2012 David W. Robertson Award for Excellence in Medicinal Chemistry: Neoclerodanes as Atypical Opioid Receptor Ligands

The neoclerodane diterpene salvinorin A is the major active component of the hallucinogenic mint plant Salvia divinorum Epling & Játiva (Lamiaceae). Since the finding that salvinorin A exerts its potent psychotropic actions through the activation of opioid receptors, the site of action of morphine and related analogues, there has been much interest in elucidating the underlying mechanisms behind its effects. These effects are particularly remarkable, because (1) salvinorin A is the first reported non-nitrogenous opioid receptor agonist, and (2) its effects are not mediated through the previously investigated targets of psychotomimetics. This perspective outlines our research program, illustrating a new direction to the development of tools to further elucidate the biological mechanisms of drug tolerance and dependence. The information gained from these efforts is expected to facilitate the design of novel agents to treat pain, drug abuse, and other CNS disorders

Chemical Methods for the Synthesis and Modification of Neoclerodane Diterpenes

Diterpenes are a structural class of molecules that are derived from four isoprene subunits and are widespread throughout nature. A number of neoclerodane diterpenes have been found to have biological activity but a limited number of chemical investigations have been conducted. Recently, the neoclerodane diterpene, salvinorin A (12) has been investigated due to its unique pharmacological profile. This review will discuss the chemical methods used to chemically modify and synthesize 12

Mitochondrial Targeted Coenzyme Q, Superoxide, and Fuel Selectivity in Endothelial Cells

Background Previously, we reported that the “antioxidant” compound “mitoQ” (mitochondrial-targeted ubiquinol/ubiquinone) actually increased superoxide production by bovine aortic endothelial (BAE) cell mitochondria incubated with complex I but not complex II substrates. Methods and Results To further define the site of action of the targeted coenzyme Q compound, we extended these studies to include different substrate and inhibitor conditions. In addition, we assessed the effects of mitoquinone on mitochondrial respiration, measured respiration and mitochondrial membrane potential in intact cells, and tested the intriguing hypothesis that mitoquinone might impart fuel selectivity in intact BAE cells. In mitochondria respiring on differing concentrations of complex I substrates, mitoquinone and rotenone had interactive effects on ROS consistent with redox cycling at multiple sites within complex I. Mitoquinone increased respiration in isolated mitochondria respiring on complex I but not complex II substrates. Mitoquinone also increased oxygen consumption by intact BAE cells. Moreover, when added to intact cells at 50 to 1000 nM, mitoquinone increased glucose oxidation and reduced fat oxidation, at doses that did not alter membrane potential or induce cell toxicity. Although high dose mitoquinone reduced mitochondrial membrane potential, the positively charged mitochondrial-targeted cation, decyltriphenylphosphonium (mitoquinone without the coenzyme Q moiety), decreased membrane potential more than mitoquinone, but did not alter fuel selectivity. Therefore, non-specific effects of the positive charge were not responsible and the quinone moiety is required for altered nutrient selectivity. Conclusions In summary, the interactive effects of mitoquinone and rotenone are consistent with redox cycling at more than one site within complex I. In addition, mitoquinone has substrate dependent effects on mitochondrial respiration, increases repiration by intact cells, and alters fuel selectivity favoring glucose over fatty acid oxidation at the intact cell level.This work was supported by Veterans Affairs Medical Research Funds and grant DK25295 from the National Institutes of Health

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

Reinstatement of methamphetamine seeking in male and female rats treated with modafinil and allopregnanolone

Background Sex differences in methamphetamine (METH) use (females>males) have been demonstrated in clinical and preclinical studies. This experiment investigated the effect of sex on the reinstatement of METH-seeking behavior in rats and to determine whether pharmacological interventions for METH-seeking behavior vary by sex. Treatment drugs were modafinil (MOD), an analeptic, and allopregnanolone (ALLO), a neuroactive steroid and progesterone metabolite. Method Male and female rats were trained to self-administer i.v. infusions of METH (0.05mg/kg/infusion). Next, rats self-administered METH for a 10-day maintenance period. METH was then replaced with saline, and rats extinguished lever-pressing behavior over 18 days. A multi-component reinstatement procedure followed where priming injections of METH (1 mg/kg) were administered at the start of each daily session, preceded 30 min by MOD (128 mg/kg, i.p.), ALLO (15 mg/kg, s.c.), or vehicle treatment. MOD was also administered at the onset of the session to determine if it would induce the reinstatement of METH-seeking behavior. Results Female rats had greater METH-induced reinstatement responding compared to male rats following control treatment injections. MOD (compared to the DMSO control) attenuated METH-seeking behavior in male and female rats; however, ALLO only reduced METH-primed responding in females. MOD alone did not induce the reinstatement of METH-seeking behavior. Conclusions These results support previous findings that females are more susceptible to stimulant abuse compared to males and ALLO effectively reduced METH-primed reinstatement in females. Further, they illustrate the utility of MOD as a potential agent for prevention of relapse to METH use in both males and females

Effect of kappa-opioid receptor agonists U69593, U50488H, spiradoline and salvinorin A on cocaine-induced drug-seeking in rats

Our previous work indicated that pretreatment with the selective kappa opioid receptor (KOPr) agonist, U69593, attenuated the ability of priming injections of cocaine to reinstate extinguished cocaine-seeking behavior. The present study expanded these initial tests to include other traditional KOPr agonists, U50488H, spiradoline (SPR), and salvinorin A (Sal A), an active constituent of the plant Salvia divinorum. Following acquisition and stabilization of cocaine self-administration, cocaine-produced drug-seeking was measured. This test was conducted in a single day and comprised an initial phase of self-administration, followed by a phase of extinguished responding. The final phase examined reinstatement of extinguished cocaine self-administration followed by a priming injection of cocaine (20.0 mg/kg, intraperitoneal (I.P.)) in combination with the various KOPr agonists. Cocaine-induced drug-seeking was attenuated by pretreatment with U69593 (0.3 mg/kg, subcutaneous (S.C.)), U50488H (30.0 mg/kg, I.P.), SPR (1.0, 3.0 mg/kg, I.P.) and Sal A (0.3, 1.0 mg/kg, I.P.). Sal A (0.3, 1.0 mg/kg, I.P.) had no effect on operant responding to obtain sucrose reinforcement or on cocaine induced hyperactivity. These findings show that Sal A, like other traditional KOPr agonists attenuates cocaine-induced drug seeking behavior

Single injection of novel kappa opioid receptor agonist salvinorin A attenuates expression of cocaine induced behavioral sensitization in rats

Kappa opioid receptor (KOPr) activation antagonizes many cocaine-related behaviors but adverse side effects such as sedation, dysphoria and depression limit their therapeutic use. Recently, salvinorin A (Sal A), a naturally occurring KOPr agonist, has been shown to attenuate cocaineinduced drug-seeking in a model of relapse in rats. The present study evaluated the effects of acute Sal A exposure on cocaine-induced hyperactivity and cocaine sensitization in rats. Acute treatment with the dose of Sal A that decreased drug-seeking in a previous study (0.3 mg/kg), significantly attenuated the expression of cocaine sensitization. This dose of Sal A failed to affect spontaneous locomotion or to produce a conditioned taste aversion to a novel-tasting saccharin solution. However, Sal A decreased climbing and swimming time and increased time spent immobile in the forced swim test. These findings indicate that Sal A, just like traditional KOPr agonists, attenuates cocaine-induced behavioral sensitization but does not produce the adverse effect of conditioned aversion, suggesting improved potential compliance. However, pro-depressive effects were also produced and these effects may limit the therapeutic potential

Cannabinoid Agonists Increase the Interaction between β-Arrestin 2 and ERK1/2 and Upregulate β-Arrestin 2 and 5-HT2A Receptors

We have recently reported that selective cannabinoid 2 (CB2) receptor agonists upregulate 5-HT2A receptors by enhancing ERK1/2 signaling in prefrontal cortex (PFCx). Increased activity of cortical 5-HT2A receptors has been associated with several neuropsychiatric disorders such as anxiety and schizophrenia. Here we examine the mechanisms involved in this enhanced ERK1/2 activation in rat PFCx and in a neuronal cell model. Sprague-Dawley rats treated with a non-selective cannabinoid agonist (CP55940, 50 μg/kg, 7 days, i.p.) showed enhanced co-immunoprecipitation of β-Arrestin 2 and ERK1/2, enhanced pERK protein levels, and enhanced expression of β-Arrestin 2 mRNA and protein levels in PFCx. In a neuronal cell line, we found that selective CB2 receptor agonists upregulate β-Arrestin 2, an effect that was prevented by selective CB2 receptor antagonist JTE-907 and CB2 shRNA lentiviral particles. Additionally, inhibition of clathrin-mediated endocytosis, ERK1/2, and the AP-1 transcription factor also prevented the cannabinoid receptor-induced upregulation of β-Arrestin 2. Our results suggest that sustained activation of CB2 receptors would enhance β-Arrestin 2 expression possibly contributing to its increased interaction with ERK1/2 thereby driving the upregulation of 5-HT2A receptors. The CB2 receptor-mediated upregulation of β-Arrestin 2 would be mediated, at least in part, by an ERK1/2-dependent activation of AP-1. These data could provide the rationale for some of the adverse effects associated with repeated cannabinoid exposure and shed light on some CB2 receptor agonists that could represent an alternative therapeutic because of their minimal effect on serotonergic neurotransmission

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

Cannabinoid 2 Receptor- and Beta Arrestin 2-Dependent Upregulation of Serotonin 2A Receptors

Recent evidence suggests that cannabinoid receptor agonists may regulate serotonin 2A (5-HT2A) receptor neurotransmission in the brain, although no molecular mechanism has been identified. Here, we present experimental evidence that sustained treatment with a non-selective cannabinoid agonist (CP 55,940) or selective CB2 receptor agonists (JWH 133 or GP 1a) upregulate 5-HT2A receptors in a neuronal cell line. Furthermore, this cannabinoid receptor agonist-induced upregulation of 5-HT2A receptors was prevented in cells stably transfected with either CB2 or β-Arrestin 2 shRNA lentiviral particles. Additionally, inhibition of clathrin-mediated endocytosis also prevented the cannabinoid receptor-induced upregulation of 5-HT2A receptors. Our results indicate that cannabinoid agonists might upregulate 5-HT2A receptors by a mechanism that requires CB2 receptors and β-Arrestin 2 in cells that express both CB2 and 5-HT2A receptors. 5-HT2A receptors have been associated with several physiological functions and neuropsychiatric disorders such as stress response, anxiety & depression and schizophrenia. Therefore, these results might provide a molecular mechanism by which activation of cannabinoid receptors might be relevant to some cognitive and mood disorders in humans