Rapid-Onset Anti-Stress Effects of a Kappa-Opioid Receptor Antagonist, LY2795050, against Immobility in an Open Space Swim Paradigm in Male and Female Mice

The kappa-opioid receptor (KOR) / dynorphin system is implicated with behavioral and neurobiological effects of stress exposure (including heavy exposure to drugs of abuse) in translational animal models. Thus some KOR-antagonists can decrease the aversive, depressant-like and anxiety-like effects caused by stress exposure. The first generation of selective KOR-antagonists have slow onsets (hours) and extremely long durations of action (days-weeks), in vivo. A new generation of KOR antagonists with rapid onset and shorter duration of action can potentially decrease the effects of stress exposure in translational models, and may be of interest for medication development. This study examined the rapid onset anti-stress effects of one of the shorter acting novel KOR-antagonists (LY2795050, (3-chloro-4-(4-(((2S)-2-pyridin-3-ylpyrrolidin-1-yl)methyl) phenoxy)benzamide)) in a single-session open space swim (OSS) stress paradigm (15 min duration), in adult male and female C57BL/6 J mice. LY2795050 (0.32 mg/kg, i.p.) had rapid onset (within 15 min) and short duration (\u3c 3 h) of KOR-antagonist effects, based on its blockade of the locomotor depressant effects of the KOR-agonist U50,488 (10 mg/kg). LY2795050 (0.32 mg/kg), when administered only 1 min prior to the OSS stress paradigm, decreased immobility in males, but not females. With a slightly longer pretreatment time (15 min), this dose of LY2795050 decreased immobility in both males and females. A 10-fold smaller dose of LY2795050 (0.032 mg/kg) was inactive in the OSS, showing dose-dependence of this anti-stress effect. Overall, these studies show that a novel KOR-antagonist can produce very rapid onset anti-immobility effects in this model of acute stress exposure

The discriminative effects of the κ-opioid hallucinogen salvinorin A in nonhuman primates: dissociation from classic hallucinogen effects

RATIONALE: The widely available hallucinogen salvinorin A is a unique example of a plant-derived compound selective for κ-opioid receptors and may produce effects distinct from those of other compounds with classic hallucinogenic or dissociative properties which are also abused in humans. OBJECTIVES: The objective of this study is to characterize the salvinorin A discriminative cue in nonhuman primates with high κ-receptor genetic homology to humans. METHODS: Adult rhesus monkeys (n=3) were trained to discriminate salvinorin A (0.015 mg/kg, s.c.) from vehicle, in a food-reinforced operant discrimination assay. Parallel studies, using unconditioned behavioral endpoints (facial relaxation and ptosis) also evaluated the κ-opioid receptor mediation of salvinorin A in vivo function. RESULTS: Monkeys trained to discriminate salvinorin A generalized structurally diverse, centrally penetrating κ-agonists (bremazocine, U69,593, and U50,488). By contrast, μ- and δ-opioid agonists (fentanyl and SNC80, respectively) were not generalized, nor were the serotonergic 5HT2 hallucinogen psilocybin or the dissociative N-methyl-D-aspartic acid antagonist, ketamine. The discriminative effects of salvinorin A were blocked by the opioid antagonist quadazocine (0.32 mg/kg), but not by the 5HT2 antagonist ketanserin (0.1 mg/kg). Consistent with these findings, salvinorin and κ-agonists (e.g., U69,593) produce effects in the unconditioned endpoints (e.g., ptosis), whereas psilocybin was inactive. Conclusions: These findings support the conclusion that the interoceptive/discriminative cue produced by salvinorin A is mediated by agonism at κ-receptors and is mechanistically distinct from that produced by a classic serotonergic hallucinogen

The Effects of Herkinorin, the First mu-Selective Ligand from a Salvinorin A-Derived Scaffold, in a Neuroendocrine Biomarker Assay in Nonhuman Primates

Herkinorin is the first μ-opioid receptor-selective ligand from the salvinorin A diterpenoid scaffold. Herkinorin has relative μ \u3e κ \u3e δ binding selectivity, and it can act as an agonist at both μ- and κ-receptors, in vitro. These studies were the first in vivo evaluation of the effects of herkinorin in nonhuman primates, using prolactin release, a neuroendocrine biomarker assay that is responsive to both μ- and κ-agonists, as well as to compounds with limited ability to cross the blood-brain barrier. In cumulative dosing studies (0.01–0.32 mg/kg i.v.), herkinorin produced only small effects in gonadally intact males (n = 4), but a more robust effect in females (n = 4). Time course studies with herkinorin (0.32 mg/kg) confirmed this greater effectiveness in females and revealed a fast onset after i.v. administration (e.g., by 5–15 min). Antagonism experiments with different doses of nalmefene (0.01 and 0.1 mg/kg) caused dose-dependent and complete prevention of the effect of herkinorin in females. This is consistent with a principal μ-agonist effect of herkinorin, with likely partial contribution by κ-agonist effects. The peripherally selective antagonist quaternary naltrexone (1 mg/kg s.c.) caused approximately 70% reduction in the peak effect of herkinorin (0.32 mg/kg) in females, indicating that this effect of herkinorin is prominently mediated outside the blood-brain barrier

Clocinnamox antagonism of opioid suppression of schedule-controlled responding in rhesus monkeys

The antagonist effects of clocinnamox were evaluated against opioid agonists, acting at μ, κ and ∂-receptors, in rhesus monkeys ( n =3–4) responding under a fixed-ratio 30 (FR 30) schedule for food delivery. Clocinnamox (0.032–0.1 mg/kg) dose-dependently antagonized fentanyl (0.001–0.32 mg/kg) after either a 3-h or 1-day pretreatment; there was substantial recovery of agonist potency by 1 week after clocinnamox. Etonitazene (0.0001–0.01 mg/kg) was also antagonized by clocinnamox (0.1 mg/kg), but to a lesser extent than fentanyl. The smaller extent of antagonism was not due to the appearance of non μ-opioid response-decreasing effects of etonitazene, since the competitive antagonist quadazocine (0.1 mg/kg) shifted the etonitazene dose-effect curve in the presence of clocinnamox (0.1 mg/kg). Clocinnamox (0.1–0.32 mg/kg) did not antagonize the rate-suppressing effects of the ∂-agonist BW373U86 (0.0.01-1.0 mg/kg) or the κ-agonist U69,593 (0.001–0.032 mg/kg). These results are consistent with previous in vivo and in vitro evidence that characterized clocinnamox as an insurmountable antagonist, with selectivity for μ-over κ- and δ-receptors.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/46352/1/213_2005_Article_BF02246641.pd

LC-MS/MS quantification of salvinorin A from biological fluids

A facile method for quantifying the concentration of the powerful and widely available hallucinogen salvinorin A (a selective kappa opioid agonist) from non-human primate cerebrospinal fluid (CSF) and human plasma has been developed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in positive electrospray ionization (ESI) mode. With CSF solid phase extraction can be avoided completely by simply diluting each sample to 10 % (v/v) acetonitrile, 1 % (v/v) formic acid and injecting under high aqueous conditions for analyte focusing. Extensive plasma sample preparation was investigated including protein precipitation, SPE column selection, and plasma particulate removal. Human plasma samples were centrifuged at 21,000 × gravity for 4 minutes to obtain clear particulate-free plasma, from which 300 μl was spiked with internal standard and loaded onto a C18 SPE column with a 100 mg mL−1 loading capacity. Guard columns (C18, hand packed 1 mm × 20 mm) were exchanged after backpressure increased above 4600psi, about 250 injections. A shallow acetonitrile/water gradient was used, 29 to 33% CH3CN over 8 minutes to elute salvinorin A. Reduction of chemical noise was achieved using tandem mass spectrometry with multiple reaction monitoring while sensitivity increases were observed using a 50 μL injection volume onto a small bore analytical column (C18, 1 mm ID × 50 mm) thus increasing peak concentration. Limits of quantification were found to be 0.0125 ng mL−1 (CSF) and 0.05 ng mL−1 (plasma) with interday precision and accuracy below 1.7 % and 9.42 % (CSF) and 3.47 % and 12.37 % (plasma) respectively. This method was used to determine the concentration of salvinorin A from an in vivo Rhesus monkey study and a trial of healthy human research participants, using behaviorally active doses

Design, Synthesis, and Preliminary Evaluation of a Potential Synthetic Opioid Rescue Agent

BACKGROUND: One of the most prominent opioid analgesics in the United States is the high potency agonist fentanyl. It is used in the treatment of acute and chronic pain and as an anesthetic adjuvant. When used inappropriately, however, ingestion of just a few milligrams of fentanyl or other synthetic opioid can cause opioid-induced respiratory depression (OIRD), often leading to death. Currently, the treatment of choice for OIRD is the opioid receptor antagonist naloxone. Recent reports, however, suggest that higher doses or repeated dosing of naloxone (due to recurrence of respiratory depression) may be required to reverse fully fentanyl-induced respiratory depression, rendering this treatment inadequate. To combat this synthetic opioid overdose crisis, this research aims at identifying a novel opioid reversal agent with enhanced efficacy towards fentanyl and other synthetic opioids. METHODS: A series of naltrexone analogues were characterized for their ability to antagonize the effects of fentanyl in vitro utilizing a modified forskolin-induced cAMP accumulation assay. Lead analogue 29 was chosen to undergo further PK studies, followed by in vivo pharmacological analysis to determine its ability to antagonize opioid-induced antinociception in the hot plate assay. RESULTS: A series of potent MOR antagonists were identified, including the highly potent analogue 29 (IC50 = 2.06 nM). Follow-up PK studies revealed 29 to possess near 100% bioavailability following IP administration. Brain concentrations of 29 surpassed plasma concentrations, with an apparent terminal half-life of ~ 80 min in mice. In the hot plate assay, 29 dose-dependently (0.01–0.1 mg/kg; IP) and fully antagonized the antinociception induced by oxycodone (5.6 mg/kg; IP). Furthermore, the dose of 29 that is fully effective in preventing oxycodone-induced antinociception (0.1 mg/kg) was ineffective against locomotor deficits caused by the KOR agonist U50,488. CONCLUSIONS: Methods have been developed that have utility to identify enhanced rescue agents for the treatment of OIRD. Analogue 29, possessing potent MOR antagonist activity in vitro and in vivo, provides a promising lead in our search for an enhanced synthetic opioid rescue agent

Semisynthetic Neoclerodanes as Kappa Opioid Receptor Probes

Modification of the furan ring of salvinorin A (1), the main active component of Salvia divinorum, has resulted in novel neoclerodane diterpenes with opioid receptor affinity and activity. Conversion of the furan ring to an aldehyde at the C-12 position (5) has allowed for the synthesis of analogues with new carbon-carbon bonds at that position. Previous methods for forming these bonds, such as Grignard and Stille conditions, have met with limited success. We report a palladium catalyzed Liebeskind-Srogl cross-coupling reaction of a thioester and a boronic acid that occurs at neutral pH and ambient temperature to produce ketone analogs at C-12. To the best of our knowledge, this is the first reported usage of the Liebeskind-Srogl reaction to diversify a natural product scaffold. We also describe a one-step protocol for the conversion of 1 to 12-epi-1 (3) through microwave irradiation. Previously, this synthetically challenging process has required multiple steps. Additionally, we report in this study that alkene 9 and aromatic analogues 12, 19, 23, 25, and 26 were discovered to retain affinity and selectivity at kappa opioid receptors (KOP). Finally, we report that the furan-2-yl analog of 1 (31) has similar affinity to 1. Collectively, these findings suggest that different aromatic groups appended directly to the decalin core may be well tolerated by KOP receptors, and may generate further ligands with affinity and activity at KOP receptors

Oral Ethanol-Reinforced Responding in Rhesus Monkeys: Effects of Opioid Antagonists Selective for the Μ-,Κ-, or Δ-Receptor

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65904/1/j.1530-0277.1998.tb03960.x.pd