Identification of 1,3,8-triazaspiro[4.5]decane-2,4-dione Derivatives as a Novel Delta Opioid Receptor-Selective Agonist Chemotype.

Delta opioid receptors hold potential as a target for neurological and psychiatric disorders, yet no delta opioid receptor agonist has proven efficacious in critical phase II clinical trials. The exact reasons for the failure to produce quality drug candidates for the delta opioid receptor is nuclear. However, it is known that certain delta opioid receptor agonists can induce seizures and exhibit tachyphylaxis. Several studies have suggested that those adverse effects are more prevalent in delta agonists that share the SNC80/BW373U86 chemotype. There is a need to find novel lead candidates for drug development that have improved pharmacological properties to differentiate them from the current failed delta agonists. Our objective in this study was to identify novel delta opioid receptor agonists. We used a beta-arrestin assay to screen a small GPCR-focused chemical library. We identified a novel chemotype of delta opioid receptor agonists, that appears to bind to the orthosteric site based of docking and molecular dynamic simulation. The most potent agonist hit compound is selective for the delta opioid receptor over a panel of 167 other GPCRs, is slightly biased towards G-protein signaling and has anti-allodynic efficacy in a complete Freund's adjuvant model of inflammatory pain in C57BL/6 male and female mice. The newly discovered chemotype contrasts with molecules like SNC80 that are highly efficacious beta-arrestin recruiters and may suggest this novel class of delta opioid receptor agonists could be expanded on to develop a clinical candidate drug. Significance Statement Delta opioid receptors are a clinical target for various neurological disorders, including migraine and chronic pain. Many of the clinically tested delta opioid agonists share a single chemotype, which carries risks during drug development. Through a small-scale high throughput screening assay, we identified a novel delta opioid receptor agonist chemotype, with anti-allodynic efficacy which may serve as alternative for the current clinical candidates.

Receptor expression and signaling properties in the brain, and structural ligand motifs that contribute to delta opioid receptor agonist-induced seizures.

The δ opioid receptor (δOR) is a therapeutic target for the treatment of various neurological disorders, such as migraines, chronic pain, alcohol use, and mood disorders. Relative to µ opioid receptor agonists, δOR agonists show lower abuse liability and may be potentially safer analgesic alternatives. However, currently no δOR agonists are approved for clinical use. A small number of δOR agonists reached Phase II trials, but ultimately failed to progress due to lack of efficacy. One side effect of δOR agonism that remains poorly understood is the ability of δOR agonists to produce seizures. The lack of a clear mechanism of action is partly driven by the fact that δOR agonists range in their propensity to induce seizure behavior, with multiple δOR agonists reportedly not causing seizures. There is a significant gap in our current understanding of why certain δOR agonists are more likely to induce seizures, and what signal-transduction pathway and/or brain area is engaged to produce these seizures. In this review we provide a comprehensive overview of the current state of knowledge of δOR agonist-mediated seizures. The review was structured to highlight which agonists produce seizures, which brain regions have been implicated and which signaling mediators have been examined in this behavior. Our hope is that this review will spur future studies that are carefully designed and aimed to solve the question why certain δOR agonists are seizurogenic. Obtaining such insight may expedite the development of novel δOR clinical candidates without the risk of inducing seizures. This article is part of the Special Issue on "Opioid-induced changes in addiction and pain circuits".

Exploration of beta-arrestin isoform signaling pathways in delta opioid receptor agonist-induced convulsions.

The δ-opioid receptor (δOR) has been considered as a therapeutic target in multiple neurological and neuropsychiatric disorders particularly as δOR agonists are deemed safer alternatives relative to the more abuse-liable µ-opioid receptor drugs. Clinical development of δOR agonists, however, has been challenging in part due to the seizure-inducing effects of certain δOR agonists. Especially agonists that resemble the δOR-selective agonist SNC80 have well-established convulsive activity. Close inspection suggests that many of those seizurogenic δOR agonists efficaciously recruit ß-arrestin, yet surprisingly, SNC80 displays enhanced seizure activity in ß-arrestin 1 knockout mice. This finding led us to hypothesize that perhaps ß-arrestin 1 is protective against, whereas ß-arrestin 2 is detrimental for δOR-agonist-induced seizures. To investigate our hypothesis, we characterized three different δOR agonists (SNC80, ADL5859, ARM390) in cellular assays and in vivo in wild-type and ß-arrestin 1 and ß-arrestin 2 knockout mice for seizure activity. We also investigated downstream kinases associated with ß-arrestin-dependent signal transduction. We discovered that δOR agonist-induced seizure activity strongly and positively correlates with ß-arrestin 2 efficacy for the agonist, but that indirect inhibition of ERK activation using the MEK inhibitor SL327 did not inhibit seizure potency and duration. Inhibition of the PI3K/AKT/mTOR signaling with honokiol but not PQR530, attenuated SNC80 seizure duration in ß-arrestin 1 knockout, but honokiol did not reduce SNC80-induced seizures in wild-type mice. Ultimately, our results indicate that ß-arrestin 2 is correlated with δOR agonist-induced seizure intensity, but that global ß-arrestin 1 knockout mice are a poor model system to investigate their mechanism of action.

Modulating ß-arrestin 2 recruitment at the δ- and µ-opioid receptors using peptidomimetic ligands.

µ-Opioid receptor agonists provide potent and effective acute analgesia; however, their therapeutic window narrows considerably upon repeated administration, such as required for treating chronic pain. In contrast, bifunctional µ/δ opioid agonists, such as the endogenous enkephalins, have potential for treating both acute and chronic pain. However, enkephalins recruit ß-arrestins, which correlate with certain adverse effects at µ- and δ-opioid receptors. Herein, we identify the C-terminus of Tyr-ψ[(Z)CF[double bond, length as m-dash]CH]-Gly-Leu-enkephalin, a stable enkephalin derivative, as a key site to regulate bias of both δ- and µ-opioid receptors. Using in vitro assays, substitution of the Leu5 carboxylate with amides (NHEt, NMe2, NCyPr) reduced ß-arrestin recruitment efficacy through both the δ-opioid and µ-opioid, while retaining affinity and cAMP potency. For this series, computational studies suggest key ligand-receptor interactions that might influence bias. These findings should enable the discovery of a range of tool compounds with previously unexplored biased µ/δ opioid agonist pharmacological profiles.

Evaluation of Kratom Opioid Derivatives as Potential Treatment Option for Alcohol Use Disorder.

Background and Purpose: Mitragyna speciosa extract and kratom alkaloids decrease alcohol consumption in mice at least in part through actions at the δ-opioid receptor (δOR). However, the most potent opioidergic kratom alkaloid, 7-hydroxymitragynine, exhibits rewarding properties and hyperlocomotion presumably due to preferred affinity for the mu opioid receptor (µOR). We hypothesized that opioidergic kratom alkaloids like paynantheine and speciogynine with reduced µOR potency could provide a starting point for developing opioids with an improved therapeutic window to treat alcohol use disorder. Experimental Approach: We characterized paynantheine, speciociliatine, and four novel kratom-derived analogs for their ability to bind and activate δOR, µOR, and κOR. Select opioids were assessed in behavioral assays in male C57BL/6N WT and δOR knockout mice. Key Results: Paynantheine (10 mg∙kg-1, i.p.) produced aversion in a limited conditioned place preference (CPP) paradigm but did not produce CPP with additional conditioning sessions. Paynantheine did not produce robust antinociception but did block morphine-induced antinociception and hyperlocomotion. Yet, at 10 and 30 mg∙kg-1 doses (i.p.), paynantheine did not counteract morphine CPP. 7-hydroxypaynantheine and 7-hydroxyspeciogynine displayed potency at δOR but limited µOR potency relative to 7-hydroxymitragynine in vitro, and dose-dependently decreased voluntary alcohol consumption in WT but not δOR in KO mice. 7-hydroxyspeciogynine has a maximally tolerated dose of at least 10 mg∙kg-1 (s.c.) at which it did not produce significant CPP neither alter general locomotion nor induce noticeable seizures. Conclusion and Implications: Derivatizing kratom alkaloids with the goal of enhancing δOR potency and reducing off-target effects could provide a pathway to develop novel lead compounds to treat alcohol use disorder with an improved therapeutic window.

Engineering Quick- and Long-acting Naloxone Delivery Systems for Treating Opioid Overdose.

PURPOSE: Opioids have been the main factor for drug overdose deaths in the United States. Current naloxone delivery systems are effective in mitigating the opioid effects only for hours. Naloxone-loaded poly(lactide-co-glycolide) (PLGA) microparticles were prepared as quick- and long-acting naloxone delivery systems to extend the naloxone effect as an opioid antidote. METHODS: The naloxone-PLGA microparticles were made using an emulsification solvent extraction approach with different formulation and processing parameters. Two PLGA polymers with the lactide:glycolide (L:G) ratios of 50:50 and 75:25 were used, and the drug loading was varied from 21% to 51%. Two different microparticles of different sizes with the average diameters of 23 µm and 50 µm were produced using two homogenization-sieving conditions. All the microparticles were critically characterized, and three of them were evaluated with ß-arrestin recruitment assays. RESULTS: The naloxone encapsulation efficiency (EE) was in the range of 70-85%. The EE was enhanced when the theoretical naloxone loading was increased from 30% to 60%, the L:G ratio was changed from 50:50 to 75:25, and the average size of the particles was reduced from 50 µm to 23 µm. The in vitro naloxone release duration ranged from 4 to 35 days. Reducing the average size of the microparticles from 50 µm to 23 µm helped eliminate the lag phase and obtain the steady-state drug release profile. The cellular pharmacodynamics of three selected formulations were evaluated by applying DAMGO, a synthetic opioid peptide agonist to a µ-opioid receptor, to recruit ß-arrestin 2. CONCLUSIONS: Naloxone released from the three selected formulations could inhibit DAMGO-induced ß-arrestin 2 recruitment. This indicates that the proposed naloxone delivery system is adequate for opioid reversal during the naloxone release duration.

The Meta-Position of Phe4 in Leu-Enkephalin Regulates Potency, Selectivity, Functional Activity, and Signaling Bias at the Delta and Mu Opioid Receptors.

As tool compounds to study cardiac ischemia, the endogenous δ-opioid receptors (δOR) agonist Leu5-enkephalin and the more metabolically stable synthetic peptide (d-Ala2, d-Leu5)-enkephalin are frequently employed. However, both peptides have similar pharmacological profiles that restrict detailed investigation of the cellular mechanism of the δOR's protective role during ischemic events. Thus, a need remains for δOR peptides with improved selectivity and unique signaling properties for investigating the specific roles for δOR signaling in cardiac ischemia. To this end, we explored substitution at the Phe4 position of Leu5-enkephalin for its ability to modulate receptor function and selectivity. Peptides were assessed for their affinity to bind to δORs and µ-opioid receptors (µORs) and potency to inhibit cAMP signaling and to recruit ß-arrestin 2. Additionally, peptide stability was measured in rat plasma. Substitution of the meta-position of Phe4 of Leu5-enkephalin provided high-affinity ligands with varying levels of selectivity and bias at both the δOR and µOR and improved peptide stability, while substitution with picoline derivatives produced lower-affinity ligands with G protein biases at both receptors. Overall, these favorable substitutions at the meta-position of Phe4 may be combined with other modifications to Leu5-enkephalin to deliver improved agonists with finely tuned potency, selectivity, bias and drug-like properties.

Repeated Use of the Psychoactive Substance Ethylphenidate Impacts Neurochemistry and Reward Learning in Adolescent Male and Female Mice.

Schedule II prescription psychostimulants, such as methylphenidate (MPH), can be misused as nootropic drugs, i.e., drugs that enhance focus and cognition. When users are unable to obtain these prescribed medications, they may seek out novel psychoactive substances (NPSs) that are not yet scheduled. An example of a NPS reportedly being abused is ethylphenidate (EPH), a close analog of MPH but with a higher preference for the dopamine transporter compared with the norepinephrine transporter. Therefore, based upon this pharmacological profile and user self-reports, we hypothesized that repeated EPH exposure in adolescent mice may be rewarding and alter cognition. Here, we report that repeated exposure to 15 mg/kg EPH decreased spatial cognitive performance as assessed by the Barnes maze spatial learning task in adolescent male C57Bl/6 mice; however, male mice did not show alterations in the expression of mature BDNF - a protein associated with increased cognitive function - in key brain regions. Acute EPH exposure induced hyperlocomotion at a high dose (15 mg/kg, i.p.), but not a low dose (5 mg/kg, i.p.). Interestingly, mice exhibited significant conditioned place preference at the low EPH dose, suggesting that even non-stimulating doses of EPH are rewarding. In both males and females, repeated EPH exposure increased expression of deltaFosB - a marker associated with increased risk of drug abuse - in the dorsal striatum, nucleus accumbens, and prefrontal cortex. Overall, our results suggest that repeated EPH use in adolescence is psychostimulatory, rewarding, increases crucial brain markers of reward-related behaviors, and may negatively impact spatial performance.