Delta-opioid Research Articles

Olanzapine/Samidorphan: A New Combination Treatment for Schizophrenia and Bipolar I Disorder Intended to Reduce Weight Gain.

Olanzapine is a second-generation antipsychotic with established efficacy in several psychiatric disease states, but its use is limited because of weight gain and metabolic side effects. Samidorphan is a novel opioid antagonist that binds to mu-opioid, kappa-opioid, and delta-opioid receptors and is hypothesized to reduce cravings for high-calorie foods thus attenuating antipsychotic-induced weight gain. The combination product olanzapine/samidorphan was approved by the US Food and Drug Administration in June 2021 for the treatment of schizophrenia and bipolar I disorder; this article reviews the pharmacological properties of oral olanzapine/samidorphan and its clinical efficacy and tolerability with a focus on mitigation of olanzapine-induced weight gain in these patient populations. In clinical trials, the combination of olanzapine/samidorphan was associated with significantly less weight gain and smaller increases in waist circumference as compared with olanzapine monotherapy. Olanzapine/samidorphan demonstrated similar efficacy as olanzapine monotherapy and was well tolerated. Weight gain and metabolic side effects associated with olanzapine monotherapy can result in tolerability issues and potentially medication nonadherence. Olanzapine/samidorphan is an effective treatment for schizophrenia and bipolar I disorder with less weight gain than olanzapine monotherapy.

In Vitro, In Vivo and In Silico Characterization of a Novel Kappa-Opioid Receptor Antagonist.

Kappa-opioid receptor (KOR) antagonists are promising innovative therapeutics for the treatment of the central nervous system (CNS) disorders. The new scaffold opioid ligand, Compound A, was originally found as a mu-opioid receptor (MOR) antagonist but its binding/selectivity and activation profile at the KOR and delta-opioid receptor (DOR) remain elusive. In this study, we present an in vitro, in vivo and in silico characterization of Compound A by revealing this ligand as a KOR antagonist in vitro and in vivo. In the radioligand competitive binding assay, Compound A bound at the human KOR, albeit with moderate affinity, but with increased affinity than to the human MOR and without specific binding at the human DOR, thus displaying a preferential KOR selectivity profile. Following subcutaneous administration in mice, Compound A effectively reverse the antinociceptive effects of the prototypical KOR agonist, U50,488. In silico investigations were carried out to assess the structural determinants responsible for opioid receptor subtype selectivity of Compound A. Molecular docking, molecular dynamics simulations and dynamic pharmacophore (dynophore) generation revealed differences in the stabilization of the chlorophenyl moiety of Compound A within the opioid receptor binding pockets, rationalizing the experimentally determined binding affinity values. This new chemotype bears the potential for favorable ADMET properties and holds promise for chemical optimization toward the development of potential therapeutics.

Kratom (Mitragyna Speciosa): a Psychoactive Plant with Opportunities and Risks

Kratom is an evergreen tree that is native to Southeast Asia. Its leafs are traditionally used as a stimulant, a remedy for various health problems and for religious purposes. Especially in the US (in a lesser extent also in Europe) kratom use is significantly prevalent. In Western countries, kratom is used predominantly as an analgesic and stimulant, for the treatment of opioid use disorders, and for improving mental health (e. g., in depression, anxiety disorders). Main molecular constituents of kratom are alkaloids of which mitragynine and 7-hydroxymitragynine appear to be most important. Pharmacodynamics and -kinetics of kratom are complex and insufficiently studied. It is known that mitragynine and 7-hydroxymitragynine are partial agonist at human μ-opioid receptors and antagonists at κ- and δ-opioid receptors with additional effects at other central receptors. Tolerability of kratom is presumably better than that of classical opioids; this is probably due to missing effects of kratom on β-arrestin and discussed as a starting point for the development of opioids with improved tolerability. Some alkaloids of kratom are inhibitors of CYP26 and to a somewhat lesser degree of CYP2C19 and CYP3A4. The addictive potential of kratom appears to be lower than that of classical opioids; however, corresponding data is limited and kratom use disorders appear to occur primarily in Western countries. Several cases of severe health-related problems and deaths are known in the US; in these cases, however, polysubstance use was usually present. Kratom use is likely associated with hepatotoxicity and cardiotoxicity. Kratom-associated mortality and morbidity in Western countries are quantitatively significantly different from Southeast Asia, where kratom use is no public health problem. The reasons for this may be the combined use of substances (which is more prevalent in Western countries), higher dosages of consumed kratom, adulterations and contaminations of commercially available kratom in Western countries, pharmacokinetic interactions, and higher concentrations of 7-hydroxymitragynine in dried kratom leafs (that are typically consumed in Western countries) in comparison to fresh leafs (that are typically consumed in Southeast Asia).

0559 Prevalence of Cannabis Use in Patients with Restless Leg Syndrome for Symptomatic Relief

Abstract Introduction Restless leg syndrome (RLS) can be debilitating and have a significant negative impact on sleep. Most patients respond well to dopamine agonists, alpha 2 delta ligands, iron supplementation, and opioids. Some patients report persistent symptoms despite using recommended therapies. Methods We investigated the prevalence of cannabis use and its potential therapeutic effects in patients with RLS through a retrospective chart review over a three-year period. All patients with diagnosis of RLS and cannabis/marijuana use listed in the electronic health record were included. Data was collected from each patient's chart pertaining to the prevalence of cannabis use including method of use and frequency, patient demographics, use of certain medications, and whether they found symptom relief when using cannabis. Results 41 patients were identified who has a diagnosis of RLS and cannabis use. 25 of 41 (61%) were using medical marijuana and 8 of 41 (20%) were using marijuana recreationally. 12 of 41 (29.3%) patients found overall relief in their sleep while 4 (9.7%) patients did not and 25 (61%) patients we were unable to determine through chart review.Among the patients who were using medial marijuana, 5 patients were using it for pain and insomnia, 4 patients were using it for RLS, and 4 patients were using it for anxiety. 3 out of 4 (75%) patients who were using medical marijuana for RLS reported regular use. 3 of the patients inhaled cannabis while the remaining patient used an ointment containing cannabis. 2 of the 4 (50%) found symptom relief of RLS when using medical marijuana. 1 patient did not find relief with marjiuana but found relief when using Methadone instead. The remaining 3 patients also used Gabapentin and 2 of the 3 (67%) patients found symptom relief with the addition of a dopamine agonist. Conclusion Patients with RLS may use Cannabis for symptomatic relief. We believe this association should be recognized by sleep physicians and documented when conducting their patient's history. Support (If Any)

Mu and Delta Opioid Receptor Targeting Reduces Connexin 43-Based Heterocellular Coupling during Neuropathic Pain.

Chronic neuropathic pain emerges from either central or peripheral lesions inducing spontaneous or amplified responses to non-noxious stimuli. Despite different pharmacological approaches to treat such a chronic disease, neuropathic pain still represents an unmet clinical need, due to long-term therapeutic regimens and severe side effects that limit application of currently available drugs. A critical phenomenon involved in central sensitization is the exchange of signalling molecules and cytokines, between glia and neurons, driving the chronicization process. Herein, using a chronic constriction injury (CCI) model of neuropathic pain, we evaluated the efficacy of the mu (M-) and delta (D-) opioid receptor (-OR) targeting agent LP2 in modulating connexin-based heterocellular coupling and cytokine levels. We found that long-term efficacy of LP2 is consequent to MOR-DOR targeting resulting in the reduction of CCI-induced astrocyte-to-microglia heterocellular coupling mediated by connexin 43. We also found that single targeting of DOR reduces TNF and IL-6 levels in the chronic phase of the disease, but the peripheral and central discharge as the primary source of excitotoxic stimulation in the spinal cord requires a simultaneous MOR-DOR targeting to reduce CCI-induced neuropathic pain.

Functional expression of opioid receptors and other human GPCRs in yeast engineered to produce human sterols

The yeast Saccharomyces cerevisiae is powerful for studying human G protein-coupled receptors as they can be coupled to its mating pathway. However, some receptors, including the mu opioid receptor, are non-functional, which may be due to the presence of the fungal sterol ergosterol instead of cholesterol. Here we engineer yeast to produce cholesterol and introduce diverse mu, delta, and kappa opioid receptors to create sensitive opioid biosensors that recapitulate agonist binding profiles and antagonist inhibition. Additionally, human mu opioid receptor variants, including those with clinical relevance, largely display expected phenotypes. By testing mu opioid receptor-based biosensors with systematically adjusted cholesterol biosynthetic intermediates, we relate sterol profiles to biosensor sensitivity. Finally, we apply sterol-modified backgrounds to other human receptors revealing sterol influence in SSTR5, 5-HTR4, FPR1, and NPY1R signaling. This work provides a platform for generating human G protein-coupled receptor-based biosensors, facilitating receptor deorphanization and high-throughput screening of receptors and effectors.

A Critical Role of δ-Opioid Receptor in Anti-microglial Activation Under Stress

Microglia are involved in the regulation of cerebral homeostasis and pathogen confrontation. There is, however, evidence showing that excessive microglia activation is implicated in various age-related cerebral diseases. On the other hand, microglia may experience complex changes of polarization in pathological insults, i.e., from a proinflammatory M1 to an anti-inflammatory M2 phenotype, which differentially contribute to the exacerbation or alleviation of cellular injury. Remolding the phenotype of microglia or inhibiting the excessive activation of microglia seems to be a promising approach against neurodegenerative pathologies. Since δ-opioid receptor (DOR) activation exhibits a strong protective capacity against various neuronal injuries, especially the hypoxic/ischemic injury, we asked if the DOR-induced neuroprotection is associated with its effect on microglia. We explored this fundamental issue by using pharmacological and genetic approaches in the BV2 cell line, a general type of microglial cells. The results showed that DOR expression significantly increased in the activated microglial M2 phenotype, but slightly decreased in the microglial M1 phenotype. Hypoxia induced dual polarizations of BV2 cells with an increase in DOR expression. Administration of a specific DOR agonist, UFP-512, largely inhibited lipopolysaccharide (LPS) or hypoxia-induced microglial M1 activation and inflammatory activity with high concentrations of UFP-512 being effective to reverse the interleukin-4 (IL4)-induced microglial activation. Consistent with these observations, inhibiting DOR or knocking-down DOR promoted the excessive activation of BV2 cells in both M1 and M2 directions, while DOR overexpression did the opposite. Furthermore, the PC12 cells exposed to the conditioned medium of BV2 cells treated by UFP-512 grew better than those treated directly with UFP-512 under LPS or hypoxic insults. DOR inhibitor naltrindole could block all the effects of DOR activation. The medium from the BV2 cells with DOR knock-down decreased the viability of PC12 cell, while the medium from the BV2 cells with DOR overexpression largely attenuated LPS or hypoxic injury in the PC12 cells. These first data suggest a close linkage between DOR expression/function and microglial polarization and a critical role of DOR in negative controlling microglial activation. Our work provides a novel clue for new protective strategies against neurodegenerative pathophysiology through DOR-mediated regulation of microglia.

Activation of δ-opioid Receptors in Anterior Cingulate Cortex Alleviates Affective Pain in Rats

The negative emotions caused by persistent pain, called affective pain, are known to seriously affect human physical and mental health. The anterior cingulate cortex (ACC), especially the rostral ACC (rACC) plays a key role in the development of this affective pain. N-methyl-d-aspartate (NMDA) receptors, which are widely distributed in the ACC, are involved in the regulation of emotional behavior. It is well known that activation of opioid receptors can relieve pain, but whether it can alleviate affective pain is not clear. In the present study, conditioned place avoidance (CPA) responses induced by complete Freund's adjuvant (CFA) were used to represent the affective pain of place aversion. The behavioral measurements were synchronously combined with multichannel electrophysiological recordings of the discharge frequency of rACC pyramidal neurons to explore whether affective pain could be alleviated by the synthetic opioid [D-Ala2, D-Leu5]-Enkefalin (DADLE), an agonist of δ-opioid receptors. To further investigate this treatment as a mechanism for the relief of affective pain in CFA-treated animals, we used whole-cell patch recordings in slice preparations of the rACC region to determine the dose-dependent effects of DADLE on NMDA receptor-mediated currents. Then, western blot was used to determine levels of phosphorylated NMDA receptor subunits GluN1, GluN2 and GluN3 as affected by the δ-opioid receptor activation. The results showed that activation of δ-opioid receptors down-regulates the phosphorylation of NMDA receptor subunits, thereby inhibiting NMDA currents, decreasing the discharge frequency of rACC pyramidal neurons, and reversing the CPA response. Thus, δ-opioid receptor activation in the rACC region can alleviate affective pain.

Functional Characterization of Sodium Channel Inhibitors at the Delta-Opioid Receptor.

Existing pharmacotherapies acting on the opioid receptor system have been extensively used to treat chronic pain and addictive disorders. Nevertheless, the adverse side effects associated with opioid therapy underscore the need for concerted measures to develop safer analgesics. A promising avenue of research stems from the characterization of a sodium-dependent allosteric regulation site housed within the delta-opioid receptor and several other G protein-coupled receptors (GPCRs), thereby revealing the presence of a cluster of sodium and water molecules lodged in a cavity thought to be present only in the inactive conformation of the receptor. Studies into the structure–function relationship of said pocket demonstrated its critical involvement in the functional control of GPCR signaling. While the sodium pocket has been proposed to be present in the majority of class A GPCRs, the shape of this allosteric cavity appears to have significant structural variation among crystallographically solved GPCRs, making this site optimal for the design of new allosteric modulators that will be selective for opioid receptors. The size of the sodium pocket supports the accommodation of small molecules, and it has been speculated that promiscuous amiloride and 5′-substituted amiloride-related derivatives could target this cavity within many GPCRs, including opioid receptors. Using pharmacological approaches, we have described the selectivities of 5′-substituted amiloride-related derivatives, as well as the hitherto undescribed activity of the NHE1 inhibitor zoniporide toward class A GPCRs. Our investigations into the structural features of the delta-opioid receptor and its ensuing signaling activities suggest a bitopic mode of overlapping interactions involving the orthosteric site and the juxtaposed Na+ pocket, but only at the active or partially active opioid receptor.

Hypoxic/Ischemic Inflammation, MicroRNAs and δ-Opioid Receptors: Hypoxia/Ischemia-Sensitive Versus-Insensitive Organs

Hypoxia and ischemia cause inflammatory injury and critically participate in the pathogenesis of various diseases in various organs. However, the protective strategies against hypoxic and ischemic insults are very limited in clinical settings up to date. It is of utmost importance to improve our understanding of hypoxic/ischemic (H/I) inflammation and find novel therapies for better prevention/treatment of H/I injury. Recent studies provide strong evidence that the expression of microRNAs (miRNAs), which regulate gene expression and affect H/I inflammation through post-transcriptional mechanisms, are differentially altered in response to H/I stress, while δ-opioid receptors (DOR) play a protective role against H/I insults in different organs, including both H/I-sensitive organs (e.g., brain, kidney, and heart) and H/I-insensitive organs (e.g., liver and muscle). Indeed, many studies have demonstrated the crucial role of the DOR-mediated cyto-protection against H/I injury by several molecular pathways, including NLRP3 inflammasome modulated by miRNAs. In this review, we summarize our recent studies along with those of others worldwide, and compare the effects of DOR on H/I expression of miRNAs in H/I-sensitive and -insensitive organs. The alternation in miRNA expression profiles upon DOR activation and the potential impact on inflammatory injury in different organs under normoxic and hypoxic conditions are discussed at molecular and cellular levels. More in-depth investigations into this field may provide novel clues for new protective strategies against H/I inflammation in different types of organs.

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.

The Effects of Activation and Blockade of Delta Opioid Receptors on the Acquisition of Responding for an Opioid‐Associated Stimulus

Drug‐associated stimuli take on conditioned reinforcing properties that promote drug‐seeking and relapse. Previously, we have demonstrated that daily systemic injections of SNC80 (delta opioid receptor agonist) resulted in dose‐dependent increases in responding for the opioid‐associated stimulus. In the current experiment, we assessed the extent to which activating and/or blocking delta opioid receptors via a systemic injection of SNC80 or naltrindole, respectively, modified the conditioned reinforcing properties of an opioid‐associated stimulus. We hypothesized that a pre‐session injection of SNC80 would increase the conditioned reinforcing properties of the remifentanil‐associated stimulus; however, an injection of naltrindole would have no effect, and an injection of naltrindole would block the ability of SNC80 to enhance the conditioned reinforcing properties of the stimulus. First, we exposed rats to 20 i.v. infusions of remifentanil (3.2 ug/kg/infusion) and 20 stimulus presentations that were delivered response‐independently each day for 5 days. For the experimental group (Paired Conditioning), the remifentanil infusions and stimulus presentations were delivered concurrently. The control group (Random control) received the same number of infusions and stimulus presentations, but they were not explicitly paired. For the next 7 sessions (acquisition), we tested the extent to which the stimulus functioned as a conditioned reinforcer by allowing rats to freely respond via a nosepoke for presentations of the remifentanil‐associated stimulus only. On the fourth acquisition session, rats received one of the following combinations of injections: (a) vehicle + vehicle, (b) vehicle + naltrindole (3.2 mg/kg), (c) vehicle + SNC80 (3.2 mg/kg), or (d) SNC80 (3.2 mg/kg) + naltrindole (3.2 mg/kg). We found that rats in the Paired Pavlovian Conditioning group responded for the remifentanil‐associated stimulus significantly more than controls (p= 0.003, hp2= 0.15). Responding for remifentanil‐paired stimuli depended on treatment condition (p < 0.001, hp2= 0.35). Post‐hoc tests revealed that SNC80 administration increased responding for the remifentanil‐associated stimulus relative to all other treatment conditions (p’s < 0.001). These findings demonstrated that a remifentanil‐associated stimulus takes on conditioned reinforcing properties and that the conditioned reinforcing properties of the stimulus was augmented by delta opioid receptor activation and these effects were blocked via naltrindole. These findings support previous work that delta opioid receptors play a role in mediating conditioned reinforcing properties of drug‐associated stimuli.

Characterizing the influence of Toxoplasma infection on opioid receptor expression using human neuroblastoma cells (SKNSH)

BackgroundToxoplasma gondii is an obligate intracellular parasite that has permanently infected billions of people. The parasite can convert to a latent form that is found preferentially in the brain and heart. Recent serological studies have linked Toxoplasma infection to many brain disorders including schizophrenia, depression, substance abuse and Alzheimer’s. However, the molecular mechanism by which Toxoplasma influences these brain disorders remains unclear. Previous studies have shown that Toxoplasmaincreases dopamine synthesis, and dopamine is an important neurotransmitter in the reward pathway, where the opioid system has been shown to play a major role in modulating this pathway. We aim to further explore these mechanisms by studying the influence of Toxoplasmainfection on opioid receptor expression.ObjectivesThe goal of our project is to elucidate the influence of Toxoplasma gondiion brain physiology through studying the impact of Toxoplasma infection on opioid receptor expression. The aim of this study is to determine the effect of Toxoplasma infection on opioid receptor expression in neuroblastoma cells (SKNSH) in the presence or absence of morphine.MethodsTo evaluate the potency of morphine against Toxoplasma, a doubling assay was performed. Briefly, Human foreskin fibroblasts (HFF) were infected with Toxoplasma. Six‐hours post‐infection, the uninvaded parasites were removed and the media was replaced with media containing 10 µM morphine, 10 µM pyrimethamine, or DMSO. The flasks were fixed with ice‐cold methanol and the number of parasites per vacuole were enumerated at 24‐ and 48‐hours post‐treatment. To evaluate the expression of opioid receptors, the neuroblastoma cell SKNSH, which has been shown to express functional mu and delta opioid receptors, was infected with Toxoplasma type 1. Four hours post‐infection, uninvaded parasites were removed and the medium was replaced with media containing DMSO or 1 µM morphine or 10 µM morphine. Thirty‐two hours post‐treatment, the cells were harvested and protein was isolated. The expression of mu and delta opioid receptors was evaluated using western blot analysis.ResultsWe observed changes in the morphology of SKNSH upon infection with Toxoplasma type 1. The results from our doubling assay demonstrate the lack of potency for morphine against Toxoplasma type 1 at 24 hours and 48 hours post treatment with the highest morphine concentration we have been using for these experiments. Preliminary results suggest that the expression of the delta opioid receptor is downregulated in the presence of Toxoplasma, independent of morphine administration. In addition, the expression of delta opioid receptor is increased in the presence of morphine alone. When pyrimethamine was added to cells containing both Toxoplasma and morphine, delta receptor expression was comparable to its expression in the presence of morphine alone.ConclusionWe have confirmed the expression of muand delta opioid receptors in SKNSH, as reported previously. In addition, we were able to successfully infect this cell line with Toxoplasma. Additionally, we have found that morphine lacks potency against Toxoplasma, unlike previously reported. Preliminary data suggest that the collective the presence of Toxoplasma can negatively influence the expression of delta opioid receptors. Future experiments are being designed to elucidate the molecular mechanism of these findings, as well as observing similar findings in the animal model of Toxoplasmosis.

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.

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.

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.

δ2-Opioid Receptors as a Target in Designing New Cardioprotective Drugs: the Role of Protein Kinase C, AMPK, and Sarcolemmal KATP Channels

In rats anesthetized with α-chloralose, coronary artery occlusion (45 min) and reperfusion (120 min) were modeled. The selective δ2-opioid receptor agonist deltorphin II was administered 5 min before reperfusion. Protein kinase C inhibitor chelerythrine, AMP-activated protein kinase inhibitor compound C, and ATP-sensitive K+ channel blockers glibenclamide, 5-hydroxydecanoate, and HMR 1098 were administered 10 min before reperfusion. It was found that the infarct-limiting effect of deltorphin II is associated with activation of protein kinase C and opening of sarcolemmal ATP-sensitive K+ channel.

Pharmacological characterization of kratom alkaloids at opiate receptors: binding affinities, in vitro and in vivo functional assessments

Kratom (Mitragyna speciosa) is consumed by 10‐15 million individuals in the US for its mood‐elevating effects, as an alternative to traditional opiates for pain relief and to attenuate opiate withdrawal symptoms. Complex natural products, such as kratom, contain a variety of chemicals whose combined effects can only be understood by the meticulous identification and characterization of the individual chemicals and their effects in preclinical model systems. Twenty‐five indole and oxindole kratom alkaloids were evaluated for mu (MOR), delta (DOR) and kappa (KOR) opiate receptors binding affinities, opiate receptor mediated G‐protein activation, inhibition of forskolin stimulated cAMP accumulation and b‐arrestin2 recruitment. Several indole and oxindole alkaloids exhibited partial agonism at the MOR (7‐hydroxymitragynine, corynoxine A > epiallo‐isopaynantheine, isospeciofoline, speciociliatine, corynoxine B > mitragynine, 3‐epicorynoxine B > paynantheine and speciogynine) – all of which also exhibited strong G protein bias. Mitraciliatine and isopaynantheine were weak MOR antagonists. Speciophylline exhibited positive allosteric modulatory activity at the MOR as evidenced by increased affinity of DAMGO binding and potentiation of [met]‐enkephalin‐induced inhibition of forskolin stimulated cAMP activity in the absence of intrinsic activity. Corynoxine A induced displayed similar affinity at the mu opiate receptor as 7 hydroxymitragynine but was less potent in the nociceptive hot plate assay and the mechanical allodynia von Frey test. Corynoxine did not significantly alter respiration or heart rate or induce a conditioned place preference. Results demonstrate the pharmacological complexity of kratom alkaloids and have important implications for determining the combined effects of less abundant indole and oxindole alkaloids with regard to the therapeutic as well as potential harmful effects of kratom.

The Effect of the Central Administration of the Neuropeptide VF on Feed Intake and Its Possible Interactions with Glutamate and Opioid Systems in Broiler Chicken

Arginine-phenylalanine-amide (RFamide)-related peptides known as neuropeptide VF (NPVF) have a crucial role in feeding regulation. The present study purposed to determine the effect of intracerebroventricular (ICV) injection of the NPVF on dietary intake and possible interactions of NPVF with glutamate and opioid systems in broiler chicken. In experiment 1, chickens received ICV administration of the control solution, NPVF (16 nmol), 15 nmol of MK-801 (NMDA glutamate receptors antagonist), and injection of the NPVF with MK-801 together. In experiments, 2–8 chickens were injected intracerebroventricularly with 390 nmol of CNQX (antagonist of AMPA glutamate receptors), 2 nmol of AIDA (an antagonist of mGLUR1 glutamate receptors), 150 nmol of LY341495 (an antagonist of mGLUR2 glutamate receptors), 2 nmol of UBP1112 (an antagonist for mGLUR3 glutamate receptors), 5 µg of β-FNA (an antagonist for mu (µ) receptors), 5 µg of NTI (an antagonist for delta (δ) receptors), nor-BNI (kappa (κ) receptors antagonist; 5 µg) instead of MK-801. Following the injection, at 30, 60, and 120 min, the chick's dietary intake was recorded. As a result, NPVF (16 nmol) reduced feed intake in broilers (P < 0.05). NPVF + MK-801 co-injection reduced the hypophagic action of the NPVF (P < 0.05). Injection of NPVF + CNQX together reduced the anorectic action of NPVF (P < 0.05). Co-injection of NPVF + β-FNA reduced the effects of NPVF injection (P < 0.05). Thus, in newborn broilers, NPVF-induced anorexia is probably mediated by NMDA/ AMPA glutamate and µ opioid receptors.

Pharmacological evaluation and forensic case series of N-pyrrolidino etonitazene (etonitazepyne), a newly emerging 2-benzylbenzimidazole ‘nitazene’ synthetic opioid

Novel synthetic opioids continue to emerge on recreational drug markets worldwide. In response to legislative bans on fentanyl analogues, non-fentanyl structural templates, such as 2-benzylbenzimidazoles ('nitazenes'), are being exploited to create new μ-opioid receptor (MOR) agonists. Here, we pharmacologically characterize an emerging cyclic analogue of etonitazene, called N-pyrrolidino etonitazene (etonitazepyne), using in vitro and in vivo methods. A series of analytically confirmed fatalities is described to complement preclinical findings. Radioligandbinding assays in rat brain tissue revealed that N-pyrrolidino etonitazene has high affinity for MOR (Ki = 4.09nM) over δ-opioid (Ki = 959nM) and κ-opioid (Ki = 980nM) receptors. In a MOR-β-arrestin2 activation assay, N-pyrrolidino etonitazene displayed high potency (EC50 = 0.348nM), similar to etonitazene (EC50 = 0.360nM), and largely exceeding the potencies of fentanyl (EC50 = 14.9nM) and morphine (EC50 = 290nM). When administered s.c. to male Sprague Dawley rats, N-pyrrolidino etonitazene induced opioid-like antinociceptive, cataleptic, and thermic effects. Its potency in the hot plate test (ED50 = 0.0017mg/kg) was tenfold and 2,000-fold greater than fentanyl (ED50 = 0.0209mg/kg) and morphine (ED50 = 3.940mg/kg), respectively. Twenty-one overdose fatalities associated with N-pyrrolidino etonitazene were found to contain low blood concentrations of the drug (median = 2.2ng/mL), commonly in the context of polysubstance use. N-Pyrrolidino etonitazene was reported as a cause of death in at least two cases, demonstrating toxicity in humans. We demonstrate that N-pyrrolidino etonitazene is an extremely potent MOR agonist that is likely to present high risk to users. Continued vigilance is required to identify and characterize emergent 2-benzylbenzimidazoles, and other non-fentanyl opioids, as they appear in the marketplace.