of biased KOR ligands that have been recently developed.Grant Funding Source: Supported by NIDA grant R01 DA031927(LMB, JA)

Effects of the kappa opioid antagonist nor‐binaltorphimine on cocaine vs. food choice and extended‐access cocaine intake in rhesus monkeys (658.6)

The dynorphin/κ opioid receptor system (KOR) has been implicated as one potential neurobiological target for treating cocaine addiction. This study determined effects of the KOR antagonist nor‐binaltorphimine (nor‐BNI) on cocaine self‐administration by rhesus monkeys responding under a novel procedure that featured two daily components: (1) 2‐h choice sessions (9‐11am) when monkeys could choose between food pellets and cocaine (0‐0.1 mg/kg/inj, IV), and (2) 20‐h “extended access” sessions (noon‐8am) when only cocaine (0.1 mg/kg/inj) was available under a fixed‐ratio schedule to promote high daily cocaine intakes. After 14 days of responding under this novel procedure, nor‐BNI (3.2 mg/kg, IM) was administered, and responding was evaluated for an additional 14 days. Consistent with previous studies, cocaine maintained a dose‐dependent increase in cocaine choice, and monkeys also self‐administered cocaine throughout the extended access component. Total daily intake averaged 7.5±0.66 mg/kg cocaine. Nor‐BNI treatment had no significant effect on cocaine choice either during or after withdrawal from extended cocaine access. These results suggest the dynorphin/KOR system does not play a major role in cocaine self‐administration under these conditions. These results also do not support the utility of KOR antagonists as medications to treat ongoing cocaine use in persons dependent on cocaine.Grant Funding Source: Funded by NIH grant R01DA026946.

Current κ Opioid Receptor Ligands and Discovery of a New Molecular Scaffold as a κ Opioid Receptor Antagonist Using Pharmacophore-Based Virtual Screening

The κ opioid receptor (KOR) plays a significant role in many physiological functions, including pain relief, stress, depression, drug abuse, anxiety and psychotic behaviors. KORs are widely distributed in the central and peripheral nervous systems, and are specifically activated by endogenous opioids derived from prodynorphin. They are members of the G protein-coupled receptor superfamily, and the crystal structure of the human KOR was recently elucidated. KORs were initially studied for their involvement in mediation of pain as stimulation of KOR produces analgesia and minimizes abuse liability and other side effects. Nowadays, the KOR is rapidly emerging as an important target for the treatment of a variety of other human disorders. Specifically, the KOR system has become increasingly implicated as a modulator of stress-related and addictive behaviors. Several selective KOR partial agonists and antagonists have been developed as potential antidepressants, anxiolytic and anti-addiction medications. Although many KOR ligands have not demonstrated desirable pharmacological properties, some have been shown to be viable drug candidates. Herein, we describe chemical and pharmacological developments on KOR ligands, advantages and challenges, and potential therapeutic applications of ligands for KORs. In the second part, recent advances in the KOR drug design utilizing computational approaches are presented, with focus on the discovery of a new naturally derived scaffold, sewarine, as a novel class of selective KOR ligands with antagonist properties, using a pharmacophore-based virtual screening strategy.

Long-Term Antagonism of κ Opioid Receptors Prevents Escalation of and Increased Motivation for Heroin Intake

The abuse of opioid drugs, both illicit and prescription, is a persistent problem in the United States, accounting for >1.2 million users who require treatment each year. Current treatments rely on suppressing immediate withdrawal symptoms and replacing illicit drug use with long-acting opiate drugs. However, the mechanisms that lead to preventing opiate dependence are still poorly understood. We hypothesized that κ opioid receptor (KOR) activation during chronic opioid intake contributes to negative affective states associated with withdrawal and the motivation to take increasing amounts of heroin. Using a 12 h long-access model of heroin self-administration, rats showed escalation of heroin intake over several weeks. This was prevented by a single high dose (30 mg/kg) of the long-acting KOR antagonist norbinaltorphimine (nor-BNI), paralleled by reduced motivation to respond for heroin on a progressive-ratio schedule of reinforcement, a measure of compulsive-like responding. Systemic nor-BNI also significantly decreased heroin withdrawal-associated anxiety-like behavior. Immunohistochemical analysis showed prodynorphin content increased in the nucleus accumbens core in all heroin-exposed rats, but selectively increased in the nucleus accumbens shell in long-access rats. Local infusion of nor-BNI (4 μg/side) into accumbens core altered the initial intake of heroin but not the rate of escalation, while local injection into accumbens shell selectively suppressed increases in heroin intake over time without altering initial intake. These data suggest that dynorphin activity in the nucleus accumbens mediates the increasing motivation for heroin taking and compulsive-like responding for heroin, suggesting that KOR antagonists may be promising targets for the treatment of opioid addiction.

Upregulated dynorphin opioid peptides mediate alcohol-induced learning and memory impairment

The dynorphin opioid peptides control glutamate neurotransmission in the hippocampus. Alcohol-induced dysregulation of this circuit may lead to impairments in spatial learning and memory. This study examines whether changes in the hippocampal dynorphin and glutamate systems are related, and contribute to impairment of spatial learning and memory in a rat model of cognitive deficit associated with alcohol binge drinking. Hippocampal dynorphins (radioimmunoassay) and glutamate (in vivo microdialysis) were analyzed in Wistar rats exposed to repeated moderate-dose ethanol bouts that impair spatial learning and memory in the Water Maze Task (WMT). The highly selective, long-acting κ-opioid receptor (KOR) antagonist nor-binaltorphimine (nor-BNI) was administered systemically or into the hippocampal CA3 region to test a role of dynorphins in alcohol-induced dysregulations in glutamate neurotransmission and behavior in the WMT. The ethanol treatment impaired learning and memory, upregulated dynorphins and increased glutamate overflow in the CA3 region. Administration of nor-BNI after cessation of ethanol exposure reversed ethanol-induced changes in glutamate neurotransmission in animals exposed to ethanol and normalized their performance in the WMT. The findings suggest that impairments of spatial learning and memory by binge-like ethanol exposure are mediated through the KOR activation by upregulated dynorphins resulting in elevation in glutamate levels. Selective KOR antagonists may correct alcohol-induced pathological processes, thus representing a novel pharmacotherapy for treating of ethanol-related cognitive deficits.

Development of κ Opioid Receptor Antagonists

κ opioid receptors (KORs) belong to the G-protein-coupled class of receptors (GPCRs). They are activated by the endogenous opioid peptide dynorphin (DYN) and expressed at particularly high levels within brain areas implicated in modulation of motivation, emotion, and cognitive function. Chronic activation of KORs in animal models has maladaptive effects including increases in behaviors that reflect depression, the propensity to engage in drug-seeking behavior, and drug craving. The fact that KOR activation has such a profound influence on behaviors often triggered by stress has led to interest in selective KOR antagonists as potential therapeutic agents. This Perspective provides a description of preclinical research conducted in the development of several different classes of selective KOR antagonists, a summary of the clinical studies conducted thus far, and recommendations for the type of work needed in the future to determine if these agents would be useful as pharmacotherapies for neuropsychiatric illness.

Corticotropin-Releasing Factor (CRF)-Induced Disruption of Attention in Rats Is Blocked by the κ-Opioid Receptor Antagonist JDTic

Stress often disrupts behavior and can lead to psychiatric illness. Considerable evidence suggests that corticotropin-releasing factor (CRF) plays an important role in regulating the effects of stress. CRF administration produces stress-like effects in humans and laboratory animals, and CRF levels are elevated in individuals with stress-related illness. Recent work indicates that κ-opioid receptor (KOR) antagonists can block CRF effects, raising the possibility that at least some of the effects of stress are mediated via KORs. Here we examined the effects of CRF on performance in the 5-choice serial reaction time task (5CSRTT), a test used to quantify attention in rodents, as well as functional interactions between CRF and KORs. Male Sprague-Dawley rats were trained in the 5CSRTT and then each was implanted with an intracerebroventricular (ICV) cannula. After recovery and restabilization of performance, they received a single intraperitoneal (IP) injection of vehicle or JDTic (10 mg/kg), a KOR antagonist with long-lasting (>14 days) effects. In subsequent sessions, rats received ICV infusions of CRF (0.25-1.0 μg) or vehicle and were tested 60 min later. CRF dose-dependently disrupted performance as reflected by decreases in correct responding, increases in omission errors, increases in latencies to respond correctly, and increases in time to complete the session. JDTic attenuated each of these CRF-induced deficits while having no effects on its own. The persistent ability of JDTic to disrupt KOR function was confirmed using the tail immersion assay. These findings indicate that KOR antagonists can prevent acute stress-related effects that degrade performance in tasks requiring attention.

Allosteric Interactions between δ and κ Opioid Receptors in Peripheral Sensory Neurons

The peripheral δ opioid receptor (DOR) is an attractive target for analgesic drug development. There is evidence that DOR can form heteromers with the κ-opioid receptor (KOR). As drug targets, heteromeric receptors offer an additional level of selectivity and, because of allosteric interactions between protomers, functionality. Here we report that selective KOR antagonists differentially altered the potency and/or efficacy of DOR agonists in primary cultures of adult rat peripheral sensory neurons and in a rat behavioral model of thermal allodynia. In vitro, the KOR antagonist nor-binaltorphimine (nor-BNI) enhanced the potency of [D-Pen(2,5)]-enkephalin (DPDPE), decreased the potency of [D-Ala(2),D-Leu(5)]-enkephalin (DADLE), and decreased the potency and efficacy of 4-[(R)-[(2S,5R)-4-allyl-2,5-dimethylpiperazin-1-yl](3-methoxyphenyl)methyl]-N,N-diethylbenzamide (SNC80) to inhibit prostaglandin E(2) (PGE(2))-stimulated adenylyl cyclase activity. In vivo, nor-BNI enhanced the effect of DPDPE and decreased the effect of SNC80 to inhibit PGE(2)-stimulated thermal allodynia. In contrast to nor-BNI, the KOR antagonist 5'-guanidinonaltrindole (5'-GNTI) reduced the response of DPDPE both in cultured neurons and in vivo. Evidence for DOR-KOR heteromers in peripheral sensory neurons included coimmunoprecipitation of DOR with KOR, a DOR-KOR heteromer selective antibody augmented the antinociceptive effect of DPDPE in vivo, and the DOR-KOR heteromer agonist 6'-GNTI inhibited adenylyl cyclase activity in vitro as well as PGE(2)-stimulated thermal allodynia in vivo. Taken together, these data suggest that DOR-KOR heteromers exist in rat primary sensory neurons and that KOR antagonists can act as modulators of DOR agonist responses most likely through allosteric interactions between the protomers of the DOR-KOR heteromer.

Quantitative PK–PD Model-Based Translational Pharmacology of a Novel Kappa Opioid Receptor Antagonist Between Rats and Humans

Pharmacokinetic-pharmacodynamic (PK-PD) modeling greatly enables quantitative implementation of the "learn and confirm" paradigm across different stages of drug discovery and development. This work describes the successful prospective application of this concept in the discovery and early development of a novel κ-opioid receptor (KOR) antagonist, PF-04455242, where PK-PD understanding from preclinical biomarker responses enabled successful prediction of the clinical response in a proof of mechanism study. Preclinical data obtained in rats included time course measures of the KOR antagonist (PF-04455242), a KOR agonist (spiradoline), and a KOR-mediated biomarker response (prolactin secretion) in plasma. Clinical data included time course measures of PF-04455242 and prolactin in 24 healthy volunteers following a spiradoline challenge and single oral doses of PF-04455242 (18 and 30 mg). In both species, PF-04455242 successfully reversed spiradoline-induced prolactin response. A competitive antagonism model was developed and implemented within NONMEM to describe the effect of PF-04455242 on spiradoline-induced prolactin elevation in rats and humans. The PK-PD model-based estimate of K(i) for PF-04455242 in rats was 414 ng/mL. Accounting for species differences in unbound fraction, in vitro K(i) and brain penetration provided a predicted human K(i) of 44.4 ng/mL. This prediction was in good agreement with that estimated via the application of the proposed PK-PD model to the clinical data (i.e., 39.2 ng/mL). These results illustrate the utility of the proposed PK-PD model in supporting the quantitative translation of preclinical studies into an accurate clinical expectation. As such, the proposed PK-PD model is useful for supporting the design, selection, and early development of novel KOR antagonists.

Pharmacological Characterization of 2-Methyl-N-((2′-(pyrrolidin-1-ylsulfonyl)biphenyl-4-yl)methyl)propan-1-amine (PF-04455242), a High-Affinity Antagonist Selective for κ-Opioid Receptors

2-Methyl-N-((2'-(pyrrolidin-1-ylsulfonyl)biphenyl-4-yl)methyl)propan-1-amine (PF-04455242) is a novel κ-opioid receptor (KOR) antagonist with high affinity for human (3 nM), rat (21 nM), and mouse (22 nM) KOR, a ∼ 20-fold reduced affinity for human μ-opioid receptors (MORs; K(i) = 64 nM), and negligible affinity for δ-opioid receptors (K(i) > 4 μM). PF-04455242 also showed selectivity for KORs in vivo. In rats, PF-04455242 blocked KOR and MOR agonist-induced analgesia with ID(50) values of 1.5 and 9.8 mg/kg, respectively, and inhibited ex vivo [(3)H](2-(benzofuran-4-yl)-N-methyl-N-((5S,7R,8R)-7-(pyrrolidin-1-yl)-1-oxaspiro[4.5]decan-8-yl)acetamide ([(3)H]CI977) and [(3)H](2S)-2-[[2-[[(2R)-2-[[(2S)-2-amino-3-(4-hydroxyphenyl) propanoyl]amino]propanoyl]amino]acetyl]-methylamino]-N-(2-hydroxyethyl)-3-phenylpropanamide ([(3)H]DAMGO) binding to KOR and MOR receptors with ID(50) values of 2.0 and 8.6 mg/kg, respectively. An in vivo binding assay was developed using (-)-4-[(3)H]methoxycarbonyl-2-[(1-pyrrolidinylmethyl]-1-[(3,4-dichlorophenyl)acetyl]-piperidine ([(3)H]PF-04767135), a tritiated version of the KOR positron emission tomography ligand (-)-4-[(11)C]methoxycarbonyl-2-[(1-pyrrolidinylmethyl]-1-[(3,4-dichlorophenyl)acetyl]-piperidine ([(11)C]GR103545) in which PF-04455242 had an ID(50) of 5.2 mg/kg. PF-04455242 demonstrated antidepressant-like efficacy (mouse forced-swim test), attenuated the behavioral effects of stress (mouse social defeat stress assay), and showed therapeutic potential in treating reinstatement of extinguished cocaine-seeking behavior (mouse conditioned place preference). KOR agonist-induced plasma prolactin was investigated as a translatable mechanism biomarker. Spiradoline (0.32 mg/kg) significantly increased rat plasma prolactin levels from 1.9 ± 0.4 to 41.9 ± 4.9 ng/ml. PF-04455242 dose-dependently reduced the elevation of spiradoline-induced plasma prolactin with an ID(50) of 2.3 ± 0.1 mg/kg, which aligned well with the ED(50) values obtained from the rat in vivo binding and efficacy assays. These data provide further evidence that KOR antagonists have potential for the treatment of depression and addiction disorders.

Kinase cascades and ligand-directed signaling at the kappa opioid receptor

The dynorphin/kappa opioid receptor (KOR) system has been implicated as a critical component of the stress response. Stress-induced activation of dynorphin-KOR is well known to produce analgesia, and more recently, it has been implicated as a mediator of stress-induced responses including anxiety, depression, and reinstatement of drug seeking. Drugs selectively targeting specific KOR signaling pathways may prove potentially useful as therapeutic treatments for mood and addiction disorders. KOR is a member of the seven transmembrane spanning (7TM) G-protein coupled receptor (GPCR) superfamily. KOR activation of pertussis toxin-sensitive G proteins leads to Galphai/o inhibition of adenylyl cyclase production of cAMP and releases Gbetagamma, which modulates the conductances of Ca(+2) and K(+) channels. In addition, KOR agonists activate kinase cascades including G-protein coupled Receptor Kinases (GRK) and members of the mitogen-activated protein kinase (MAPK) family: ERK1/2, p38 and JNK. Recent pharmacological data suggests that GPCRs exist as dynamic, multi-conformational protein complexes that can be directed by specific ligands towards distinct signaling pathways. Ligand-induced conformations of KOR that evoke beta-arrestin-dependent p38 MAPK activation result in aversion; whereas ligand-induced conformations that activate JNK without activating arrestin produce long-lasting inactivation of KOR signaling. In this review, we discuss the current status of KOR signal transduction research and the data that support two novel hypotheses: (1) KOR selective partial agonists that do not efficiently activate p38 MAPK may be useful analgesics without producing the dysphoric or hallucinogenic effects of selective, highly efficacious KOR agonists and (2) KOR antagonists that do not activate JNK may be effective short-acting drugs that may promote stress-resilience.

Comparison of the kappa-opioid receptor antagonist DIPPA in tests of anxiety-like behavior between Wistar Kyoto and Sprague Dawley rats

Recent evidence suggests a role for the dynorphin/kappa-opioid receptor (KOR) system in the expression of stress-induced behaviors. Wistar Kyoto (WKY) rats exhibit increased depression-like and anxiety-like responses in behavioral tests compared to other strains and may be a model of comorbid depression and anxiety characterized by increased activity within the dynorphin/KOR system. Though KOR antagonists produce antidepressant-like effects in WKY rats, their effects in tests of anxiety-like behavior have not been examined in the WKY strain. The aim of the current study was to investigate the effects of the KOR antagonist 2-(3,4-dichlorophenyl)-N-methyl-N-[(1S)-1-(3-isothiocyanatophenyl)-2-(1-pyrrolidinyl)ethyl]acetamide hydrochloride (DIPPA) on the behavior of WKY rats and Sprague Dawley (SD) rats in tests of anxiety-like behavior. The novelty-induced hypophagia and defensive burying tests were used to measure anxiety-like behavior in WKY and SD rats and determine the effects of DIPPA on anxiety-like behavior in both strains. WKY rats displayed greater amounts of anxiety-like behavior compared to SD rats. DIPPA produced anxiolytic-like effects in both tests in both strains. WKY rats display more anxiety-like behavior at baseline compared to SD rats, and DIPPA produced anxiolytic-like effects in both WKY and SD rats. These findings support previous research suggesting that KOR antagonists possess anxiolytic-like properties and may potentially represent a novel class of treatments for mood disorders.

Desipramine Reduces Stress-Activated Dynorphin Expression and CREB Phosphorylation in NAc Tissue

The nucleus accumbens (NAc) is a critical brain area for reward and motivated behavior. Accumulating evidence suggests that altered function of the transcription factor cAMP response element binding protein (CREB) within the NAc is involved in depressive behavior. In rats, stress activates CREB within the NAc, and elevation of CREB expression in this region produces depressive-like behaviors that are accompanied by activation of CREB-regulated target genes. The depressive-like behaviors seem to be due, at least in part, to CREB-mediated increases in dynorphin function, because they are mimicked by kappa-opioid receptor (KOR) agonists and attenuated by KOR antagonists. We hypothesized that if CREB-mediated dynorphin expression in the NAc contributes to depressive behavior, then antidepressants might reduce dynorphin function in this region. Here, we demonstrate that desipramine (DMI), a norepinephrine reuptake inhibitor that has been used for decades to treat clinical depression, blocks swim stress-induced activation of prodynorphin (encodes dynorphin) in the NAc. In primary cultures of NAc and striatum, DMI decreases basal and stimulated CREB phosphorylation by causing reductions in intracellular calcium (Ca(2+)) availability that are independent of norepinephrine or other monoaminergic inputs, identifying a potential mechanism for alterations in CREB-mediated gene expression. Fluoxetine (FLX), a selective serotonin reuptake inhibitor, has similar effects in culture, suggesting a common intracellular effect of these antidepressants. These findings raise the possibility that a therapeutically relevant mechanism of action of DMI occurs through attenuation of CREB-mediated gene transcription, which is mediated via previously uncharacterized mechanisms that occur directly within the NAc.

Long-Acting κ Opioid Antagonists Disrupt Receptor Signaling And Produce Noncompetitive Effects By Activating C-Jun N-Terminal Kinase

Norbinaltorphimine (NorBNI), guanidinonaltrindole, and atrans-(3R,4R)-dimethyl-4-(3-hydroxyphenyl) piperidine (JDTic) are selective kappa opioid receptor (KOR) antagonists having very long durations of action in vivo despite binding non-covalently in vitro and having only moderately high affinities. Consistent with this, we found that antagonist treatment significantly reduced the subsequent analgesic response of mice to the KOR agonist U50,488 in the tail-withdrawal assay for 14-21 days. Receptor protection assays were designed to distinguish between possible explanations for this anomalous effect, and we found that mice pretreated with the readily reversible opioid antagonists naloxone or buprenorphine before norBNI responded strongly in the tail-flick analgesia assay to a subsequent challenge with U50,488 1 week later. Protection by a rapidly cleared reagent indicates that norBNI did not persist at the site of action. In vitro binding of [(3)H]U69,593 to KOR showed that K(d) and Bmax values were not significantly affected by prior in vivo norBNI exposure, indicating that the agonist binding site was intact. Consistent with the concept that the long-lasting effects might be caused by a functional disruption of KOR signaling, both norBNI and JDTic were found to stimulate c-Jun N-terminal kinase (JNK) phosphorylation in HEK293 cells expressing KOR-GFP but not in untransfected cells. Similarly, norBNI increased phospho-JNK in both the striatum and spinal cord in wild type mice but not in KOR knock-out mice. Pretreatment of mice with the JNK inhibitor SP600125 before norBNI attenuated the long acting antagonism. Together, these results suggest that the long duration KOR antagonists disrupt KOR signaling by activating JNK.

Effects of Butorphanol on Morphine-induced Itch and Analgesia in Primates

Butorphanol is an opioid analgesic with partial agonist actions at micro- and kappa-opioid receptors (MOR and KOR). Previous studies have demonstrated that both MOR antagonists and KOR agonists are effective in alleviating intrathecal morphine-induced itch in primates. The aim of the study was to investigate the effectiveness of butorphanol as an antipruritic and to elucidate the receptor mechanisms underlying butorphanol's antipruritic effect in primates. Adult rhesus monkeys were used in the behavioral assays for measuring itch/scratching and analgesia. The dose-response curves of butorphanol were studied using selective MOR and KOR antagonists. In addition, the effect of butorphanol as an antipruritic was studied on subcutaneous and intrathecal morphine-induced itch and analgesia. KOR-selective antagonists were further used to compare the degrees of MOR and KOR activation underlying the antipruritic effect of butorphanol. Butorphanol alone produced analgesia with slight itch responses, and both effects were blocked by a MOR antagonist, clocinnamox (0.1 mg/kg). In contrast, a KOR antagonist, 5'-guanidinylnaltrindole (1 mg/kg), increased butorphanol-elicited itch. Systemic butorphanol (0.0032-0.032 mg/kg) dose-dependently attenuated systemic or intrathecal morphine-induced itch. In addition, butorphanol either potentiated or maintained morphine-induced analgesia without producing sedation. KOR-selective antagonists, 5'-guanidinylnaltrindole (1 mg/kg) and nor-binaltorphimine (3.2 mg/kg), only partially reversed the antipruritic effect of butorphanol with different durations of KOR antagonism. Butorphanol is effective in attenuating systemic or spinal morphine-induced itch without reducing morphine analgesia. This study provides functional evidence that both partial MOR and KOR agonist actions contribute to the effectiveness of butorphanol as an antipruritic in primates.