Delta-opioid Receptor Antagonist Research Articles

Coexpressed δ-, μ-, and κ-Opioid Receptors Modulate Voltage-Gated Ca2+ Channels in Gastric-Projecting Vagal Afferent Neurons.

Opioid analgesics are frequently associated with gastrointestinal side effects, including constipation, nausea, dysphagia, and reduced gastric motility. Though it has been shown that stimulation of opioid receptors expressed in enteric motor neurons contributes to opioid-induced constipation, it remains unclear whether activation of opioid receptors in gastric-projecting nodose ganglia neurons contributes to the reduction in gastric motility and emptying associated with opioid use. In the present study, whole-cell patch-clamp recordings were performed to determine the mechanism underlying opioid receptor-mediated modulation of Ca2+ currents in acutely isolated gastric vagal afferent neurons. Our results demonstrate that CaV2.2 channels provide the majority (71% ± 16%) of Ca2+ currents in gastric vagal afferent neurons. Furthermore, we found that application of oxycodone, U-50488, or deltorphin II on gastric nodose ganglia neurons inhibited Ca2+ currents through a voltage-dependent mechanism by coupling to the Gα i/o family of heterotrimeric G-proteins. Because previous studies have demonstrated that the nodose ganglia expresses low levels of δ-opioid receptors, we also determined the deltorphin II concentration-response relationship and assessed deltorphin-mediated Ca2+ current inhibition following exposure to the δ-opioid receptor antagonist ICI 174,864 (0.3 µM). The peak mean Ca2+ current inhibition following deltorphin II application was 47% ± 24% (EC50 = 302.6 nM), and exposure to ICI 174,864 blocked deltorphin II-mediated Ca2+ current inhibition (4% ± 4% versus 37% ± 20%). Together, our results suggest that analgesics targeting any opioid receptor subtype can modulate gastric vagal circuits. SIGNIFICANCE STATEMENT: This study demonstrated that in gastric nodose ganglia neurons, agonists targeting all three classical opioid receptor subtypes (μ, δ, and κ) inhibit voltage-gated Ca2+ channels in a voltage-dependent mechanism by coupling to Gαi/o. These findings suggest that analgesics targeting any opioid receptor subtype would modulate gastric vagal circuits responsible for regulating gastric reflexes.

Abstract P2159: Naltrindole Attenuates Phorbol 12-myristate 13-acetate Induced Superoxide Release In Polymorphonuclear Leukocytes Independently Of Delta Opioid Receptor Antagonism

Naltrindole (NTI) is a selective delta opioid receptor antagonist that recently has been shown to be cardioprotective in both in vivo and ex vivo studies. In vivo, NTI showed cardioprotective effects that were dose-dependent significantly reducing infarct size up to 82% compared to controls subjected to ischemia-reperfusion (I/R) injury (p<0.05). In ex vivo isolated rat I/R hearts, NTI significantly reduced infarct size compared to controls (p<0.01). NTI restored cardiac left ventricular end diastolic pressure to near baseline values compared to controls or other general opioid receptor antagonists in ex vivo hearts, suggesting a tissue salvaging mechanism independent of opioid delta receptor antagonism. Polymorphonuclear leukocytes (PMNs) do not express opioid receptors. In this study, we investigated NTI attenuation of PMN superoxide (SO) release in vitro . We hypothesized that NTI would attenuate PMN SO release via a calcium handling mechanism, a similar mechanism that explained cardioprotective effects of NTI pretreatment mitigating ischemic hypercontracture due to an increase in intracellular calcium. Rat PMNs (5x10 6 ) were incubated for 15 min at 37 o C in the presence or absence (dH 2 O vehicle controls) of NTI (10μM, 50μM, or 200μM). PMN SO release was calculated by the change in absorbance at 550 nm over 420 sec via ferricytochrome c reduction after phorbol 12-myristate-13 acetate (PMA) stimulation (100nM). Cell viability was determined microscopically by 0.2% trypan blue exclusion at the end of the assay. Data were analyzed using ANOVA Fisher’s PLSD post-hoc test. NTI significantly decreased PMN SO release from 30 sec to 420 sec. Changes in final absorbance of NTI 200μM (0.244±0.07, n=8, p<0.05) and 50μM (0.293±0.06, n=4, p<0.05), but not NTI 10μM (0.412±0.07, n=5), were significant compared to controls (0.487±0.12, n=9). Cell viability was not significantly different compared to PMA across all NTI concentrations. These results suggest that NTI reduces I/R injury and PMN SO release, by a means independent of delta opioid receptor antagonism. Future studies will assess NTI SO attenuation in human umbilical vein endothelial cells. We will use different concentrations to investigate optimal concentration response.

Efficacy of dual enkephalinase inhibition in a preclinical migraine model is mediated by activation of peripheral delta opioid receptors.

The aim of this study was to evaluate whether elevating levels of enkephalin by inhibiting their degradation can attenuate stress-induced migraine-like behaviors in mice. Previous studies in animals have suggested the delta opioid receptor (DOR) as a novel migraine target. The primary endogenous ligands for DOR are enkephalins and their levels can be increased by pharmacological inhibition of enkephalinases; however, it is not clear whether enkephalinase inhibition can be efficacious in preclinical migraine models through activation of DOR or whether other opioid receptors might be involved. Further, it is not clear whether opioid receptors in the central nervous system are necessary for these effects. This study used a model of repetitive restraint stress in mice that induces periorbital hypersensitivity and priming to the nitric oxide donor sodium nitroprusside (SNP; 0.1 mg/kg). Von Frey filaments were used to measure periorbital mechanical thresholds and grimace scores were evaluated by observing mouse facial features. Animals were treated with the dual enkephalinase inhibitor (DENKI) PL37. On day two post-stress, PL37 given to mice via either intravenous injection (10 mg/kg) or oral gavage (20 mg/kg) significantly attenuated stress-induced periorbital hypersensitivity and facial grimace responses. Additionally, both intravenous (10 mg/kg) and oral gavage (20 mg/kg) of PL37 prior to SNP (0.1 mg/kg) administration on day 14 post-stress significantly reduced SNP-induced facial hypersensitivity. Injection of the DOR antagonist naltrindole (0.1 mg/kg) but not the mu-opioid receptor antagonist CTAP (1 mg/kg) prior to PL37 treatment blocked the effects. Finally, pretreatment of mice with the peripherally restricted opioid receptor antagonist naloxone methiodide (5 mg/kg) blocked the effects of PL37. These data demonstrate that inhibiting enkephalinases, and thus protecting enkephalins from degradation, attenuates stress-induced migraine-like behavior via activation of peripheral DOR. Peripheral targeting of endogenous opioid signaling may be an effective therapeutic strategy for migraine.

Effects of the acute administration of delta-opioid receptor ligands on the excitability of rat hippocampal glutamate and brainstem monoamine neurons in vivo.

It was previously reported that the delta opioid receptor (DOR) agonist SNC80 and antagonist naltrindole modulate the excitability of hippocampal glutamate neurons in primary cultures. The present study aimed to investigate the acute effects of these ligands on the firing activity of hippocampal cornu ammonis 1/3 (CA1/3) glutamate, dorsal raphe nucleus (DRN) serotonin (5-HT), locus coeruleus (LC) noradrenaline, and ventral tegmental area (VTA) dopamine neurons in in vivo conditions. Adult Wistar male rats were used. SNC80 and naltrindole were administered intravenously. Neuronal firing activity was assessed using extracellular single-unit electrophysiology. SNC80, administered first at 1-3 mg/kg, dose-dependently inhibited CA1/3 glutamate, DRN 5-HT, and VTA dopamine neurons. Naltrindole, administered at 1-3 mg/kg after SNC80, did not have any additional effect. Naltrindole, administered first at 1-3 mg/kg, stimulated DRN 5-HT neurons in a dose-dependent manner; this stimulation was dose-dependently reversed by 1-3 mg/kg of SNC80. SNC80 and naltrindole inhibited LC noradrenaline neurons when only they were co-administered at 3 mg/kg, and only when SNC80 was administered first. In conclusion, DOR ligands alter the firing activity of hippocampal glutamate and brainstem monoamine neurons in in vivo conditions. The psychoactive effects of DOR ligands, reported in previous studies, might be explained, at least in part, by their ability to modulate the firing activity of hippocampal glutamate and brainstem monoamine neurons.

Baicalein exerts anxiolytic and antinociceptive effects in a mouse model of posttraumatic stress disorder: Involvement of the serotonergic system and spinal delta-opioid receptors

Post-traumatic stress disorder (PTSD) is a serious mental disease featured by a stress dysfunction that occurs after an individual has faced intense mental stress, often accompanied by anxiety and chronic pain. Currently, the mainstream drug for PTSD is serotonin reuptake inhibitors (SSRIs), however, their pain management for patients is limited. Baicalein, a Chinese traditional herbal medicine, has shown promising results in treating anxiety, depression, and pain. In this study, we found that baicalein may alleviate single prolonged stress (SPS)-induced PTSD-like behaviors in mice without altering baseline nociceptive sensitivity or activity. Meanwhile, baicalein increased the noradrenaline (NE) and serotonin (5-HT) content and decreased the ratio of 5-hydroxyindoleacetic acid (5-HIAA)/5-HT by inhibiting the activity of monoamine oxidase A (MAO-A) in SPS-induce mice. The anxiolytic and antinociceptive effects induced by baicalein were totally abolished by 5-HT depleting agents. Moreover, the anxiolytic effects of baicalein could be abolished by the 5-HT1A receptor antagonist WAY-100635, and the analgesic effects could be abolished by delta-opioid receptor antagonists in the spinal. Taken together, our study provides compelling evidence that baicalein reversed anxiety-like behaviors and neuropathic pain in PTSD through serotonergic system and spinal delta-opioid receptors.

The Delta-Opioid Receptor Bidirectionally Modulates Itch

Opioid signaling has been shown to be critically important in the neuromodulation of sensory circuits in the superficial spinal cord. Agonists of the mu-opioid receptor (MOR) elicit itch, whereas agonists of the kappa-opioid receptor (KOR) have been shown to inhibit itch. Despite the clear roles of MOR and KOR for the modulation itch, whether the delta-opioid receptor (DOR) is involved in the regulation of itch remained unknown. Here, we show that intrathecal administration of DOR agonists suppresses chemical itch and that intrathecal application of DOR antagonists is sufficient to evoke itch. We identify that spinal enkephalin neurons co-express neuropeptide Y (NPY), a peptide previously implicated in the inhibition of itch. In the spinal cord, DOR overlapped with both the NPY receptor (NPY1R) and KOR, suggesting that DOR neurons represent a site for convergent itch information in the dorsal horn. Lastly, we found that neurons co-expressing DOR and KOR showed significant Fos induction following pruritogen-evoked itch. These results uncover a role for DOR in the modulation of itch in the superficial dorsal horn. PerspectiveThis article reveals the role of the delta-opioid receptor in itch. Intrathecal administration of delta agonists suppresses itch whereas the administration of delta antagonists is sufficient to induce itch. These studies highlight the importance of delta-opioid signaling for the modulation of itch behaviors, which may represent new targets for the management of itch disorders.

Activation of Delta-Opioid Receptor Protects ARPE19 Cells against Oxygen-Glucose Deprivation/Reoxygenation-Induced Necroptosis and Apoptosis by Inhibiting the Release of TNF-α.

Retinal ischemia-reperfusion injury (RIRI) is the basis of the pathology that leads to many retinal diseases and induces necroptosis and apoptosis. Tumor necrosis factor-α (TNF-α) is critically involved in necroptosis and apoptosis. Delta-opioid receptor (DOR) activation inhibits TNF-α release in our previous studies, it might prevent necroptosis and apoptosis by inhibiting the release of TNF-α. However, the role of TNF-α and DOR in necroptosis and apoptosis of retinal pigment epithelial (RPE) cells remains largely unknown. Here, we explored the mechanisms of TNF-α and DOR in necroptosis and apoptosis using an oxygen-glucose deprivation/reoxygenation (OGD/R) model of adult retinal pigment epithelial cell line-19 (ARPE19) cells. ARPE19 cells were exposed to OGD/R conditions to mimic RIRI in vitro. Cell viability was quantified using the Cell Counting Kit-8 (CCK-8) assay. Morphological changes were observed by inverted microscopy. TNF-α protein levels in cell lysates were measured by enzyme-linked immunosorbent assay (ELISA). The DOR agonist TAN-67 and antagonist naltrindole (NTI) were used to pretreat cells for 1 or 2 hours before OGD24/R36 administration. Calcein acetoxymethylester/propidium iodide (Calcein-AM/PI) and Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining were used to detect necroptotic and apoptotic ARPE19 cells, respectively. The protein expression of DOR, p-RIP1 (RIP1), p-RIP3 (RIP3), p-MLKL (MLKL), and cleaved Caspase3 (Caspase3) was measured by western blotting. OGD severely damaged ARPE19 cells. Prolonged reoxygenation significantly increased TNF-α level and decreased DOR expression in ARPE19 cells. Pretreatment with the DOR agonist TAN-67 (10 µM) significantly improved ARPE19 cell viability after OGD24/R36 by reducing the number of necroptotic and apoptotic cells. Furthermore, DOR activation significantly inhibited TNF-α release and suppressed the expression of proteins related to necroptosis and apoptosis, including p-RIP1, p-RIP3, p-MLKL, and cleaved Caspase3, after OGD24/R36. This effect was reversed by the DOR antagonist NTI. These results strongly suggest that DOR activation inhibits necroptosis and apoptosis by decreasing TNF-α release, leading to the prevention of OGD/R-induced injury in ARPE19 cells. This study provides an innovative idea for clinical treatment strategies for retinal damage and vision loss due to RIRI.

Medial prefrontal cortex nitric oxide modulates neuropathic pain behavior through mu opioid receptors in rats.

BackgroundThe neocortex, including the medial prefrontal cortex (mPFC), contains many neurons expressing nitric oxide synthase (NOS). In addition, increasing evidence shows that the nitric oxide (NO) and opioid systems interact in the brain. However, there have been no studies on the interaction of the opioid and NO systems in the mPFC. The objective of this study was to investigate the effects of administrating L-arginine (L-Arg, a precursor of NO) and N(gamma)-nitro-L-arginine methyl ester (L-NAME, an inhibitor of NOS) into the mPFC for neuropathic pain in rats. Also, we used selective opioid receptor antagonists to clarify the possible participation of the opioid mechanism.MethodsComplete transection of the peroneal and tibial branches of the sciatic nerve was applied to induce neuropathic pain, and seven days later, the mPFC was cannulated bilaterally. The paw withdrawal threshold fifty percent (50% PWT) was recorded on the 14th day.ResultsMicroinjection of L-Arg (2.87, 11.5 and 45.92 nmol per 0.25 µL) increased 50% PWT. L-NAME (17.15 nmol per 0.25 µL) and naloxonazine (an antagonist of mu opioid receptors, 1.54 nmol per 0.25 µL) inhibited anti-allodynia induced by L-Arg (45.92 nmol per 0.25 µL). Naltrindole (a delta opioid receptor antagonist, 2.45 nmol per 0.25 µL) and nor-binaltorphimine (a kappa opioid receptor antagonist, 1.36 nmol per 0.25 µL) were unable to prevent L-Arg (45.92 nmol per 0.25 µL)-induced antiallodynia.ConclusionsOur results indicate that the NO system in the mPFC regulates neuropathic pain. Mu opioid receptors of this area might participate in pain relief caused by L-Arg.

Crotalphine Modulates Microglia M1/M2 Phenotypes and Induces Spinal Analgesia Mediated by Opioid-Cannabinoid Systems.

Pain is a worldwide public health problem and its treatment is still a challenge since clinically available drugs do not completely reverse chronic painful states or induce undesirable effects. Crotalphine is a 14 amino acids synthetic peptide that induces a potent and long-lasting analgesic effect on acute and chronic pain models, peripherally mediated by the endogenous release of dynorphin A and the desensitization of the transient receptor potential ankyrin 1 (TRPA1) receptor. However, the effects of crotalphine on the central nervous system (CNS) and the signaling pathway have not been investigated. Thus, the central effect of crotalphine was evaluated on the partial sciatic nerve ligation (PSNL)-induced chronic neuropathic pain model. Crotalphine (100 µg/kg, p.o.)-induced analgesia on the 14th day after surgery lasting up to 24 h after administration. This effect was prevented by intrathecal administration of CB1 (AM251) or CB2 (AM630) cannabinoid receptor antagonists. Besides that, crotalphine-induced analgesia was reversed by CTOP, nor-BNI, and naltrindole, antagonists of mu, kappa, and delta-opioid receptors, respectively, and also by the specific antibodies for β-endorphin, dynorphin-A, and met-enkephalin. Likewise, the analgesic effect of crotalphine was blocked by the intrathecal administration of minocycline, an inhibitor of microglial activation and proliferation. Additionally, crotalphine decreased the PSNL-induced IL-6 release in the spinal cord. Importantly, in vitro, crotalphine inhibited LPS-induced CD86 expression and upregulated CD206 expression in BV-2 cells, demonstrating a polarization of microglial cells towards the M2 phenotype. These results demonstrated that crotalphine, besides activating opioid and cannabinoid analgesic systems, impairs central neuroinflammation, confirming the neuromodulatory mechanism involved in the crotalphine analgesic effect.

Supraspinal melatonin MT2 receptor agonism alleviates pain via a neural circuit that recruits mu opioid receptors.

Melatonin, through its G protein-coupled receptor (GPCR) (MTNR1B gene)MT2 , is implicated in analgesia, but the relationship between MT2 receptors and the opioid system remains elusive. In a model of rodent neuropathic pain (spared nerve injured [SNI]), the selective melatonin MT2 agonist UCM924 reversed the allodynia (a pain response to a non-noxious stimulus), and this effect was nullified by the pharmacological blockade or genetic inactivation of the mu opioid receptor (MOR), but not the delta opioid receptor (DOR). Indeed, SNI MOR, but not DOR knockout mice, did not respond to the antiallodynic effects of the UCM924. Similarly, the nonselective opioid antagonist naloxone and the selective MOR antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP) blocked the effects of UCM924 in SNI rats, but not the DOR antagonist naltrindole (NTI). Electrophysiological recordings in the rostral-ventromedial medulla (RVM) revealed that the typical reduction of the firing activity of pronociceptive ON-cells, and the enhancement of the firing of the antinociceptive OFF-cells, induced by the microinjection of the MT2 agonist UCM924 into the ventrolateral periaqueductal gray (vlPAG) were blocked by MOR, but not DOR, antagonism. Immunohistochemistry studies showed that MT2 receptors are expressed in both excitatory (CaMKIIα+ ) and inhibitory (GAD65+ ) neuronal cell bodies in the vlPAG (~2.16% total), but not RVM. Only 0.20% of vlPAG neurons coexpressed MOR and MT2 receptors. Finally, UCM924 treatment induced an increase in the enkephalin precursor gene (PENK) in the PAG of SNI mice. Collectively, the melatonin MT2 receptor agonism requires MORs to exert its antiallodynic effects, mostly through an interneuronal circuit involving MOR and MT2 receptors.

D-Ala2, D-Leu5] Enkephalin Attenuates Hepatic Ischemia-Reperfusion Injury in Cirrhotic Rats.

Background and AimsHepatic ischemia–reperfusion injury (IRI) is a common phenomenon that occurs after liver transplantation and liver tumor surgery. It can cause liver dysfunction and recovery failure after liver surgery, even leading to acute liver failure. Our aim is to investigate the protective effect and related potential mechanism of [D-Ala2, D-Leu5] enkephalin (DADLE) treatment on hepatic IRI in cirrhotic livers of rats.MethodsThe models of liver cirrhosis and hepatic IRI were established with male Sprague–Dawley rats. DADLE at a dose series of 0.5, 1, or 5 mg·kg−1 was injected intravenously to rats 10 min prior hepatic ischemia, followed by a 6- h reperfusion. The serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), histological changes, and liver cell apoptosis were used to assess liver IRI. The optimal dose of DADLE was assessed by using the Suzuki score and ALT and AST levels. We repeated the hepatic IRI procedure on the optimal dose of the DADLE group and the delta opioid receptor (DOR) antagonist natrindole hydrochloride (NTD) injection group. Serum ALT and AST levels, histological staining, hepatic apoptosis, and serum levels of tumor necrosis factor alpha (TNF-α) and interleukin 1 β (IL-1β) were measured. The expression of protein kinase B (Akt) and its downstream proteins were evaluated by using quantitative real-time polymerase chain action (qRT-PCR) and Western blotting.ResultsCompared with the control group, DADLE treatment at a dose of 5 mg·kg−1 reduced the Suzuki score (mean: 5.8, range: 5.0–6.6 vs. mean: 8.0, range: 7.0–8.9), the ALT level (134.3 ± 44.7 vs. 247.8 ± 104.6), and the AST (297.1 ± 112.7 vs. 660.8 ± 104.3) level. DOR antagonist NTD aggravated hepatic IRI. Compared with the control group, DADLE treatment decreased the number of apoptosis cells and microphages and neutrophils, increased the expression of Akt and its mRNA to much higher levels, and upregulated the mRNA and protein expression of Bcl-2 and Bcl-2-associated death promoter (BAD).ConclusionDADLE treatment at a dose of 5 mg·kg−1 injected intravenously 10 min prior hepatic ischemia could contain rats’ hepatic IRI by activating DOR in cirrhotic livers. The effects of DADLE could be offset by NTD. The potential molecular mechanism seems to be involved in the phosphatidylinositol-3-kinase (PI3K)/Akt pathway.

SRI 22136, A Novel Delta Opioid Receptor (DOR) Antagonist for the Treatment of Alzheimer’s Disease

Alzheimer’s disease (AD) is the most common form of dementia, which affects 47 million people worldwide. There are over five million AD patients over the age of 65 in the U.S. which is predicted to increase to 16 million by 2050. There are currently no approved disease‐modifying therapies for AD and attempts to prevent or slow the progression of the formation of beta‐amyloid plaques by targeting both the beta‐secretase 1 (BACE1) and gamma‐secretase enzymes have not yet achieved clinical success.Studies suggest that indirect modulation of the function of these enzymes via G‐protein coupled receptors (GPCRs) may provide a novel strategy to reduce A‐beta peptide production with potentially less side effects. Among GPCRs that influence amyloidogenesis, the delta opioid receptor (DOR), in particular, has been shown to play an important role in the trafficking and function of BACE1 and gamma‐secretase and in the production of A‐beta peptide. DOR activation increases BACE1 and gamma‐secretase activity in vitro and in a mouse model of AD, and antagonism of DOR specifically blocks the amyloidogenic pathway and efficaciously prevents AD progression in mice. These effects were demonstrated using a known DOR antagonist, naltrindole. However, the potential of DOR antagonists as therapeutic agents for AD has yet to be explored.The aim of this project is to create novel DOR antagonists via medicinal chemistry and identify the most promising lead compound based on binding, selectivity, and functional profile in vitro. Further, select a small set of the most promising compounds and evaluate the compound’s ability to mitigate AD‐like pathology in vivo using APP/PS double‐transgenic mice. We report here the identification of several selective DOR antagonists with low nanomolar affinity/potency that have been shown to block BACE1 activity in vitro. Lead compound SRI‐22136, showed systemic stability and blood brain barrier penetration using the in vivo tail flick assay in CD‐1 mice. SRI‐22136 also showed efficacy in Novel Object Recognition (NOR) behavioral studies and in brain histological analysis studies we observed greater reduction in the AD pathological markers at a dose of 1mg/kg twice daily for 90 days continuous treatment in vivo using the APP/PS AD model double‐transgenic mice. Also, in our brain histological analysis the compound exhibited greater efficacy in reduction of the AD pathology markers such as Aβ signal, and secondary inflammation and activated glia detected by GFAP (astrocytes) and CD11b (microglia) in APP/PS AD model double‐transgenic mice. We further report our progress in testing this compound’s effect on biochemical marker evaluation using the cortex and hippocampus regions of the APP/PS AD model double‐transgenic mice.

Preconditioning Using Naltrindole or its Analogues Exerts Robust Infarct‐Sparing Effects in Rat Myocardial Ischemia/Reperfusion

Ischemic heart disease remains the leading cause of death worldwide. Pharmacological agents that mimic the cardioprotective effects of ischemic preconditioning may have therapeutic potential as a secondary prevention strategy to resist infarction from subsequent cardiovascular events. Increased left ventricular end diastolic pressure (LVEDP) during ischemia, or ischemic peak pressure (IPP), is known to be correlated to infarct size. Recently our laboratory demonstrated that naltrindole (NTI), a selective delta opioid receptor antagonist, reduces IPP and infarct size when given prior to ischemia (preconditioning) in a Langendorff rat heart model. The purpose of this study was to examine the effects of NTI analogues naltriben (NTB, delta opioid receptor antagonist) and guanidinonaltrindole (GNTI, kappa opioid receptor antagonist) compared to NTI. Nor‐binaltrophine (BNI, kappa opioid receptor antagonist) and naloxone (NX, broad‐spectrum opioid receptor antagonist) were tested to evaluate cardioprotection by other opioid receptor antagonists.Isolated hearts from male Sprague‐Dawley rats (~300g) were subjected to 30‐min global ischemia (I)/45‐min reperfusion (R) with treatments infused for 5 min before I and during the first 5 min of R. LV cardiac function was measured using a pressure transducer. At the end of reperfusion, infarct size was assessed using 1% triphenyltetrazolium chloride staining and defined as infarcted tissue/total area at risk. Data were evaluated using ANOVA Student‐Neuman‐Keuls post‐hoc analysis.Control I/R hearts demonstrated an IPP of 39±3 mmHg compared to pre‐ischemic LVEDP of 9±1 mmHg at baseline (n=12, p<0.01), resulting in substantial infarct at the end of 45 min R (32±4%). NTI (n=7) and NTB (n=6) elicited cardiodepressive effects during preconditioning by reducing the maximal rate in the rise of LV pressure (dP/dt max) to 1581±379 mmHg/s and 929±243 mmHg/s, respectively, compared to control (2471±72 mmHg/s, p<0.01). IPP was reduced by NTI (18±3 mmHg/s) and NTB (15±3 mmHg/s) compared to all groups (p<0.05). Post‐reperfused dP/dt max and infarct size was most improved following NTI (1830±90 mmHg/s, 7±2%) and NTB (1846±140 mmHg/s, 7±2%) pretreatment, compared to control (777±142 mmHg/s, 32±4%, p<0.01). GNTI reduced infarct size (17 ± 4%, n=6, p<0.05), but did not exert a negative inotropic effect. Cardiac function and infarct size did not improve with BNI (n=7) or NX (n=6) pretreatment.These results suggest that NTI and analogues, GNTI and NTB, are cardioprotective against myocardial I/R injury. The negative inotropic effects of NTI and NTB were associated with ~75% reduction in infarct size compared to control. GNTI decreased infarct size by ~50% and these results suggest that NTI, NTB, and GNTI exert tissue‐salvaging effects independent of delta or kappa opioid receptor antagonism. In future studies, we will examine different ischemic time points to administer NTI and its analogues to determine optimal cardioprotection and investigate downstream effects on calcium handling.

Naltrindole Pretreatment Exhibits Robust Cardioprotection in an Acute In Vivo Model of Ischemia/Reperfusion

Myocardial infarctions (MI), remain prevalent in the U.S. with over one million individuals experiencing this cardiac event every year. Delta opioid receptor activation is known to mimic pharmacologic preconditioning to reduce infarct size post‐MI. Surprisingly, results from our laboratory have shown that naltrindole (NTI), a selective delta opioid receptor antagonist, elicits unexpected cardioprotective effects ex vivo when given prior to 30 min global ischemia (I) followed by 45 min reperfusion (R). Whether NTI’s cardioprotective effects can be translated in vivo with intact neurohormonal compensatory mechanisms, has not yet been elucidated. In this study, we aim to determine the cardioprotective effects of NTI in an in vivo model that more closely mimics acute MI clinically.Female Sprague‐Dawley rats (~250g) were assigned into three cohorts: non‐treated control (n=15), high dose (HD, 4.0‐8.0 mg/kg, n=5) NTI, and low dose (LD, 0.2‐1.0 mg/kg, n=18) treatment groups. All three cohorts were anesthetized with ketamine‐xylazine (90 mg/kg, 9mg/kg). Control rats received normal saline (0.9% NaCl), while treatment groups received either HD or LD NTI. NTI was infused through the jugular vein (0.4mL/min) over 5 mins before inducing regional ischemia (pretreatment). The proximal left coronary artery was occluded for 30 min followed by 3 hr reperfusion. At the end of the experiment, blue dye was administered to determine the area at risk (AR). Planimetered photographs of heart slices were then stained with 1% triphenyltetrazolium chloride to determine area of necrosis (AN) and infarct size (AN/AR). Cardiac function was measured via a pressure transducer placed into the left ventricle (LV) in a small cohort of the LD NTI (0.4‐1.0 mg/kg, n=3) and HD NTI groups (n=5). Data were analyzed via ANOVA Student Newman Keuls post‐hoc analysis (infarct size) and unpaired t‐test (cardiac function), respectively, with p<0.05 considered statistically significant.HD NTI exhibited a robust ~60% reduction in infarct size (21±7 %; n=4, p<0.01) compared to control (57±2 %; n=15) and LD NTI (55±4 %; n=18) hearts. HD NTI (n=5) also exerted a significant depression of the maximal and minimal rate of LV pressure change on dP/dt max (6167±489 mmHg/s vs. 3714±645 mmHg/s) and dP/dt min (‐7015±942 mmHg/s vs. ‐4034±776 mmHg/s) respectively and heart rate (261±20 BPM vs. 208±3 BPM) at the end of pretreatment compared to LD NTI (0.4‐1.0 mg/kg, n=3 all p<0.05). This preconditioning effect was correlated with the robust decrease in infarct size compared to LD NTI and control groups. These results were similar to our ex‐vivo studies, although a 20x dose was needed in vivo to presumably overcome compensatory neurohormonal responses (i.e. 4mg/kg vs 0.2mg/kg). Results suggest that NTI pretreatment can protect against I/R injury. Future studies will examine the effects of drug infusion at different ischemic time points for optimal cardioprotection.

Subanalgesic morphine doses augment fentanyl analgesia by interacting with delta opioid receptors in male rats

Opioids are commonly used for the treatment of postoperative and post-traumatic pain; however, their therapeutic effectiveness is limited by undesirable and life-threatening side effects. Researchers have long attempted to develop opioid co-administration therapies that enhance analgesia, but the complexity of opioid analgesia and our incomplete mechanistic understanding has made this a daunting task. We discovered that subanalgesic morphine doses (100ng/kg-10µg/kg) augmented the acute analgesic effect of fentanyl (20µg/kg) following subcutaneous drug co-administration to male rats. In addition, administration of equivalent drug ratios to naïve rat spinal cord membranes induced a twofold increase in G protein activation. The rate of GTP hydrolysis remained unchanged. We demonstrated that these behavioral and biochemical effects were mediated by the delta opioid receptor (DOP). Subanalgesic doses of the DOP-selective agonist SNC80 also augmented the acute analgesic effect of fentanyl. Furthermore, co-administration of the DOP antagonist naltrindole with both fentanyl-morphine and fentanyl-SNC80 combinations prevented augmentation of both analgesia and G protein activation. The mu opioid receptor (MOP) antagonist cyprodime did not block augmentation. Confocal microscopy of the substantia gelatinosa of rats treated with fentanyl, subanalgesic morphine, or this combination showed that changes in MOP internalization did not account for augmentation effects. Together, these findings suggest that augmentation of fentanyl analgesia by subanalgesic morphine is mediated by increased G protein activation resulting from a synergistic interaction between or heterodimerization of MOPs and DOPs. This finding is of great therapeutic significance because it suggests a strategy for the development of DOP-selective ligands that can enhance the therapeutic index of clinically used MOP drugs.

Abstract P500: Naltrindole And Naltrindole Derivatives Exhibit Potent Cardioprotection In Myocardial Ischemia Reperfusion Injury

Previously, naltrindole (NTI; selective delta opioid receptor antagonist) was shown to improve post-reperfused cardiac function and reduced infarct size when given prior to ischemia (I)/ reperfusion (R) in ex-vivo rat hearts. Conversely, naloxone (NX, broad-spectrum opioid antagonist) and nor-binaltrophine (BNI, selective kappa receptor antagonist) were similar to control hearts. In this study, the effects of NTI derivatives naltriben (NTB, delta receptor antagonist) and guanidonaltrindole (GNTI, kappa receptor antagonist) were compared to NTI, BNI, and NX. Isolated hearts from male SD rats (300g) were subjected to global I(30min)/R(45min). Treatments were given 5 min before I (preconditioning) and during the first 5 min of R. Left ventricular (LV) cardiac function was measured using a pressure transducer. At the end of reperfusion, infarcted heart tissue was compared to total tissue weight. Data were evaluated using ANOVA. As shown in Table 1, NTI, NTB, and GNTI significantly improved post-reperfused cardiac function and reduced infarct size compared to control hearts. NTI and NTB elicited direct effects on cardiac function when given during preconditioning in contrast to all other study groups and were the most robust at reducing infarct size and restoring post reperfusion cardiac function. The negative inotropic effects of NTI and NTB were correlated with a decrease in the rise of ischemic pressure. GNTI also elicited significant improvement in post-reperfused cardiac function and reduction of infarct size compared to BNI which suggests a separate cardioprotective mechanism that this NTI derivative may exert in contrast to kappa opioid receptor inhibition. Results suggest that NTI and derivatives, GNTI and NTB, are cardioprotective against I/R injury resulting in reduced ischemic peak pressure (NTI/NTB) and infarct size. In future studies, we will examine the mechanism of the protective effects of NTI and derivatives in hearts subjected to I/R injury.

Delta Opioid Receptor (DOR) Agonist Activity of Buprenorphine in Mice

Buprenorphine is an approved method of treatment for opioid use disorder (OUD), and it is under investigation for cocaine use disorder-- for which there are no FDA-approved, pharmacological treatments. Buprenorphine is considered a mixed efficacy opioid ligand, and its partial agonist activity at the mu opioid receptor (MOR) is thought to mediate the therapeutic effects for OUD. There is also evidence that it acts as a kappa opioid receptor (KOR) and delta opioid receptor (DOR) antagonist and a nociception/orphanin FQ receptor (NOPR) agonist. However, one recent report found buprenorphine to act as a partial DOR agonist in cells expressing human opioid receptors (Bidlack et al., 2018). The goal of the present study was to investigate the in vivo activity of buprenorphine at DOR in rodents. Male C57BL6/N mice were used to evaluate the effects of buprenorphine on pentylenetetrazol (PTZ)-induced convulsive activity and in the mouse forced swim test. Buprenorphine did not produce overt convulsions in mice but dose-dependently increased PTZ-induced convulsions with 32 mg/kg buprenorphine as the maximally effective dose, and these effects were attenuated by the DOR antagonist naltrindole. Also, buprenorphine (0.01-0.1 mg/kg) decreased immobility in the mouse forced swim test, which was blocked by pretreatment with naltrindole. Together, these data demonstrate that buprenorphine produces DOR-mediated antidepressant-like effects and potentiates PTZ-induced convulsions, suggesting that buprenorphine acts as a partial DOR agonist in mice. Interestingly, there is a large separation between doses that produce potential antidepressant-like effects and pro-convulsive effects. Future studies will investigate whether this large separation between DOR-mediated effects are due to the complex pharmacology of buprenorphine. Further, these data offer additional insight into the activity of buprenorphine, which could help elucidate the mechanism needed for successful treatment of opioid or cocaine use disorder. These studies were supported in part by R21 DA041565 and DA007315.

Naltrindole Exhibits Robust Cardioprotection in Myocardial Ischemia Reperfusion Injury

The predominant opioid receptor subtypes in heart tissue are delta and kappa opioid receptors. Activation of delta and kappa opioid receptors during preconditioning have been shown to reduce infarct size in myocardial ischemia reperfusion (I/R) injury. This cardioprotective effect has been abolished with naloxone (NX), a broad-spectrum opioid receptor antagonist (e.g., delta, kappa, and mu). Previously, we demonstrated that a novel tripeptide, structurally similar to other delta or kappa opioid peptide receptor agonists, when given prior to global ischemia, significantly reduced infarct size and improved post-reperfused cardiac function using a Langendorf rat I/R heart ex vivo model. To identify the opioid receptor subtype responsible for tripeptide's cardioprotective effects, we used naltrindole (NTI), a selective delta opioid receptor antagonist. The cardioprotective effects of tripeptide were blocked by NX, but not NTI. To determine whether the cardioprotective effects of the combination was due to tripeptide or NTI, the effects of NTI and NX were evaluated individually in the same model. Male Sprague-Dawley rats (~300g) were subjected to global I(30min)/R(50min). NX (10 μM, n=6) or NTI (5 μM, n=8) or H2O vehicle (control n= 10) were given to hearts 5 min prior to ischemia and during the first 5 min of reperfusion in a Langendorf perfused model. Left ventricular (LV) cardiac function indices were measured using a pressure transducer. At the end of reperfusion (50min), hearts were frozen, sectioned (2 mm), and stained with 1% triphenyltetrazolium chloride (TTC). Infarcted heart tissue was compared to total tissue weight. Data were evaluated using ANOVA Student-Newman-Keuls post-hoc analysis. Control hearts (n=10) exhibited increased final LV end diastolic pressure (LVEDP) of 60±5 mmHg from baseline values (~8mmHg for all groups) and a mean infarct size of 36 ± 3% at 50 min post-reperfusion. NX showed no significant cardioprotective effect, since final LVEDP (64 ± 5%) and infarct size (35 ± 5%) was similar to control hearts. Surprisingly, the selective delta opioid antagonist, NTI, remarkably improved final LVEDP to 17 ± 3mmHg to near baseline values and reduced infarct size to 7 ± 2% compared to control and NX hearts (both p<0.01). The results indicate that NTI can prevent ischemic injury and restore post-reperfused heart function to pre-ischemic levels. In future studies, we will examine the intracellular mechanisms of NTI responsible for preventing myocardial ischemic injury.

Endogenous opioid and cannabinoid systems modulate the muscle pain: A pharmacological study into the peripheral site

The participation of the peripheral opioid and cannabinoid endogenous systems in modulating muscle pain and inflammation has not been fully explored. Thus, the aim of this study was to investigate the involvement of these endogenous systems during muscular-tissue hyperalgesia induced by inflammation. Hyperalgesia was induced by carrageenan injection into the tibialis anterior muscles of male Wistar rats. We padronized an available Randal-Sellito test adaptation to evaluate nociceptive behavior elicited by mechanical insult in muscles. Western blot analysis was performed to evaluate the expression levels of opioid and cannabinoid receptors in the dorsal root ganglia. The non-selective opioid peptide receptor antagonist (naloxone) and the selective mu opioid receptor MOP (clocinnamox) and kappa opioid receptor KOP (nor-binaltorphimine) antagonists were able to intensify carrageenan-induced muscular hyperalgesia. On the other hand, the selective delta opioid receptor (DOP) antagonist (naltrindole) did not present any effect on nociceptive behavior. Moreover, the selective inhibitor of aminopeptidases (Bestatin) provoked considerable dose-dependent analgesia when intramuscularly injected into the hyperalgesic muscle. The CB1 receptor antagonist (AM251), but not the CB2 receptor antagonist (AM630), intensified muscle hyperalgesia. All irreversible inhibitors of anandamide hydrolase (MAFP), the inhibitor for monoacylglycerol lipase (JZL184) and the anandamide reuptake inhibitor (VDM11) decreased carrageenan-induced hyperalgesia in muscular tissue. Lastly, MOP, KOP and CB1 expression levels in DRG were baseline even after muscular injection with carrageenan. The endogenous opioid and cannabinoid systems participate in peripheral muscle pain control through the activation of MOP, KOP and CB1 receptors.

Delta-Opioid Receptors Play a Role in the Control of Selected Parameters Related to Stress and Brain Plasticity Under Non-stress and/or Stress Conditions.

There is some evidence that delta-opioid receptors may be involved in the brain processes related to neuroprotection. The aim of the present studies was to test the hypothesis that endogenous opioid peptides acting via delta-opioid receptors can protect against stress-induced changes in factors related to brain plasticity and stress hormone release. Forty male adult Wistar rats were used. Half of the animals were exposed to sustained partial restraint stress (hypokinesis) lasting48h. Rats were treated with vehicle (isotonic saline) or the delta-opioid receptor antagonist naltrindole (3mg/kg/ml, s.c.) six times a day. The stressfulness of the model was confirmed by increased plasma concentrations of corticosterone and prolactin, the increase in anxiety behavior in the open field test, as well as the reduction of BrdU incorporation into newly formed DNA in the hippocampus. Treatment with naltrindole potentiated the stress-induced rise in aldosterone concentrations. The blockade of delta-opioid receptors resulted in a decrease in hippocampal BDNF gene expression independently of control or stress conditions. Treatment with naltrindole enhanced plasma concentrations of copeptin, a stable precursor of vasopressin. In conclusion, these results suggest that endogenous opioid peptides might play an inhibitory role in aldosterone release under stress conditions and in the control of vasopressin release independently of stress exposure. Endogenous opioids might stimulate hippocampal gene expression of the important neurotrophic factor BDNF via delta-opioid receptors.