Structure-Activity Relationships of the Fentanyl Scaffold: Identification of Antagonists as Potential Opioid Overdose Reversal Agents.

Opioid-related overdoses account for almost half of all drug overdose deaths in the United States and cause more preventable deaths every year than car crashes. Fentanyl, a highly potent mu opioid receptor (MOR) agonist and its analogues (fentalogues) are increasingly found in illicit drug samples, both where the primary drug of abuse is an opioid and where it is not. The prevalence of fentalogues in the illicit drug market is thought to be the primary driver of the increased number of opioid-related overdose deaths since 2016. In fact, fentanyl and its analogues are involved in more than 70% of opioid-related overdoses. The standard opioid overdose rescue therapy naloxone is often insufficient to reverse opioid overdoses caused by fentalogue agonists under current treatment paradigms. However, the pharmacology of many fentalogues is unknown. Moreover, within the fentalogue series of compounds, it is possible that antagonists could be identified that might be superior to naloxone as opioid overdose reversal agents. In this report, we explore the pharmacology of 70 fentalogues and identify compounds that behave as MOR antagonists in vitro and demonstrate with one of these reversals of fentanyl-induced respiratory depression in the mouse. Such compounds could provide leads for the development of effective agents for the reversal of opioid overdose.

PACAP and acetylcholine cause distinct Ca2+ signals and secretory responses in chromaffin cells.

The adrenomedullary chromaffin cell transduces chemical messages into outputs that regulate end organ function throughout the periphery. At least two important neurotransmitters are released by innervating preganglionic neurons to stimulate exocytosis in the chromaffin cell-acetylcholine (ACh) and pituitary adenylate cyclase activating polypeptide (PACAP). Although PACAP is widely acknowledged as an important secretagogue in this system, the pathway coupling PACAP stimulation to chromaffin cell secretion is poorly understood. The goal of this study is to address this knowledge gap. Here, it is shown that PACAP activates a Gαs-coupled pathway that must signal through phospholipase C ε (PLCε) to drive Ca2+ entry and exocytosis. PACAP stimulation causes a complex pattern of Ca2+ signals in chromaffin cells, leading to a sustained secretory response that is kinetically distinct from the form stimulated by ACh. Exocytosis caused by PACAP is associated with slower release of peptide cargo than exocytosis stimulated by ACh. Importantly, only the secretory response to PACAP, not ACh, is eliminated in cells lacking PLCε expression. The data show that ACh and PACAP, acting through distinct signaling pathways, enable nuanced and variable secretory outputs from chromaffin cells.

Delta Opioid Receptor-Mediated Antidepressant-Like Effects of Diprenorphine in Mice.

Major depressive disorder is a highly common disorder, with a lifetime prevalence in the United States of approximately 21%. Traditional antidepressant treatments are limited by a delayed onset of action and minimal efficacy in some patients. Ketamine is effective and fast-acting, but there are concerns over its abuse liability. Thus, there is a need for safe, fast-acting antidepressant drugs. The opioid buprenorphine shows promise but also has abuse liability due to its mu-agonist component. Preclinical evidence indicates that the delta-opioid system contributes to mood disorders, and delta-opioid agonists are effective in preclinical models of depression- and anxiety-like states. In this study, we test the hypothesis that the mu-opioid antagonist diprenorphine by virtue of its partial delta opioid agonist activity may offer a beneficial profile for an antidepressant medication without abuse liability. Diprenorphine was confirmed to bind with high affinity to all three opioid receptors, and functional experiments for G protein activation verified diprenorphine to be a partial agonist at delta- and kappa-opioid receptors and a mu-antagonist. Studies in C57BL/6 mice demonstrated that an acute dose of diprenorphine produced antidepressant-like effects in the tail suspension test and the novelty-induced hypophagia test that were inhibited in the presence of the delta-selective antagonist, naltrindole. Diprenorphine did not produce convulsions, a side effect of many delta agonists but rather inhibited convulsions caused by the full delta agonist SNC80; however, diprenorphine did potentiate pentylenetetrazole-induced convulsions. Diprenorphine, and compounds with a similar pharmacological profile, may provide efficient and safe rapidly acting antidepressants. SIGNIFICANCE STATEMENT: The management of major depressive disorder, particularly treatment-resistant depression, is a significant unmet medical need. Here we show that the opioid diprenorphine, a compound with mu-opioid receptor antagonist activity and delta- and kappa-opioid receptor partial agonist activities, has rapid onset antidepressant-like activity in animal models. Diprenorphine and compounds with a similar pharmacological profile to diprenorphine should be explored as novel antidepressant drugs.

The Buprenorphine Analogue BU10119 Attenuates Drug-Primed and Stress-Induced Cocaine Reinstatement in Mice.

There are no Food and Drug Administration-approved medications for cocaine use disorder, including relapse. The µ-opioid receptor (MOPr) partial agonist buprenorphine alone or in combination with naltrexone has been shown to reduce cocaine-positive urine tests and cocaine seeking in rodents. However, there are concerns over the abuse liability of buprenorphine. Buprenorphine's partial agonist and antagonist activity at the nociception receptor (NOPr) and κ-opioid receptor (KOPr), respectively, may contribute to its ability to inhibit cocaine seeking. Thus, we hypothesized that a buprenorphine derivative that exhibits antagonist activity at MOPr and KOPr with enhanced agonist activity at the NOPr could provide a more effective treatment. Here we compare the pharmacology of buprenorphine and two analogs, BU10119 and BU12004, in assays for antinociception and for cocaine- and stress-primed reinstatement in the conditioned place preference paradigm. In vitro and in vivo assays showed that BU10119 acts as an antagonist at MOPr, KOPr, and δ-opioid receptor (DOPr) and a partial agonist at NOPr, whereas BU12004 showed MOPr partial agonist activity and DOPr, KOPr, and NOPr antagonism. BU10119 and buprenorphine but not BU12004 lessened cocaine-primed reinstatement. In contrast, BU10119, BU12004, and buprenorphine blocked stress-primed reinstatement. The selective NOPr agonist SCH221510 but not naloxone decreased cocaine-primed reinstatement. Together, these findings are consistent with the concept that NOPr agonism contributes to the ability of BU10119 and buprenorphine to attenuate reinstatement of cocaine-conditioned place preference in mice. The findings support the development of buprenorphine analogs lacking MOPr agonism with increased NOPr agonism for relapse prevention to cocaine addiction. SIGNIFICANCE STATEMENT: There are no Food and Drug Administration-approved medications for cocaine use disorder. Buprenorphine has shown promise as a treatment for cocaine relapse prevention; however, there are concerns over the abuse liability of buprenorphine. Here we show a buprenorphine analogue, BU10119, which lacks µ-opioid receptor agonism and inhibits cocaine-primed and stress-primed reinstatement in a conditioned place-preference paradigm. The results suggest the development of BU10119 for the management of relapse to cocaine seeking.

In vivo modeling of docosahexaenoic acid and eicosapentaenoic acid-mediated inhibition of both platelet function and accumulation in arterial thrombi.

Omega-3 polyunsaturated fatty acids (n-3 PUFAs) are associated with a variety of cellular alterations that mitigate cardiovascular disease. However, pinpointing the positive therapeutic effects is challenging due to inconsistent clinical trial results and overly simplistic in vitro studies. Here we aimed to develop realistic models of n-3 PUFA effects on platelet function so that preclinical results can better align with and predict clinical outcomes. Human platelets incubated with the n-3 PUFAs docosahexaenoic acid and eicosapentaenoic acid were stimulated with agonist combinations mirroring distinct regions of a growing thrombus. Platelet responses were then monitored in a number of ex-vivo functional assays. Furthermore, intravital microscopy was used to monitor arterial thrombosis and fibrin deposition in mice fed an n-3 PUFA-enriched diet. We found that n-3 PUFA treatment had minimal effects on many basic ex-vivo measures of platelet function using agonist combinations. However, n-3 PUFA treatment delayed platelet-derived thrombin generation in both humans and mice. This impaired thrombin production paralleled a reduced platelet accumulation within thrombi formed in either small arterioles or larger arteries of mice fed an n-3 PUFA-enriched diet, without impacting P-selectin exposure. Despite an apparent lack of robust effects in many ex-vivo assays of platelet function, increased exposure to n-3 PUFAs reduces platelet-mediated thrombin generation and attenuates elements of thrombus formation. These data support the cardioprotective value of-3 PUFAs and strongly suggest that they modify elements of platelet function in vivo.

Dissection of complex adult traits in a mouse synthetic population.

Finding the causative genetic variations that underlie complex adult traits is a significant experimental challenge. The unbiased search strategy of genome-wide association (GWAS) has been used extensively in recent human population studies. These efforts, however, typically find only a minor fraction of the genetic loci that are predicted to affect variation. As an experimental model for the analysis of adult polygenic traits, we measured a mouse population for multiple phenotypes and conducted a genome-wide search for effector loci. Complex adult phenotypes, related to body size and bone structure, were measured as component phenotypes, and each subphenotype was associated with a genomic spectrum of candidate effector loci. The strategy successfully detected several loci for the phenotypes, at genome-wide significance, using a single, modest-sized population (N = 505). The effector loci each explain 2%-10% of the measured trait variation and, taken together, the loci can account for over 25% of a trait's total population variation. A replicate population (N = 378) was used to confirm initially observed loci for one trait (femur length), and, when the two groups were merged, the combined population demonstrated increased power to detect loci. In contrast to human population studies, our mouse genome-wide searches find loci that individually explain a larger fraction of the observed variation. Also, the additive effects of our detected mouse loci more closely match the predicted genetic component of variation. The genetic loci discovered are logical candidates for components of the genetic networks having evolutionary conservation with human biology.