Selective Kappa-opioid Receptor Antagonist Research Articles

Kappa-opioid receptor blockade in the inferior colliculus of prey threatened by pit vipers decreases anxiety and panic-like behaviour.

The dorsal midbrain comprises dorsal columns of the periaqueductal grey matter and corpora quadrigemina. These structures are rich in beta-endorphinergic and leu-enkephalinergic neurons and receive GABAergic inputs from substantia nigra pars reticulata. Although the inferior colliculus (IC) is mainly involved in the acoustic pathways, the electrical and chemical stimulation of central and pericentral nuclei of the IC elicits a vigorous defensive behaviour. The defensive immobility and escape elicited by IC activation is commonly related to panic-like emotional states. To investigate the role of κ-opioid receptor of the IC in the antiaversive effects of endogenous opioid receptor blockade in a dangerous situation, male Wistar rats were pretreated in the IC with the κ-opioid receptor-selective antagonist nor-binaltorphimine at different concentrations and submitted to the non-enriched polygonal arena for a snake panic test in the presence of a rattlesnake and, after 24 h, prey were resubmitted to the experimental context. The snakes elicited in prey a set of antipredatory behaviours, such as the anxiety-like responses of defensive attention and risk assessment, and the panic-like reactions of defensive immobility and either escape or active avoidance during the elaboration of unconditioned and conditioned fear-related responses. Pretreatment of the IC with microinjections of nor-binaltorphimine at higher concentrations significantly decreased the frequency and duration of both anxiety- and panic-attack-like behaviours. These findings suggest that κ-opioid receptor blockade in the IC causes anxiolytic- and panicolytic-like responses in threatening conditions, and that kappa-opioid receptor-selective antagonists can be a putative coadjutant treatment for panic syndrome treatment.

Kappa Opioid Receptor Activation Induces Epigenetic Silencing of Brain-Derived Neurotropic Factor via HDAC5 in Depression.

Treatment-resistant depression (TRD) occurs in almost 50% of the depressed patients. Central kappa opioid receptor (KOR) agonism has been demonstrated to induce depression and anxiety, while KOR antagonism alleviates depression-like symptoms in rodent models and TRD in clinical studies. Previously, we have shown that sustained KOR activation leads to a TRD-like phenotype in mice, and modulation of brain-derived neurotrophic factor (BDNF) expression in the prefrontal cortex (PFC) appears to be one of the molecular determinants of the antidepressant response. In the present study, we observed that sustained KOR activation by a selective agonist, U50488, selectively reduced the levels of Bdnf transcripts II, IV, and Bdnf CDS (protein-coding Exon IX) in the PFC and cultured primary cortical neurons, which was blocked by selective KOR antagonist, norbinaltorphimine. Considering the crucial role of epigenetic pathways in BDNF expression, we further investigated the role of various epigenetic markers in KOR-induced BDNF downregulation in mice. We observed that treatment with U50488 resulted in selective and specific downregulation of acetylation at the ninth lysine residue of the histone H3 protein (H3K9ac) and upregulation of histone deacetylase 5 (HDAC5) expression in the PFC. Further, using anti-H3K9ac and anti-HDAC5 antibodies in the chromatin immune precipitation assay, we detected decreased enrichment of H3K9ac and increased HDAC5 binding at Bdnf II and IV transcripts after U50488 treatment, which were blocked by a selective KOR antagonist, norbinaltorphimine. Further mechanistic studies using HDAC5 selective inhibitor, LMK235, in primary cortical neurons and adeno-associated viral shRNA-mediated HDAC5-knockdown in the PFC of mice demonstrated an essential role of HDAC5 in KOR-mediated reduction of Bdnf expression in the PFC and in depression-like symptoms in mice. These results suggest that KOR engages multiple pathways to induce depression-like symptoms in mice and provide novel insights into the mechanisms by which activation of KOR regulates major depressive disorders.

S102 - NMRA-140, a Novel and Selective Kappa Opioid Receptor Antagonist: Mode of Action Profiling Studies Demonstrate No Opioid Agonist Properties Implicated in Opioid-Related Abuse

S102 - NMRA-140, a Novel and Selective Kappa Opioid Receptor Antagonist: Mode of Action Profiling Studies Demonstrate No Opioid Agonist Properties Implicated in Opioid-Related Abuse

A Bicyclic Analog of the Linear Peptide Arodyn Is a Potent and Selective Kappa Opioid Receptor Antagonist.

Kappa opioid receptor (KOR) antagonists have potential therapeutic applications in the treatment of stress-induced relapse to substance abuse and mood disorders. The dynorphin A analog arodyn (Ac[Phe1,2,3,Arg4,D-Ala8]dynorphin A-(1-11)-NH2) exhibits potent and selective kappa opioid receptor antagonism. Multiple cyclizations in longer peptides, such as dynorphin and its analogs, can extend the conformational constraint to additional regions of the peptide beyond what is typically constrained by a single cyclization. Here, we report the design, synthesis, and pharmacological evaluation of a bicyclic arodyn analog with two constraints in the opioid peptide sequence. The peptide, designed based on structure-activity relationships of monocyclic arodyn analogs, was synthesized by solid-phase peptide synthesis and cyclized by sequential ring-closing metathesis (RCM) in the C- and N-terminal sequences. Molecular modeling studies suggest similar interactions of key aromatic and basic residues in the bicyclic peptide with KOR as found in the cryoEM structure of KOR-bound dynorphin, despite substantial differences in the backbone conformations of the two peptides. The bicyclic peptide's affinities at KOR and mu opioid receptors (MOR) were determined in radioligand binding assays, and its KOR antagonism was determined in the [35S]GTPγS assay in KOR-expressing cells. The bicyclic analog retains KOR affinity and selectivity (Ki = 26 nM, 97-fold selectivity over MOR) similar to arodyn and exhibits potent KOR antagonism in the dynorphin-stimulated [35S]GTPγS assay. This bicyclic peptide represents a promising advance in preparing cyclic opioid peptide ligands and opens avenues for the rational design of additional bicyclic opioid peptide analogs.

Navacaprant, a novel and selective kappa opioid receptor antagonist, has no agonist properties implicated in opioid-related abuse

Kappa opioid receptors (KORs) are implicated in the pathophysiology of various psychiatric and neurological disorders creating interest in targeting the KOR system for therapeutic purposes. Accordingly, navacaprant (NMRA-140) is a potent, selective KOR antagonist being evaluated as a treatment for major depressive disorder. In the present report, we have extended the pharmacological characterization of navacaprant by further demonstrating its selective KOR antagonist properties and confirming its lack of agonist activity at KORs and related targets involved in opioid-related abuse. Using CHO–K1 cells expressing human KOR, mu (MOR), or delta (DOR) opioid receptors, navacaprant demonstrated selective antagonist properties at KOR (IC50 = 0.029 μM) versus MOR (IC50 = 3.3 μM) and DOR (IC50 > 10 μM) in vitro. In vivo, navacaprant (10–30 mg/kg, i.p.) dose-dependently abolished KOR-agonist induced analgesia in the mouse tail-flick assay. Additionally, navacaprant (10, 30 mg/kg, p.o.) significantly reduced KOR agonist–stimulated prolactin release in mice and rats, confirming KOR antagonism in vivo. Navacaprant showed no agonist activity at any opioid receptor subtype (EC50 > 10 μM) in vitro and exhibited no analgesic effect in the tail-flick assays at doses ≤100 mg/kg, p.o. thereby confirming a lack of opioid receptor agonist activity in vivo. Importantly, navacaprant did not alter extracellular dopamine concentrations in the nucleus accumbens shell of freely-moving rats following doses ≤100 mg/kg, p.o., whereas morphine (10, 20 mg/kg, i.p.) significantly increased dopamine levels. These results demonstrate that navacaprant is a KOR-selective antagonist with no pharmacological properties implicated in opioid-related abuse.

Identification and In Vivo Evaluation of Myelination Agent PIPE-3297, a Selective Kappa Opioid Receptor Agonist Devoid of β-Arrestin-2 Recruitment Efficacy.

Structure-activity relationship studies led to the discovery of PIPE-3297, a fully efficacious and selective kappa opioid receptor (KOR) agonist. PIPE-3297, a potent activator of G-protein signaling (GTPγS EC50 = 1.1 nM, 91% Emax), did not elicit a β-arrestin-2 recruitment functional response (Emax < 10%). Receptor occupancy experiments performed with the novel KOR radiotracer [3H]-PIPE-3113 revealed that subcutaneous (s.c.) administration of PIPE-3297 at 30 mg/kg in mice achieved 90% occupancy of the KOR in the CNS 1 h post dose. A single subcutaneous dose of PIPE-3297 in healthy mice produced a statistically significant increase of mature oligodendrocytes (P < 0.0001) in the KOR-enriched striatum, an effect that was not observed in animals predosed with the selective KOR antagonist norbinaltorphimine. An equivalent dose given to mice in an open-field activity-monitoring system revealed a small KOR-independent decrease in total locomotor activity versus vehicle measured between 60 and 75 min post dose. Daily doses of PIPE-3297 at both 3 and 30 mg/kg s.c. reduced the disease score in the mouse experimental autoimmune encephalomyelitis (EAE) model. Visually evoked potential (VEP) N1 latencies were also significantly improved versus vehicle in both dose groups, and latencies matched those of untreated animals. Taken together, these findings highlight the potential therapeutic value of functionally selective G-protein KOR agonists in demyelinating disease, which may avoid the sedating side effects typically associated with classical nonbiased KOR agonists.

Preclinical Models of Attention Deficit Hyperactivity Disorder: Neurobiology, Drug Discovery, and Beyond

Objective: We offer an overview of ADHD research using mouse models of nicotine exposure. Method: Nicotine exposure of C57BL/6 or Swiss Webster mice occurred during prenatal period only or during the prenatal and the pre-weaning periods. Behavioral, neuroanatomical and neurotransmitter assays were used to investigate neurobiological mechanisms of ADHD and discover candidate ADHD medications. Results: Our studies show that norbinaltorphimine, a selective kappa opioid receptor antagonist is a candidate novel non-stimulant ADHD treatment and that a combination of methylphenidate and naltrexone has abuse deterrent potential with therapeutic benefits for ADHD. Other studies showed transgenerational transmission of ADHD-associated behavioral traits and demonstrated that interactions between untreated ADHD and repeated mild traumatic brain injury produced behavioral traits not associated with either condition alone. Conclusion: Preclinical models contribute to novel insights into ADHD neurobiology and are valuable tools for drug discovery and translation to benefit humans with ADHD.

225. NMRA-140, a Novel and Selective Kappa Opioid Receptor Antagonist: Mode of Action Profiling Studies Demonstrate No Opioid Agonist Properties Implicated in Opioid-Related Abuse

225. NMRA-140, a Novel and Selective Kappa Opioid Receptor Antagonist: Mode of Action Profiling Studies Demonstrate No Opioid Agonist Properties Implicated in Opioid-Related Abuse

Alleviation of Cocaine Withdrawal and Pertinent Interactions between Salvinorin-Based Antagonists and Kappa Opioid Receptor.

The kappa opioid receptor (KOR) is involved in the regulation of both the reward and mood processes. Recent reports find that the use of drugs of abuse increases the production of dynorphin and the overall activation of KOR. Long-acting KOR antagonists, such as norbinaltorphimine (nor-BNI), JDTic, and 5'-guanidinonaltrindole (GNTI), have been shown to stop depressive and anxiety-related disorders, which are the common side effects of withdrawal that can lead to a relapse in drug use. Unfortunately, these prototypical KOR antagonists are known to induce selective KOR antagonism that is delayed by hours and extremely prolonged, and their use in humans comes with serious safety concerns because they possess a large window for potential drug-drug interactions. Furthermore, their persistent pharmacodynamic activities can hinder the ability to reverse unanticipated side effects immediately. Herein, we report our studies of the lead selective, salvinorin-based KOR antagonist (1) as well as nor-BNI on C57BL/6N male mice for spontaneous cocaine withdrawal. Assessment of pharmacokinetics showed that 1 is a short-acting compound with an average half-life of 3.75 h across different compartments (brain, spinal cord, liver, and plasma). Both 1 (5 mg/kg) and nor-BNI (5 mg/kg) were shown to reduce spontaneous withdrawal behavior in mice, with 1 producing additional anti-anxiety-like behavior in a light-dark transition test (however, no mood-related effects of 1 or nor-BNI were observed at the current dosing in an elevated plus maze or a tail suspension test). Our results support the study of selective, short-acting KOR antagonists for the treatment of psychostimulant withdrawal and the associated negative mood states that contribute to relapse. Furthermore, we identified pertinent interactions between 1 and KOR via computational studies, including induced-fit docking, mutagenesis, and molecular dynamics simulations, to gain insight into the design of future selective, potent, and short-acting salvinorin-based KOR antagonists.

Kappa opioid receptor antagonism protects working memory performance from mild stress exposure in Rhesus macaques

Extensive preclinical and emerging clinical evidence point to an involvement of the kappa opioid receptor (KOR) in brain networks that promotes neurobehavioral stability. KOR expression in mesolimbic and mesocortical pathways has been the basis for characterizing the role of this receptor system in regulating motivation and emotion; however, the involvement of the KOR system in higher-order executive processes such as working memory (WM) is not well-understood. WM is readily impaired with uncontrollable stress exposure and is dysregulated in many neurobehavioral disorders. To empirically evaluate the role of the KOR system on WM performance, we administered a selective KOR antagonist, NMRA-140 (0, 0.1, 0.3, 1.0 mg/kg, intramuscular) to monkeys under both stress and non-stress conditions. In this study, NMRA-140 was co-administered with FG7142, a benzodiazepine inverse agonist, known to produce a mild stress response and to impair WM function in monkeys. NMRA-140 protected WM performance from the detrimental effects of FG7142-induced stress and exhibited no significant effect under non-stress conditions. Collectively, these data highlight the functional influence of the KOR system in mediating stress-induced dysfunction of executive processes and suggest that modulating KOR activity could offer therapeutic benefit in stress-related neurobehavioral disorders.

Antinociceptive Effects and Interaction Mechanisms of Intrathecal Pentazocine and Neostigmine in Two Different Pain Models in Rats

Background Pentazocine produces a wide variety of actions in the treatment of perioperative analgesia. Neostigmine is a cholinesterase inhibitor used to antagonize the residual effects of muscle relaxants and also produces an analgesic effect. Objectives To investigate the analgesic effects of intrathecally injected pentazocine and neostigmine and their interaction. Methods Sprague–Dawley rats were used to test the analgesic effect of pentazocine and neostigmine using the paw formalin pain model and the incision mechanical allodynia model. Pentazocine (3, 10, 30, and 100 μg), neostigmine (0.3, 1, 3, and 10 μg) or a pentazocine-neostigmine mixture were separately injected to evaluate their antinociceptive effects alone on the treatment groups. The corresponding control group received an intrathecal injection containing the same volume of saline. The formalin pain test, or the plantar incision pain behavior test were performed 30 minutes later. Isobolographic analysis was used to evaluate the interaction between pentazocine and neostigmine. Intrathecally administered selective mu-opioid receptor antagonist CTAP, selective kappa-opioid receptor antagonist nor-Binaltorphimine (nor-BNI), nonselective opioid receptor antagonist naloxone, and muscarinic acetylcholine receptor antagonist atropine were also used to test the possible interaction mechanism. These antagonists were used 30 minutes before the pentazocine and neostigmine mixtures which were intrathecally injected. Results Intrathecally administered pentazocine (3, 10, 30, and 100 μg) and neostigmine (0.3, 1, 3, and 10 μg) alone had a marked dose-related impact on suppressing the biphasic responses in the formalin test. Pentazocine (3, 10, 30, and 100 μg) and neostigmine (0.3, 1, 3, and 10 μg) alone attenuated the mechanical allodynia in a plantar incision model in a dose-dependent manner. Isobolographic analysis revealed that the mixture of intrathecal pentazocine and neostigmine synergistically decreased both phase I and II activity in the formalin test and mechanical allodynia in the plantar incision model. Pretreatment of intrathecally administered nor-BNI, naloxone, atropine, but not CTAP, antagonized the analgesic effect of the pentazocine-neostigmine mixture. Conclusions All of these results suggest that the combined application of pentazocine and neostigmine is an effective way to relieve pain from formalin and acute incision mechanical allodynia. The synergistic effect between pentazocine and neostigmine is mostly attributed to the kappa-opioid receptor and the cholinergic receptor in the spinal cord.

Aticaprant (Clinically Developed Kappa-Opioid Receptor Antagonist) Combined With Naltrexone Prevents Alcohol "Relapse" Drinking.

Alcohol relapse is the treatment target for medications development for alcohol dependence. Aticaprant, a selective and short-acting kappa-opioid receptor (KOR) antagonist, has recently been under development for new clinical implications (depression or anhedonia). Recent studies have also found that aticaprant reduces alcohol intake and prevents stress- triggered alcohol seeking in rodents via a KOR-mediated mechanism. Here, we further investigated whether aticaprant alone or in combination with naltrexone (mu-opioid receptor [MOR] antagonist) altered alcohol relapse-like drinking using a mouse alcohol deprivation effect (ADE) paradigm to mimic the relapse episodes in human alcoholics. A long-acting and selective KOR antagonist nor-BNI was used as a reference compound for the effects of the KOR antagonism on the ADE. After 3-week intermittent-access alcohol drinking (two-bottle choice, 24-h access every other day), male and female mice displayed excessive alcohol intake and then pronounced ADE after 1-week abstinence. Aticaprant alone decreased alcohol ADE in a dose- dependent manner (1–3 mg/kg) in both males and females. Aticaprant at a lower dose (0.3 mg/kg) than the effective one (3 mg/kg) combined with a low dose of naltrexone (1 mg/kg) reduced the ADE in both sexes, and the combination was effective after a multi-dosing regimen (5 daily injections during the abstinence) without development of tolerance, suggesting synergistic effects of the combination. In contrast, nor-BNI alone or with naltrexone had no effect on the ADE in either sex. Our present study suggests that a combination of clinically developed, short-acting KOR antagonist aticaprant with low-dose naltrexone has therapeutic potential in alcohol “relapse” treatment.

Evaluation of Specific Binding of [11C]RTI-97 to Kappa Opioid Receptor by Autoradiography and PET Imaging in Rat.

Kappa opioid receptor (KOR) PET imaging remains attractive to understand the role of KOR in health and diseases and to help the development of drugs especially for psychiatric disorders such as depression, anxiety, and addiction. The potent and selective KOR antagonist RTI-97 labeled with carbon-11 was previously demonstrated to display specific KOR binding in mouse brain by ex vivo autoradiography studies. Herein, we evaluated [11C]RTI-97 in rat by in vitro autoradiography and by in vivo PET imaging. The radiosynthesis of [11C]RTI-97 was optimized to obtain high molar activities. Despite a low cerebral uptake, the overall results showed a heterogeneous repartition and specific KOR binding of [11C]RTI-97 in brain and a high and specific accumulation of [11C]RTI-97 in pituitary in accordance with KOR expression.

Release of endogenous dynorphin opioids in the prefrontal cortex disrupts cognition.

Following repeated opioid use, some dependent individuals experience persistent cognitive deficits that contribute to relapse of drug-taking behaviors, and one component of this response may be mediated by the endogenous dynorphin/kappa opioid system in neocortex. In C57BL/6 male mice, we find that acute morphine withdrawal evokes dynorphin release in the medial prefrontal cortex (PFC) and disrupts cognitive function by activation of local kappa opioid receptors (KORs). Immunohistochemical analyses using a phospho-KOR antibody confirmed that both withdrawal-induced and optically evoked dynorphin release activated KOR in PFC. Using a genetically encoded sensor based on inert KOR (kLight1.2a), we revealed the in vivo dynamics of endogenous dynorphin release in the PFC. Local activation of KOR in PFC produced multi-phasic disruptions of memory processing in an operant-delayed alternation behavioral task, which manifest as reductions in response number and accuracy during early and late phases of an operant session. Local pretreatment in PFC with the selective KOR antagonist norbinaltorphimine (norBNI) blocked the disruptive effect of systemic KOR activation during both early and late phases of the session. The early, but not late phase disruption was blocked by viral excision of PFC KORs, suggesting an anatomically dissociable contribution of pre- and postsynaptic KORs. Naloxone-precipitated withdrawal in morphine-dependent mice or optical stimulation of pdynCre neurons using Channelrhodopsin-2 disrupted delayed alternation performance, and the dynorphin-induced effect was blocked by local norBNI. Our findings describe a mechanism for control of cortical function during opioid dependence and suggest that KOR antagonism could promote abstinence.

Kappa-opioid receptor-mediated thermal analgesia evoked by the intrathecal administration of the chemokine CCL1 in mice.

The chemokine CC motif ligand 1 (CCL1) participates in immune cell recruitment and, as other chemokines, is also involved in nociceptive processing. In contrast with previous reports indicating its participation in allodynia and cold hypernociception when spinally administered, its ability to evoke heat thermal analgesia, mediated by circulating leukocytes and endocannabinoids, after systemic administration has recently been reported. Aiming to explore the role played by CCL1 on spinal nociception, we study here the effect of its intrathecal administration on thermal nociception in mice. Behavioral nociceptive assays, immunohistochemical experiments, white cell blood depletion procedures and qRT-PCR experiments were performed. The intrathecal administration of CCL1 (0.3-30 ng) produced analgesia as measured by the unilateral hot plate test. This effect peaked 1 h after injection, was prevented by the CCR8 antagonist R243 and was accompanied by a reduction of c-Fos expression in spinal neurons. Whereas blood leukocyte depletion did not modify it, analgesia was abolished by the microglial inhibitor minocycline, but not the astroglial inhibitor aminoadipate. Furthermore, antinociception remained unmodified by the coadministration of cannabinoid type 1 or 2 receptors antagonists. However, it was reversed by naloxone but not by selective blockade of mu- or delta-opioid receptors. The inhibitory effect induced by the selective kappa-opioid receptor antagonist, nor-binaltorphimine, and by an anti-dynorphin A 1-17 antibody indicates that analgesia evoked by spinal CCL1 is mediated by endogenous dynorphins acting on kappa-opioid receptors. Endogenous dynorphin and microglia behave as key players in heat thermal analgesia evoked by spinal CCL1 in mice.

A Functional Interaction Between LMO4 And Kappa Opioid Receptors In The Basolateral Amygdala Regulates Alcohol Consumption and Reward

Repeated alcohol exposure leads to changes in gene expression that are thought to underlie the transition from moderate to excessive alcohol drinking. However, the mechanisms by which these gene expression changes are mobilized to a maladaptive response are not well understood. One mechanism could involve the recruitment of transcriptional co-regulators that bind and modulate the activity of several transcription factors. Our results indicate that the transcription cofactor LMO4 is one such candidate regulator. Previous results demonstrate that knock down of LMO4 in the basolateral amygdala (BLA) decreased alcohol consumption and preference as well as reduced conditioned place preference to alcohol. To identify transcriptional targets of LMO4 in the BLA we carried out unbiased transcriptome profiling of the BLA in WT and Lmo4gt/+ mice, in which LMO4 expression is reduced by approximately 50%. Our analysis revealed a number of genes whose expression was significantly up or downregulated in the BLA of Lmo4gt/+ mice compared to WT. We took a systems approach to identify gene networks that were responsive to LMO4 downregulation in the BLA. Weighted gene co-expression network analysis revealed genes related to the extracellular matrix and the kappa opioid receptor (KOR) as putative transcriptional targets of LMO4 in the BLA. Chromatin immunoprecipitation studies indicated that LMO4 bound to the KOR promoter elements in the amygdala and that infusion of Nor-BNI, a selective KOR antagonist into the BLA reduced alcohol consumption. Further, knockdown of LMO4 in the BLA attenuated the ability of U50,488, a KOR agonist to enhance alcohol consumption. Studies are currently underway to determine if restricting LMO4 knockdown to KOR cells in the BLA using intersectional strategies will reduce alcohol consumption and reward. In summary, our results reveal a functional interaction between LMO4 and KOR in the BLA in regulating alcohol consumption.

Conformational Constraint between Aromatic Residue Side Chains in the "Message" Sequence of the Peptide Arodyn Using Ring Closing Metathesis Results in a Potent and Selective Kappa Opioid Receptor Antagonist.

Kappa opioid receptor (KOR) antagonists have recently shown potential for treating drug addiction and mood disorders. The linear acetylated dynorphin A analog arodyn (Ac[Phe1,2,3,Arg4,d-Ala8]dynorphin A-(1-11)NH2), synthesized in our laboratory, demonstrated potent and selective KOR antagonism. Cyclization of arodyn could potentially stabilize the bioactive conformation and enhance its metabolic stability. The cyclization strategy employed involved ring closing metathesis between adjacent meta- or para-substituted Tyr(allyl) residues in the "message" sequence that were predicted in a docking study to yield analogs that would bind to the KOR with binding poses similar to arodyn. Consistent with the modeling, the resulting analogs retained KOR affinity similar to arodyn; the peptides involving cyclization between para O-allyl groups also retained high KOR selectivity, with one analog exhibiting KOR antagonist potency (KB = 15 nM) similar to arodyn. These promising cyclized analogs with constrained aromatic residues represent novel leads for further exploration of KOR pharmacology.

Effects of kappa opioid receptor agonists on fentanyl vs. food choice in male and female rats: contingent vs. non-contingent administration

Strategies are needed to decrease the abuse liability of mu opioid receptor (MOR) agonists. One strategy under consideration is to combine MOR agonists with kappa opioid receptor (KOR) agonists. The effects of KOR agonists (U50488, nalfurafine) on fentanyl-vs.-food choice were compared under conditions where the KOR agonists were added to the intravenously self-administered fentanyl (contingent delivery) or administered as subcutaneous pretreatments (non-contingent delivery) in male and female rats. Rats were trained to respond under a concurrent schedule of fentanyl (0, 0.32-10μg/kg/infusion) and food reinforcement. In experiment 1, U50488 and nalfurafine were co-administered with fentanyl as fixed-proportion mixtures (contingent administration). In experiment 2, U50488 (1-10mg/kg) and nalfurafine (3.2-32μg/kg) were administered as acute pretreatments (non-contingent administration). The selective KOR antagonist, nor-BNI (32mg/kg), was administered prior to contingent and non-contingent KOR-agonist treatment in experiment 3. Both U50488 and nalfurafine decreased fentanyl choice when administered contingently, demonstrating that KOR agonists punish opioid choice. However, evidence for punishment corresponded with an elimination of operant responding in the majority of rats. Non-contingent U50488 and nalfurafine administration only decreased the number of choices made during the behavioral session without altering fentanyl choice. Contingent and non-contingent KOR-agonist effects on fentanyl choice were both attenuated by nor-BNI. These results illustrate that the effects of KOR agonists on fentanyl reinforcement are dependent upon the contingencies under which they are administered.

Differential effects of novel kappa opioid receptor antagonists on dopamine neurons using acute brain slice electrophysiology.

Activation of the kappa opioid receptor (KOR) contributes to the aversive properties of stress, and modulates key neuronal circuits underlying many neurobehavioral disorders. KOR agonists directly inhibit ventral tegmental area (VTA) dopaminergic neurons, contributing to aversive responses (Margolis et al. 2003, 2006); therefore, selective KOR antagonists represent a novel therapeutic approach to restore circuit function. We used whole cell electrophysiology in acute rat midbrain slices to evaluate pharmacological properties of four novel KOR antagonists: BTRX-335140, BTRX-395750, PF-04455242, and JNJ-67953964. Each compound concentration-dependently reduced the outward current induced by the KOR selective agonist U-69,593. BTRX-335140 and BTRX-395750 fully blocked U-69,593 currents (IC50 = 1.2 ± 0.9 and 1.2 ± 1.3 nM, respectively). JNJ-67953964 showed an IC50 of 3.0 ± 4.6 nM. PF-04455242 exhibited partial antagonist activity asymptoting at 55% blockade (IC50 = 6.7 ± 15.1 nM). In 3/8 of neurons, 1 μM PF-04455242 generated an outward current independent of KOR activation. BTRX-335140 (10 nM) did not affect responses to saturating concentrations of the mu opioid receptor (MOR) agonist DAMGO or the delta opioid receptor (DOR) agonist DPDPE, while JNJ-67953964 (10 nM) partially blocked DAMGO and DPDPE responses. Importantly, BTRX-335140 (10 nM) rapidly washed out with complete recovery of U-69,593 responses within 10 min. Collectively, we show electrophysiological evidence of key differences amongst KOR antagonists that could impact their therapeutic potential and have not been observed using recombinant systems. The results of this study demonstrate the value of characterizing compounds in native neuronal tissue and within circuits implicated in the neurobehavioral disorders of interest.

Pre-training naltrexone increases conditioned fear learning independent of adolescent alcohol consumption history

Our previous research has shown a relationship between low voluntary alcohol consumption and high conditioned fear in male Long Evans rats. Here, we examined whether differences in the endogenous opioid systems might be responsible for these differences. Rats received 6 weeks of chronic intermittent to 20% alcohol (v/v) or water-only from PND 26–66. Based on their consumption during the last 2 weeks of alcohol access, the alcohol-access rats were divided into high drinking (>2.5 g/kg/24-h) or low drinking (<2 g/kg/24-h). Rats were then given injections of the preferential mu opioid receptor antagonist naltrexone (1 mg/kg, s.c.) or the selective kappa opioid receptor antagonist LY2456302 (10 mg/kg, s.c.) prior to fear conditioning and were then tested for conditioned fear 2 days later. Pre-training naltrexone increased conditioned suppression of lever-pressing during training and testing, with no differences between high versus low alcohol drinkers or between water-only versus alcohol access groups (averaged across drinking levels). There was no effect of LY2456302 on conditioned fear in any comparison. We also found no differences between high and low alcohol drinkers and no reliable effect of prior alcohol access (averaged across drinking levels) on conditioned fear. Our experiment replicates and extends previous demonstrations that a preferential MOR antagonist can increase fear learning using conditioned suppression of lever-pressing as a fear measure. However, additional research is needed to determine the cause of the differences in conditioned fear that we previously observed (as they were not observed in the current experiments).