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.

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.

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.

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.

Sex Differences in Kappa Opioid Receptor Agonist Mediated Attenuation of Chemotherapy-Induced Neuropathic Pain in Mice.

Chemotherapy-induced neuropathic pain is a common side effect for cancer patients which has limited effective treatment options. Kappa opioid receptor (KOR) agonists are a promising alternative to currently available opioid drugs due to their low abuse potential. In the current study, we have investigated the effects of Salvinorin A (SalA) analogues, 16-Ethynyl SalA, 16-Bromo SalA and ethyoxymethyl ether (EOM) SalB, and in a preclinical model of paclitaxel-induced neuropathic pain in male and female C57BL/6J mice. Using an acute dose-response procedure, we showed that compared to morphine, 16-Ethynyl SalA was more potent at reducing mechanical allodynia; and SalA, 16-Ethynyl SalA, and EOM SalB were more potent at reducing cold allodynia. In the mechanical allodynia testing, U50,488 was more potent in males and SalA was more potent in females. There were no sex differences in the acute cold allodynia testing. In the chronic administration model, treatment with U50,488 (10 mg/kg) reduced the mechanical and cold allodynia responses to healthy levels over 23 days of treatment. Overall, we have shown that KOR agonists are effective in a model of chemotherapy-induced neuropathic pain, indicating that KOR agonists could be further developed to treat this debilitating condition.

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.

Intranasal Salvinorin A Improves Long-term Neurological Function via Immunomodulation in a Mouse Ischemic Stroke Model.

Salvinorin A (SA), a highly selective kappa opioid receptor agonist, has been shown to reduce brain infarct volume and improve neurological function after ischemic stroke. However, the underlying mechanisms have not been fully understood yet. Therefore, we explored whether SA provides neuroprotective effects by regulating the immune response after ischemic stroke both in the central nervous system (CNS) and peripheral circulation. In this study, adult male mice were subjected to transient Middle Cerebral Artery Occlusion (tMCAO) and then were treated intranasally with SA (50 µg/kg) or with the vehicle dimethyl sulfoxide (DMSO). Multiple behavioral tests were used to evaluate neurofunction. Flow cytometry and immunofluorescence staining were used to evaluate the infiltration of peripheral immune cells into the brain. The tracer cadaverine and endogenous immunoglobulin G (IgG) extravasation were used to detect blood brain barrier leakage. We observed that SA intranasal administration after ischemic stroke decreased the expression of pro-inflammatory factors in the brain. SA promoted the polarization of microglia/macrophages into a transitional phenotype and decreased the pro-inflammatory phenotype in the brain after tMCAO. Interestingly, SA treatment scarcely altered the number of peripheral immune cells but decreased the macrophage and neutrophil infiltration into the brain at 24 h after tMCAO. Furthermore, SA treatment also preserved BBB integrity, reduced long-term brain atrophy and white matter injury, as well as improved the long-term neurofunctional outcome in mice. In this study, intranasal administration of SA improved long-term neurological function via immuno-modulation and by preserving blood-brain barrier integrity in a mouse ischemic stroke model, suggesting that SA could potentially serve as an alternative treatment strategy for ischemic stroke.

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 inhibits ovalbumin-stimulated allergic rhinitis and RBL-2H3 cells degranulation.

Allergic rhinitis (AR) is a long-term noncommunicable inflammatory disease of the nasal mucosa mediated by immunoglobulin E and is mainly caused by exposure of genetically susceptible individuals to environmental allergens. Mast cells contribute to the pathogenesis of allergic and nonallergic inflammatory diseases. Salvinorin A has been previously shown to inhibit leukotriene production and mast cell degranulation to suppress airway hyperresponsiveness caused by sensitization; thus, we hypothesized that salvinorin A has an anti-AR effect. We tested this hypothesis using monoclonal anti-2,4,6-dinitrophenyl immunoglobulin E/human serum albumin-induced rat basophilic leukemia cells (RBL-2H3 cells) and ovalbumin (OVA)-induced AR in mice as in vivo and in vitro AR models, respectively. The expression levels of histamine, ß-hexosaminidase, interleukin-4 and tumor necrosis factor-α were decreased by salvinorin A in vitro. Granule release and F-actin organization were also suppressed by salvinorin A. Furthermore, salvinorin A inhibited OVA-induced features of AR in mice, including nasal rubbing and sneezing, as well as increased OVA-specific immunoglobulin E, histamine, tumor necrosis factor-α and interleukin-4 levels. In addition, salvinorin A decreased the phosphorylation of phosphoinositide 3-kinase/Akt in vitro and in vivo. Our work suggests that salvinorin A suppresses AR caused by sensitization by inhibiting the inflammatory responses of mast cells; thus, salvinorin A may have potential for treatment of AR.

Regulation of blood-brain barrier permeability by Salvinorin A via alleviating endoplasmic reticulum stress in brain endothelial cell after ischemia stroke.

Inhibition of endoplasmic reticulum (ER) stress reduces blood-brain barrier (BBB) injury caused by ischemia/reperfusion (I/R), with indistinct mechanisms. Salvinorin A (SA) relieves I/R-induced BBB leakage; however, whether it is related to the suppression of ER stress is yet unclear. To address this question, we have used both a rat model of middle cerebral artery occlusion (MCAO) and human brain microvascular endothelial cells (HBMECs) with oxygen-glucose deprivation (OGD). SA was injected by tail vein at the terminal of ischemia; Norbinaltorphimine (NB), a kappa opioid antagonist, was administered 30 min prior to SA; 4-phenylbutyric acid (4-PBA), an ER stress inhibitor, was injected intraperitoneally after the onset of ischemia; adenylate-activated protein kinase (AMPK)-specific small interfering RNAs (siRNAs) were transfected to HBMECs before OGD. The assessment was as follows: infarct volume, brain water gain, Evans blue leakage, and modified neurological severity score (mNSS) after MCAO; HBMECs apoptosis rate and permeability, ER stress-related protein, and reactive oxygen species (ROS) and calcium levels after OGD. The results showed that SA significantly reduced the BBB leakage in vivo; SA relieved the apoptotic rates and ER stress in HBMECs, protected the permeability of HBMECs, and reduced ROS and calcium ion level after OGD. Moreover, the SA function was blocked by NB in vivo and AMPK- siRNAs in vitro. We conclude that SA mitigated BBB damage and HBMEC injury after I/R and alleviated ER stress in endothelial cells via AMPK pathway.

The Salvinorin Analogue, Ethoxymethyl Ether Salvinorin B, Promotes Remyelination in Preclinical Models of Multiple Sclerosis.

Multiple sclerosis is a neurodegenerative disease associated with demyelination and neuroinflammation in the central nervous system. There is an urgent need to develop remyelinating therapies to better treat multiple sclerosis and other demyelinating diseases. The kappa opioid receptor (KOR) has been identified as a potential target for the development of remyelinating therapies; however, prototypical KOR agonists, such as U50,488 have side effects, which limit clinical use. In the current study, we investigated a Salvinorin A analog, ethoxymethyl ether Salvinorin B (EOM SalB) in two preclinical models of demyelination in C57BL/6J mice. We showed that in cellular assays EOM SalB was G-protein biased, an effect often correlated with fewer KOR-mediated side effects. In the experimental autoimmune encephalomyelitis model, we found that EOM SalB (0.1-0.3 mg/kg) effectively decreased disease severity in a KOR-dependent manner and led to a greater number of animals in recovery compared to U50,488 treatment. Furthermore, EOM SalB treatment decreased immune cell infiltration and increased myelin levels in the central nervous system. In the cuprizone-induced demyelination model, we showed that EOM SalB (0.3 mg/kg) administration led to an increase in the number of mature oligodendrocytes, the number of myelinated axons and the myelin thickness in the corpus callosum. Overall, EOM SalB was effective in two preclinical models of multiple sclerosis and demyelination, adding further evidence to show KOR agonists are a promising target for remyelinating therapies.

Intranasal salvinorin A improves neurological outcome in rhesus monkey ischemic stroke model using autologous blood clot.

Salvinorin A (SA) exerts neuroprotection and improves neurological outcomes in ischemic stroke models in rodents. In this study, we investigated whether intranasal SA administration could improve neurological outcomes in a monkey ischemic stroke model. The stroke model was induced in adult male rhesus monkeys by occluding the middle cerebral artery M2 segment with an autologous blood clot. Eight adult rhesus monkeys were randomly administered SA or 10% dimethyl sulfoxide as control 20 min after ischemia. Magnetic resonance imaging was used to confirm the ischemia and extent of injury. Neurological function was evaluated using the Non-Human Primate Stroke Scale (NHPSS) over a 28-day observation period. SA significantly reduced infarct volume (3.9 ± 0.7 cm3 vs. 7.2 ± 1.0 cm3; P = 0.002), occupying effect (0.3 ± 0.2% vs. 1.4 ± 0.3%; P = 0.002), and diffusion limitation in the lesion (-28.2 ± 11.0% vs. -51.5 ± 7.1%; P = 0.012) when compared to the control group. SA significantly reduced the NHPSS scores to almost normal in a 28-day observation period as compared to the control group (P = 0.005). Intranasal SA reduces infarct volume and improves neurological outcomes in a rhesus monkey ischemic stroke model using autologous blood clot.

Salvinorin A protects against methicillin resistant staphylococcus aureus-induced acute lung injury via Nrf2 pathway.

Salvinorin A (SA), a neoclerodane diterpene, is isolated from the dried leaves ofSalvia divinorum. SA has traditionally been used treatments for chronic pain diseases. Recent research has demonstrated that SA possesses the anti-inflammatory property. The present study aim to explore the effects and potentialmechanisms ofSA in protection against Methicillin Resistant Staphylococcus aureus (MRSA)-induced acute lung injury (ALI). Here, we firstly found that verylowdosesof SA (50 µg/kg) could markedly decrease the infiltration of pulmonary neutrophils, mRNA expression of pro-inflammatory cytokines (TNF-α, IL-1ß and IL-6) and then attenuated ALI cause by MRSA infection in mice. In vitro findings revealed that SA attenuated lipoteichoicacid-induced apoptosis, inflammation and oxidative stress in RAW264.7 cells. Mechanism research revealed that SA increased both mRNA levels and protein levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and up-regulated mRNA expression of its downstream genes (HO-1, Gclm, Trx-1, SOD1 and SOD2). Additionally, Nrf2 knockout mice abolished the inhibitory effect of SA on neutrophil accumulation and oxidative stress in MRSA-induced ALI. In conclusion, SA attenuates MRSA-induced ALI via Nrf2 signaling pathways.

Salvinorin A alleviating cerebral vasospasm after subarachnoid hemorrhage through phosphatidylinositol 3-kinase/protein kinase B/endothelial nitric oxide synthase pathway / 解剖学报

Objective To investigate the effect of salvinorin A (SA) on alleviating cerebral vasospasm (CVS) after subarachnoid hemorrhage (SAH). Methods The SAH models were established by endovascular perforation method. Adult male SD rats (n = 91) were randomly divided into the sham group (sham), SAH model group (SAH), control group (SAH+DMSO) and drug administration group (SAH + SA). SA and DMSO were diluted with saline, and injected intraperitoneally at hour 24, hour 48 and hour 72 after SAH. At hour 72 after SAH, the neurological score was evaluated. The diameter and wall thickness of the internal carotid artery were observed through HE staining. Endothelin 1 (ET-1) ELISA kit and nitric oxide (NO) kit were used to observe the ET-1 concentration and NO content on the blood vessels of Willis circle. The expression of phosphorglated PI3K (p-PI3K), PI3K, phosphorylated Akt (p-Akt), Akt and endothelial nitric oxide synthase (eNOS) proteins were detected by Western blotting and the location of eNOS protein was observed by immunofluorescent staining. Results At hour 72 after SAH, SA could increase the neurological score, increase the vessel diameter and reduce the wall thickness of internal carotid artery. SA could reduce the ET-1 concentration and increase NO content in the blood vessels of Willis circle at hour 72 after SAH. SA could increase the ratio of p-PI3K/PI3K, p-Akt/Akt and the expression of eNOS proteins, which could be inhibited by PI3K inhibitor wortmannin and eNOS inhibitor L-NAME. eNOS expressed in vascular endothelial cells was detected by the immunofluorescence staining. Conclusion SA can alleviate CVS after SAH through PI3K/Akt/eNOS pathway.

Kappa Opioid Receptor on Pulmonary Macrophages and Immune Function.

BACKGROUND: Respiratory failure significantly increases mortality in critically ill patients. While opioids are often used during the perioperative period and in critically ill situations, little is known about how opioids are involved in pulmonary immune function and the inflammatory response. There is currently no clear information on the role of the kappa opioid receptor (KOR) in pulmonary inflammation. Here we evaluate whether KORs are involved in the modulation of lung macrophages by the use of selective KOR agonists in lipopolysaccharide (LPS) activated alveolar macrophages. METHOD: The inflammatory response in rat NR8383 macrophages was induced by stimulation with LPS (100 ng/ml) at different time-points. The effects of the KOR agonists Salvinorin A (SA) and U50488 on inflammatory factors such as nitrite, TNF-α, IL-1ß, iNOS and COX-2 were investigated. Nor-binaltorphimine, a selective KOR antagonist, was used to investigate the specific role of KOR. RESULTS: Stimulation of NR8383 cells with LPS (100 ng/ml) significantly increased the level of TNF-α at 1h, 2h and 6h compared to un-stimulated cells. SA attenuated the inflammatory response by reducing the levels of TNF-α and IL-1ß after LPS treatment. SA co-treatment reduced the elevated levels of NO induced by LPS and also alleviated the over-expression of iNOS and COX-2 within 2 hours after LPS activation, and such effects can be partially blocked by KOR antagonist, nor-binaltorphimine. Similar results from U50488 were observed. CONCLUSION: Our results indicate that KORs may play a critical role in the modulation of the pulmonary inflammatory process by their activation in macrophages. Selective KOR agonists exert their anti-inflammatory effects acutely on lung macrophages, within 1-2 hours of LPS-stimulated inflammation in vitro.

DARK Classics in Chemical Neuroscience: Salvinorin A.

Salvinorin A is the main bioactive compound in Salvia divinorum, an endemic plant with ancestral use by the inhabitants of the Mazateca mountain range (Sierra Mazateca) in Oaxaca, México. The main use of la pastora, as locally known, is in spiritual rites due to its extraordinary hallucinogenic effects. Being the first known nonalkaloidal opioid-mediated psychotropic molecule, salvinorin A set new research areas in neuroscience. The absence of a protonated amine group, common to all previously known opioids, results in a fast metabolism with the concomitant fast elimination and swift loss of activity. The worldwide spread and psychotropic effects of salvinorin A account for its misuse and classification as a drug of abuse. Consequently, salvinorin A and Salvia divinorum are now banned in many countries. Several synthetic efforts have been focused on the improvement of physicochemical and biological properties of salvinorin A: from total synthesis to hundreds of analogues. In this Review, we discuss the impact of salvinorin A in chemistry and neuroscience covering the historical relevance, isolation from natural sources, synthetic efforts, and pharmacological and safety profiles. Altogether, the chemistry behind and the taboo that encloses salvinorin A makes it one of the most exquisite naturally occurring drugs.

Evaluation of Biased and Balanced Salvinorin A Analogs in Preclinical Models of Pain.

In the search for safer, non-addictive analgesics, kappa opioid receptor (KOPr) agonists are a potential target, as unlike mu-opioid analgesics, they do not have abuse potential. Salvinorin A (SalA) is a potent and selective KOPr agonist, however, clinical utility is limited by the short duration of action and aversive side effects. Biasing KOPr signaling toward G-protein activation has been highlighted as a key cellular mechanism to reduce the side effects of KOPr agonists. The present study investigated KOPr signaling bias and the acute antinociceptive effects and side effects of two novel analogs of SalA, 16-Bromo SalA and 16-Ethynyl SalA. 16-Bromo SalA showed G-protein signaling bias, whereas 16-Ethynyl SalA displayed balanced signaling properties. In the dose-response tail-withdrawal assay, SalA, 16-Ethynyl SalA and 16-Bromo SalA were more potent than the traditional KOPr agonist U50,488, and 16-Ethynyl SalA was more efficacious. 16-Ethynyl SalA and 16-Bromo SalA both had a longer duration of action in the warm water tail-withdrawal assay, and 16-Ethynyl had greater antinociceptive effect in the hot-plate assay, compared to SalA. In the intraplantar 2% formaldehyde test, 16-Ethynyl SalA and 16-Bromo SalA significantly reduced both nociceptive and inflammatory pain-related behaviors. Moreover, 16-Ethynyl SalA and 16-Bromo SalA had no anxiogenic effects in the marble burying task, and 16-Bromo SalA did not alter behavior in the elevated zero maze. Overall, 16-Ethynyl SalA significantly attenuated acute pain-related behaviors in multiple preclinical models, while the biased KOPr agonist, 16-Bromo SalA, displayed modest antinociceptive effects, and lacked anxiogenic effects.

Synthetic Studies of Neoclerodane Diterpenes from Salvia divinorum: Design, Synthesis, and Evaluation of Analogues with Improved Potency and G-protein Activation Bias at the µ-Opioid Receptor.

Previous structure-activity relationship (SAR) studies identified the first centrally acting, non-nitrogenous µ-opioid receptor (MOR) agonist, kurkinorin (1), derived from salvinorin A. In an effort to further probe the physiological effects induced upon activation of MORs with this nonmorphine scaffold, a variety of analogues were synthesized and evaluated in vitro for their ability to activate G-proteins and recruit ß-arrestin-2 upon MOR activation. Through these studies, compounds that are potent agonists at MORs and either biased toward ß-arrestin-2 recruitment or biased toward G-protein activation have been identified. One such compound, 25, has potent activity and selectivity at the MOR over KOR with bias for G-protein activation. Impressively, 25 is over 100× more potent than morphine and over 5× more potent than fentanyl in vitro and elicits antinociception with limited tolerance development in vivo. This is especially significant given that 25 lacks a basic nitrogen and other ionizable groups present in other opioid ligand classes.

Kappa opioid receptors internalization is protective against oxygen-glucose deprivation through ß-arrestin activation and Akt-mediated signaling pathway.

Hypoxia induces reversible κ-opioid receptor (KOR) internalization similar to the internalization that is induced by KOR agonists. In the current study, we demonstrate that this KOR internalization is a protective mechanism via the ß-arrestin specific pathway in an oxygen-glucose deprivation (OGD) model. Mouse neuroblastoma Neuro2A cells were stably transfected with mouse KOR-tdTomato fusion protein (N2A-mKOR-tdT cells). Various concentrations of salvinorin A (SA), a highly selective KOR agonist, were given in the presence and absence of norbinaltorphimine (norBNI), which is a KOR antagonist, or Dyngo-4a (internalization inhibitor) or API-2 (Akt/Protein kinase B signaling inhibitor-2). Various concentrations of SA and RB-64 (22-thiocyanatosalvinorin A, selective for the G protein signaling pathway) were administered both in normoxic and hypoxic conditions. Autophagosomes and ultrastructural components of cells were observed using transmission electron microscopy (TEM). Cell viability, severity of cell injury, and levels of proteins related to the Akt signaling pathway were evaluated using live cell counting (by Cell Counting Kit-8), the lactic acid dehydrogenase (LDH) release rate, and Western blot analysis, respectively. SA promoted cell survival and attenuated OGD-induced cell injury. The Akt signaling pathway is activated by SA. KOR internalization, when blocked by norBNI or Dyngo-4a, increased LDH release and decreased cell viability under OGD. Treatment with SA significantly inhibited autophagy, and the effects of SA on autophagy were reversed by API-2 pretreatment. RB-64 in a low concentration without ß-arrestin recruitment did not reduce LDH release and increase cell viability as observed with SA. KOR internalization through ß-arrestin activation is a protective mechanism against OGD. The Akt pathway might play a critical role in modulating these protective effects by inhibiting autophagy.

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.