Solving an Old Puzzle: Elucidation and Evaluation of the Binding Mode of Salvinorin A at the Kappa Opioid Receptor.

The natural product Salvinorin A (SalA) was the first nitrogen-lacking agonist discovered for the opioid receptors and exhibits high selectivity for the kappa opioid receptor (KOR) turning SalA into a promising analgesic to overcome the current opioid crisis. Since SalA's suffers from poor pharmacokinetic properties, particularly the absence of gastrointestinal bioavailability, fast metabolic inactivation, and subsequent short duration of action, the rational design of new tailored analogs with improved clinical usability is highly desired. Despite being known for decades, the binding mode of SalA within the KOR remains elusive as several conflicting binding modes of SalA were proposed hindering the rational design of new analgesics. In this study, we rationally determined the binding mode of SalA to the active state KOR by in silico experiments (docking, molecular dynamics simulations, dynophores) in the context of all available mutagenesis studies and structure-activity relationship (SAR) data. To the best of our knowledge, this is the first comprehensive evaluation of SalA's binding mode since the determination of the active state KOR crystal structure. SalA binds above the morphinan binding site with its furan pointing toward the intracellular core while the C2-acetoxy group is oriented toward the extracellular loop 2 (ECL2). SalA is solely stabilized within the binding pocket by hydrogen bonds (C210ECL2, Y3127.35, Y3137.36) and hydrophobic contacts (V1182.63, I1393.33, I2946.55, I3167.39). With the disruption of this interaction pattern or the establishment of additional interactions within the binding site, we were able to rationalize the experimental data for selected analogs. We surmise the C2-substituent interactions as important for SalA and its analogs to be experimentally active, albeit with moderate frequency within MD simulations of SalA. We further identified the non-conserved residues 2.63, 7.35, and 7.36 responsible for the KOR subtype selectivity of SalA. We are confident that the elucidation of the SalA binding mode will promote the understanding of KOR activation and facilitate the development of novel analgesics that are urgently needed.

The Kappa Opioid Receptor Agonist 16-Bromo Salvinorin A Has Anti-Cocaine Effects without Significant Effects on Locomotion, Food Reward, Learning and Memory, or Anxiety and Depressive-like Behaviors.

Kappa opioid receptor (KOR) agonists have preclinical antipsychostimulant effects; however, adverse side effects have limited their therapeutic development. In this preclinical study, conducted in Sprague Dawley rats, B6-SJL mice, and non-human primates (NHPs), we evaluated the G-protein-biased analogue of salvinorin A (SalA), 16-bromo salvinorin A (16-BrSalA), for its anticocaine effects, side effects, and activation of cellular signaling pathways. 16-BrSalA dose-dependently decreased the cocaine-primed reinstatement of drug-seeking behavior in a KOR-dependent manner. It also decreased cocaine-induced hyperactivity, but had no effect on responding for cocaine on a progressive ratio schedule. Compared to SalA, 16-BrSalA had an improved side effect profile, with no significant effects in the elevated plus maze, light-dark test, forced swim test, sucrose self-administration, or novel object recognition; however, it did exhibit conditioned aversive effects. 16-BrSalA increased dopamine transporter (DAT) activity in HEK-293 cells coexpressing DAT and KOR, as well as in rat nucleus accumbens and dorsal striatal tissue. 16-BrSalA also increased the early phase activation of extracellular-signal-regulated kinases 1 and 2, as well as p38 in a KOR-dependent manner. In NHPs, 16-BrSalA caused dose-dependent increases in the neuroendocrine biomarker prolactin, similar to other KOR agonists, at doses without robust sedative effects. These findings highlight that G-protein-biased structural analogues of SalA can have improved pharmacokinetic profiles and fewer side effects while maintaining their anticocaine effects.

The Kappa Opioid Receptor and the Sleep of Reason: Cortico-Subcortical Imbalance Following Salvinorin-A.

BACKGROUND: The mechanisms through which kappa opioid receptor (KOR) agonists induce psychotomimetic effects are largely unknown, although the modulation of this receptor has attracted attention for its clinical use. In this work, we characterize the neuropharmacological effects of salvinorin-A, a highly selective KOR agonist. METHODS: Changes in multimodal electroencephalography, single-photon emission computed tomography, and subjective effects following the acute administration of salvinorin-A are reported. The study included 2 sub-studies that employed a double-blind, crossover, randomized, placebo-controlled design. RESULTS: The electroencephalography measures showed a marked increase in delta and gamma waves and a decrease in alpha waves while subjects were under the effect of salvinorin-A. Regarding single-photon emission computed tomography measures, significant decreases in regional cerebral blood flow were detected in multiple regions of the frontal, temporal, parietal, and occipital cortices. Significant regional cerebral blood flow increases were observed in some regions of the medial temporal lobe, including the amygdala, the hippocampal gyrus, and the cerebellum. The pattern of subjective effects induced by salvinorin-A was similar to those observed in relation to other psychotomimetic drugs but with an evidently dissociative nature. No dysphoric effects were reported. CONCLUSION: The salvinorin-A-mediated KOR agonism induced dramatic psychotomimetic effects along with a generalized decrease in cerebral blood flow and electric activity within the cerebral cortex.

Salvinorin A Does Not Affect Seizure Threshold in Mice.

The κ-opioid receptor has recently gained attention as a new molecular target in the treatment of many psychiatric and neurological disorders including epilepsy. Salvinorin A is a potent plant-derived hallucinogen that acts as a highly selective κ-opioid receptor agonist. It has unique structure and pharmacological properties, but its influence on seizure susceptibility has not been studied so far. Therefore, the aim of the present study was to investigate the effect of salvinorin A on seizure thresholds in three acute seizure tests in mice. We also examined its effect on muscular strength and motor coordination. The obtained results showed that salvinorin A (0.1-10 mg/kg, i.p.) did not significantly affect the thresholds for the first myoclonic twitch, generalized clonic seizure, or forelimb tonus in the intravenous pentylenetetrazole seizure threshold test in mice. Likewise, it failed to affect the thresholds for tonic hindlimb extension and psychomotor seizures in the maximal electroshock- and 6 Hz-induced seizure threshold tests, respectively. Moreover, no changes in motor coordination (assessed in the chimney test) or muscular strength (assessed in the grip-strength test) were observed. This is a preliminary report only, and further studies are warranted to better characterize the effects of salvinorin A on seizure and epilepsy.

Collybolide is a novel biased agonist of κ-opioid receptors with potent antipruritic activity.

Among the opioid receptors, the κ-opioid receptor (κOR) has been gaining considerable attention as a potential therapeutic target for the treatment of complex CNS disorders including depression, visceral pain, and cocaine addiction. With an interest in discovering novel ligands targeting κOR, we searched natural products for unusual scaffolds and identified collybolide (Colly), a nonnitrogenous sesquiterpene from the mushroom Collybia maculata. This compound has a furyl-δ-lactone core similar to that of Salvinorin A (Sal A), another natural product from the plant Salvia divinorum Characterization of the molecular pharmacological properties reveals that Colly, like Sal A, is a highly potent and selective κOR agonist. However, the two compounds differ in certain signaling and behavioral properties. Colly exhibits 10- to 50-fold higher potency in activating the mitogen-activated protein kinase pathway compared with Sal A. Taken with the fact that the two compounds are equipotent for inhibiting adenylyl cyclase activity, these results suggest that Colly behaves as a biased agonist of κOR. Behavioral studies also support the biased agonistic activity of Colly in that it exhibits ∼10-fold higher potency in blocking non-histamine-mediated itch compared with Sal A, and this difference is not seen in pain attenuation by these two compounds. These results represent a rare example of functional selectivity by two natural products that act on the same receptor. The biased agonistic activity, along with an easily modifiable structure compared with Sal A, makes Colly an ideal candidate for the development of novel therapeutics targeting κOR with reduced side effects.

Biosynthesis of the psychotropic plant diterpene salvinorin A: Discovery and characterization of the Salvia divinorum clerodienyl diphosphate synthase.

Salvia divinorum commonly known as diviner's sage, is an ethnomedicinal plant of the mint family (Lamiaceae). Salvia divinorum is rich in clerodane-type diterpenoids, which accumulate predominantly in leaf glandular trichomes. The main bioactive metabolite, salvinorin A, is the first non-nitrogenous natural compound known to function as an opioid-receptor agonist, and is undergoing clinical trials for potential use in treating neuropsychiatric diseases and drug addictions. We report here the discovery and functional characterization of two S. divinorum diterpene synthases (diTPSs), the ent-copalyl diphosphate (ent-CPP) synthase SdCPS1, and the clerodienyl diphosphate (CLPP) synthase SdCPS2. Mining of leaf- and trichome-specific transcriptomes revealed five diTPSs, two of which are class II diTPSs (SdCPS1-2) and three are class I enzymes (SdKSL1-3). Of the class II diTPSs, transient expression in Nicotiana benthamiana identified SdCPS1 as an ent-CPP synthase, which is prevalent in roots and, together with SdKSL1, exhibits a possible dual role in general and specialized metabolism. In vivo co-expression and in vitro assays combined with nuclear magnetic resonance (NMR) analysis identified SdCPS2 as a CLPP synthase. A role of SdCPS2 in catalyzing the committed step in salvinorin A biosynthesis is supported by its biochemical function, trichome-specific expression and absence of additional class II diTPSs in S. divinorum. Structure-guided mutagenesis revealed four catalytic residues that enabled the re-programming of SdCPS2 activity to afford four distinct products, thus advancing our understanding of how neo-functionalization events have shaped the array of different class II diTPS functions in plants, and may promote synthetic biology platforms for a broader spectrum of diterpenoid bioproducts.

Salvinorin A ameliorates cerebral vasospasm through activation of endothelial nitric oxide synthase in a rat model of subarachnoid hemorrhage.

OBJECTIVE: This study aimed to demonstrate the potential of salvinorin A (SA) for cerebral vasospasm after subarachnoid hemorrhage (SAH) and investigate mechanisms of therapeutic effect using rat SAH model. METHODS: Salvinorin A was injected intraperitoneally, and the neurobehavioral changes were observed at 12 hours, 24 hours, 48 hours, and 72 hours after SAH. Basilar artery was observed by magnetic resonance imaging (MRI). The inner diameter and thickness of basilar artery were measured. The morphological changes and the apoptosis in CA1 area of hippocampus were detected. Endothelin-1 (ET-1) and nitric oxide (NO) levels were detected by ELISA kit. The protein expression of endothelial NO synthase (eNOS) and aquaporin-4 (AQP-4) was determined by Western blot for potential mechanism exploration. RESULTS: Salvinorin A administration could relieve neurological deficits, decrease the neuronal apoptosis, and alleviate the morphological changes in CA1 area of hippocampus. SA alleviated CVS by increasing diameter and decreasing thickness of basilar artery, and such changes were accompanied by the decreased concentration of ET-1 and increased level of NO. Meanwhile, SA increased the expression of eNOS and decreased the expression of AQP-4 protein in the basilar artery and hippocampus. CONCLUSIONS: Salvinorin A attenuated CVS and alleviated brain injury after SAH via increasing expression of eNOS and NO content, and decreasing ET-1 concentration and AQP-4 protein expression.

Kappa Opioid Receptor Agonist Mesyl Sal B Attenuates Behavioral Sensitization to Cocaine with Fewer Aversive Side-Effects than Salvinorin A in Rodents.

The acute activation of kappa opioid receptors (KOPr) produces antinociceptive and anti-cocaine effects, however, their side-effects have limited further clinical development. Mesyl Sal B is a potent and selective KOPr analogue of Salvinorin A (Sal A), a psychoactive natural product isolated from the plant Salvia divinorum. We assessed the antinociceptive, anti-cocaine, and side-effects of Mesyl Sal B. The anti-cocaine effects are evaluated in cocaine-induced hyperactivity and behavioral sensitization to cocaine in male Sprague Dawley rats. Mesyl Sal B was assessed for anhedonia (conditioned taste aversion), aversion (conditioned place aversion), pro-depressive effects (forced swim test), anxiety (elevated plus maze) and learning and memory deficits (novel object recognition). In male B6.SJL mice, the antinociceptive effects were evaluated in warm-water (50 °C) tail withdrawal and intraplantar formaldehyde (2%) assays and the sedative effects measured with the rotarod performance task. Mesyl Sal B (0.3 mg/kg) attenuated cocaine-induced hyperactivity and behavioral sensitization to cocaine without modulating sucrose self-administration and without producing aversion, sedation, anxiety, or learning and memory impairment in rats. However, increased immobility was observed in the forced swim test indicating pro-depressive effects. Mesyl Sal B was not as potent as Sal A at reducing pain in the antinociceptive assays. In conclusion, Mesyl Sal B possesses anti-cocaine effects, is longer acting in vivo and has fewer side-effects when compared to Sal A, however, the antinociceptive effects are limited.

A (-)-kolavenyl diphosphate synthase catalyzes the first step of salvinorin A biosynthesis in Salvia divinorum.

Salvia divinorum (Lamiaceae) is an annual herb used by indigenous cultures of Mexico for medicinal and ritual purposes. The biosynthesis of salvinorin A, its major bioactive neo-clerodane diterpenoid, remains virtually unknown. This investigation aimed to identify the enzyme that catalyzes the first reaction of salvinorin A biosynthesis, the formation of (-)-kolavenyl diphosphate [(-)-KPP], which is subsequently dephosphorylated to afford (-)-kolavenol. Peltate glandular trichomes were identified as the major and perhaps exclusive site of salvinorin accumulation in S. divinorum. The trichome-specific transcriptome was used to identify candidate diterpene synthases (diTPSs). In vitro and in planta characterization of a class II diTPS designated as SdKPS confirmed its activity as (-)-KPP synthase and its involvement in salvinorin A biosynthesis. Mutation of a phenylalanine into histidine in the active site of SdKPS completely converts the product from (-)-KPP into ent-copalyl diphosphate. Structural elements were identified that mediate the natural formation of the neo-clerodane backbone by this enzyme and suggest how SdKPS and other diTPSs may have evolved from ent-copalyl diphosphate synthase.

An immunochromatographic assay for rapid etection of salvinorin A.

We developed an immunochromatographic assay (ICA) that enables rapid analysis of salvinorin A (Sal A) in Salvia divinorum within 10 min. The result shows that no Sal A in other samples of Lamiaceae plants was detected, but it could recognize Sal A among other substances in complex samples. The main advantage of the ICA is its high performance in combination with low cost, simplicity, and speed. Our newly developed combined ICA/indirect competitive ELISA(icELISA) system enables analysis of large numbers of samples over short periods of time without cumbersome pretreatments in complex mixtures. This method can complement other instrumental analyses for salvinorins, and could be used to deter S. divinorum abuse.

Naltrexone but Not Ketanserin Antagonizes the Subjective, Cardiovascular, and Neuroendocrine Effects of Salvinorin-A in Humans.

BACKGROUND: Salvinorin-A is a terpene found in the leaves of the plant Salvia divinorum. When administered to humans, salvinorin-A induces an intense but short-lasting modified state of awareness, sharing features with those induced by the classical serotonin-2A receptor agonist psychedelics. However, unlike substances such as psilocybin or mescaline, salvinorin-A shows agonist activity at the kappa-opioid receptor rather than at the serotonin-2A receptor. Here, we assessed the involvement of kappa-opioid receptor and serotonin-2A agonism in the subjective, cardiovascular, and neuroendocrine effects of salvinorin-A in humans. METHODS: We conducted a placebo-controlled, randomized, double-blind study with 2 groups of 12 healthy volunteers with experience with psychedelic drugs. There were 4 experimental sessions. In group 1, participants received the following treatment combinations: placebo+placebo, placebo+salvinorin-A, naltrexone+placebo, and naltrexone+salvinorin-A. Naltrexone, a nonspecific opioid receptor antagonist, was administered at a dose of 50mg orally. In group 2, participants received the treatment combinations: placebo+placebo, placebo+salvinorin-A, ketanserin+placebo, and ketanserin+salvinorin-A. Ketanserin, a selective serotonin-2A antagonist, was administered at a dose of 40mg orally. RESULTS: Inhalation of 1mg of vaporized salvinorin-A led to maximum plasma concentrations at 1 and 2 minutes after dosing. When administered alone, salvinorin-A severely reduced external sensory perception and induced intense visual and auditory modifications, increased systolic blood pressure, and cortisol and prolactin release. These effects were effectively blocked by naltrexone, but not by ketanserin. CONCLUSIONS: Results support kappa opioid receptor agonism as the mechanism of action underlying the subjective and physiological effects of salvinorin-A in humans and rule out the involvement of a serotonin-2A-mediated mechanism.

The hallucinogenic diterpene salvinorin A inhibits leukotriene synthesis in experimental models of inflammation.

Leukotrienes (LTs) are lipid mediators derived from arachidonic acid (AA) involved in a number of autoimmune/inflammatory disorders including asthma, allergic rhinitis and cardiovascular diseases. Salvinorin A (SA), a diterpene isolated from the hallucinogenic plant Salvia divinorum, is a well-established analgesic compound, but its anti-inflammatory properties are under-researched and its effects on LT production is unknown to date. Here, we studied the possible effect of SA on LT production and verified its actions on experimental models of inflammation in which LTs play a prominent role. Peritoneal macrophages (PM) stimulated by calcium ionophore A23187 were chosen as in vitro system to evaluate the effect of SA on LT production. Zymosan-induced peritonitis in mice and carrageenan-induced pleurisy in rats were selected as LT-related models to evaluate the effect of SA on inflammation as well as on LT biosynthesis. SA inhibited, in a concentration-dependent manner, A23187-induced LTB4 biosynthesis in isolated PM. In zymosan-induced peritonitis, SA inhibited cell infiltration, myeloperoxidase activity, vascular permeability and LTC4 production in the peritoneal cavity without decreasing the production of prostaglandin E2. In carrageenan-induced pleurisy in rats, a more sophisticated model of acute inflammation related to LTs, SA significantly inhibited LTB4 production in the inflammatory exudates, along with reducing the phlogistic process in the lung. In conclusion, SA inhibited LT production and it was effective in experimental models of inflammation in which LTs play a pivotal role. SA might be considered as a lead compound for the development of drugs useful in LTs-related diseases.

Molecular Signaling Pathways Behind the Biological Effects of Salvia Species Diterpenes in Neuropharmacology and Cardiology.

The genus Salvia, from the Lamiaceae family, has diverse biological properties that are primarily attributable to their diterpene contents. There is no comprehensive review on the molecular signaling pathways of these active components. In this review, we investigated the molecular targets of bioactive Salvia diterpenes responsible for the treatment of nervous and cardiovascular diseases. The effects on different pathways, including apoptosis signaling, oxidative stress phenomena, the accumulation of amyloid beta plaques, and tau phosphorylation, have all been considered to be mechanisms of the anti-Alzheimer properties of Salvia diterpenes. Additionally, effects on the benzodiazepine and kappa opioid receptors and neuroprotective effects are noted as neuropharmacological properties of Salvia diterpenes, including tanshinone IIA, salvinorin A, cryptotanshinone, and miltirone. Tanshinone IIA, as the primary diterpene of Salvia miltiorrhiza, has beneficial activities in heart diseases because of its ability to scavenge free radicals and its effects on transcription factors, such as nuclear transcription factor-kappa B (NF-κB) and the mitogen-activated protein kinases (MAPKs). Additionally, tanshinone IIA has also been proposed to have cardioprotective properties including antiarrhythmic activities and effects on myocardial infarction. With respect to the potential therapeutic effects of Salvia diterpenes, comprehensive clinical trials are warranted to evaluate these valuable molecules as lead compounds. Copyright © 2016 John Wiley & Sons, Ltd.

Salvinorin-A Induces Intense Dissociative Effects, Blocking External Sensory Perception and Modulating Interoception and Sense of Body Ownership in Humans.

BACKGROUND: Salvinorin-A is a terpene with agonist properties at the kappa-opioid receptor, the binding site of endogenous dynorphins. Salvinorin-A is found in Salvia divinorum, a psychoactive plant traditionally used by the Mazatec people of Oaxaca, Mexico, for medicinal and spiritual purposes. Previous studies with the plant and salvinorin-A have reported psychedelic-like changes in perception, but also unusual changes in body awareness and detachment from external reality. Here we comprehensively studied the profiles of subjective effects of increasing doses of salvinorin-A in healthy volunteers, with a special emphasis on interoception. METHODS: A placebo and three increasing doses of vaporized salvinorin-A (0.25, 0.50, and 1mg) were administered to eight healthy volunteers with previous experience in the use of psychedelics. Drug effects were assessed using a battery of questionnaires that included, among others, the Hallucinogen Rating Scale, the Altered States of Consciousness, and a new instrument that evaluates different aspects of body awareness: the Multidimensional Assessment for Interoceptive Awareness. RESULTS: Salvinorin-A led to a disconnection from external reality, induced elaborate visions and auditory phenomena, and modified interoception. The lower doses increased somatic sensations, but the highest dose led to a sense of a complete loss of contact with the body. CONCLUSIONS: Salvinorin-A induced intense psychotropic effects characterized by a dose-dependent gating of external audio-visual information and an inverted-U dose-response effect on body awareness. These results suggest a prominent role for the kappa opioid receptor in the regulation of sensory perception, interoception, and the sense of body ownership in humans.

The Association of Salvia divinorum and Psychotic Disorders: A Review of the Literature and Case Series.

The association of substance abuse and psychotic disorders is of interest to clinicians, academics, and lawmakers. Commonly abused substances, such as cannabis, cocaine, amphetamines, and alcohol, have all been associated with substance-induced psychosis. Hallucinogens can induce desired psychedelic effects and undesirable psychomimetic reactions. These are usually transient and resolve once the duration of action is over. Sometimes, these effects persist, causing distress and requiring intervention. This article focuses on the hallucinogenic substance Salvia divinorum, the use of which has been observed, particularly among youth worldwide. We present background information based on a review of the literature and on our own clinical encounters, as highlighted by two original case reports. We hypothesize that consumption of Salvia divinorum could be associated with the development of psychotic disorders. We propose that clinicians routinely inquire about the use of Salvia in patients with substance use disorders or psychotic illnesses. More research is required to assess any relationship between Salvia divinorum and psychosis. Additionally, we advocate increased public and medical awareness of this substance and other emerging drugs of abuse.

Acute Effects of Hallucinogens on Functional Connectivity: Psilocybin and Salvinorin-A.

The extent of changes in functional connectivity (FC) within functional networks as a common feature across hallucinogenic drug classes is under-explored. This work utilized fMRI to assess the dissociative hallucinogens Psilocybin, a classical serotonergic psychedelic, and Salvinorin-A, a kappa-opioid receptor (KOR) agonist, on resting-state FC in nonhuman primates. We highlight overlapping and differing influence of these substances on FC relative to the thalamus, claustrum, prefrontal cortex (PFC), default mode network (DMN), and DMN subcomponents. Analysis was conducted on a within-subject basis. Findings support the cortico-claustro-cortical network model for probing functional effects of hallucinogens regardless of serotonergic potential, with a potential key paradigm centered around the claustrum, PFC, anterior cingulate cortices (ACC), and angular gyrus relationship. Thalamo-cortical networks are implicated but appear dependent on 5-HT2AR activation. Acute desynchronization relative to the DMN for both drugs was also shown. Our findings provide a framework to understand broader mechanisms at which hallucinogens in differing classes may impact subjects regardless of the target receptor.

Salvia divinorum: from Mazatec medicinal and hallucinogenic plant to emerging recreational drug.

Salvia divinorum is a sage endemic to a small region of Mexico and has been traditionally used by the Mazatec Indians for divination and spiritual healing. Recently, it has gained increased popularity as a recreational drug, used by adolescents and young adults as an alternative to marijuana and LSD. Salvinorin A, the major active ingredient of the plant, is considered to be the most potent known hallucinogen of natural origin. This review surveys the current state of knowledge on the neurochemical, pharmacokinetic, and pharmacological properties of salvinorin A, the trends and motivation behind S. divinorum use, and the health problems among users of the plant's products. S. divinorum induces intense, but short-lived, psychedelic-like changes in mood and perception, with concomitant hallucinations and disorientation. Many websites have misinterpreted the limited existing research-based information on the side effects of salvia as evidence for its safety. However, data accumulated over the last few years indicate that potential health risks are associated with the use of S. divinorum, especially by teenagers, users of other substances of abuse, and individuals with underlying psychotic disturbances. Taken together, the data presented in this review point to the need for further basic and clinical studies to create a basis for the development of well-addressed prevention and treatment strategies.

Studies toward bivalent κ opioids derived from salvinorin A: heteromethylation of the furan ring reduces affinity.

The recent crystal structure of the κ-opioid receptor (κ-OR) revealed, unexpectedly, that the antagonist JDTic is a bivalent ligand: in addition to the orthosteric pocket occupied by morphinans, JDTic also occupies a distinct (allotopic) pocket. Mutagenesis data suggest that salvinorin A (1) also binds to this allotopic pocket, adjacent to the aspartate residue that anchors the basic nitrogen atom of classical opiates (Asp138). It has been suggested that an H-bond donor appended to 1 might interact with Asp138, increasing affinity. Such a bivalent ligand might also possess altered functional selectivity. Based on modeling and known N-furanylmethyl opioid antagonists, we appended H-bond donors to the furan ring of 1. (Dimethylamino)methyl groups at C-15 or C-16 abolished affinity for κ-OR. Hydroxymethylation at C-16 was tolerated, but 15,16-bis-hydroxymethylation was not. Since allosteric modulators may go undetected in binding assays, we also tested these and other low-affinity derivatives of 1 for allosteric modulation of dynorphin A in the [(35)S]GTPγS assay. No modulation was detected. As an alternative attachment point for bivalent derivatives, we prepared the 2-(hydroxyethoxy)methyl ether, which retained high affinity for κ-OR. We discuss alternative design strategies for linked, fused or merged bivalent derivatives of 1.

Pharmacologic hyperreactivity of kappa opioid receptors in periaqueductal gray matter during alcohol withdrawal syndrome in rats.

BACKGROUND: Periaqueductal gray matter (PAG) is a brain region rich in kappa-opioid receptors (KOR). KOR in PAG mediates behavioral responses related to pain integration, and panic response, among others. Its participation in the addiction phenomena has been poorly studied. Hence, this preliminary study explored the pharmacological effects of KOR stimulation/blockade in dorsal-PAG (D-PAG) during alcohol withdrawal on anxiety-type behaviors and alcohol intake/preference. METHODS: Juvenile male Wistar rats were unexposed (A-naïve group) or exposed to alcohol for 5 weeks and then restricted (A-withdrawal group). Posteriorly, animals received intra D-PAG injections of vehicle (10% DMSO), salvinorin A (SAL-A; a selective KOR agonist), or 2-Methyl-N-((2'-(pyrrolidin-1-ylsulfonyl)biphenyl-4-yl)methyl)propan-1-amine (PF-04455242; a highly selective KOR-antagonist). Subsequently, the defensive burying behavior (DBB) and alcohol intake/preference paradigms were evaluated. RESULTS: SAL-A markedly increased burying time, the height of bedding, and alcohol consumption/preference in A-withdrawal, while slightly increased the height of bedding in A-näive rats. PF-04455242 decreased both burying and immobility duration, whereas increases latency to burying, frequency of rearing, and the number of stretches attempts with no action on alcohol intake/preference in A-withdrawal rats. CONCLUSIONS: In general, stimulation/blockade of KOR in A-withdrawal animals exert higher responses compared to A-naïve ones. SAL-A produced anxiety-like behaviors and increased alcohol consumption/preference, especially/solely in the alcohol-withdrawal condition, while PF-04455242 augmented exploration with no effects on alcohol intake/preference. Our findings suggest a possible pharmacologic hyperreactivity of the KOR in PAG during alcohol withdrawal.

Formal Total Synthesis of Salvinorin A.

The generation of the quaternary stereocenter at the C9 position of salvinorin A precursors by the Claisen rearrangement was investigated. The required allyl alcohol was prepared from a Wieland-Miescher ketone using a known γ-hydroxylation, reduction of the enone double bond, cyanohydrin formation, and elimination, yielding an unsaturated nitrile. A two-step reduction led to the required allyl alcohol. The subsequent Johnson-Claisen rearrangement provided a mixture of two diastereomeric 1,4-unsaturated esters in a ratio of around 2.6 : 1. The major isomer could be converted to a key intermediate of the Hagiwara synthesis of salvinorin A.