Comparison of an Addictive Potential of μ-Opioid Receptor Agonists with G Protein Bias: Behavioral and Molecular Modeling Studies

Among different approaches to the search for novel—safer and less addictive—opioid analgesics, biased agonism has received the most attention in recent years. Some μ-opioid receptor agonists with G protein bias, including SR compounds, were proposed to induce diminished side effects. However, in many aspects, behavioral effects of those compounds, as well as the mechanisms underlying differences in their action, remain unexplored. Here, we aimed to evaluate the effects of SR-14968 and SR-17018, highly G protein-biased opioid agonists, on antinociception, motor activity and addiction-like behaviors in C57BL/6J mice. The obtained results showed that the compounds induce strong and dose-dependent antinociception. SR-14968 causes high, and SR-17018 much lower, locomotor activity. Both agonists develop reward-associated behavior and physical dependence. The compounds also cause antinociceptive tolerance, however, developing more slowly when compared to morphine. Interestingly, SR compounds, in particular SR-17018, slow down the development of antinociceptive tolerance to morphine and inhibit some symptoms of morphine withdrawal. Therefore, our results indicate that SR agonists possess rewarding and addictive properties, but can positively modulate some symptoms of morphine dependence. Next, we have compared behavioral effects of SR-compounds and PZM21 and searched for a relationship to the substantial differences in molecular interactions that these compounds form with the µ-opioid receptor.

Investigating the Effects of Novel Kappa Opioid Receptor Agonists on the Dopamine Transporter

<p>Classic kappa opioid receptor (KOPr) agonists have shown anti-addictive properties in rat models of addiction (Heidbreder et al. 1998; Schenk et al. 1999; Sun et al. 2010), and this has been shown to be partially through modulation of dopamine and serotonin in the synapse (Thompson et al. 2000; Zhang et al. 2004; Zakharova et al. 2008a). However, they have side effects such as depression and dysphoria and therefore have not been moved into the clinic. The novel KOPr agonist salvinorin A has a completely different structure compared to the classic agonists, and along with its novel analogues has opened up a new family of KOPr agonists which may possess anti-addictive properties and have the potential to have decreased side effects. Salvinorin A has also demonstrated anti-addictive properties (Morani et al. 2009). In this study the novel KOPr agonist salvinorin A and its analogues DS-1-240 and DS-3-216 were investigated, along with the classic agonists U50,488H and U69,593. Their effects on the dopamine transporter (DAT) were measured using isolated rat brain tissue and cell models. The effects of U50,488H and salvinorin A on the serotonin transporter (SERT) was also measured in rat striatum using rotating disk electrode voltammetry, which was established to measure serotonin uptake in our lab during this study. We found that all of the kappa opioid receptor agonists studied in isolated rat brain tissue caused dose dependent increases in uptake of dopamine by the dopamine transporter and a decrease in uptake of serotonin by the serotonin transporter. The effect on the serotonin transporter was observed after a 15 min incubation with the agonists. Salvinorin A had a faster effect on the dopamine transporter than the other compounds investigated, with increases measured at 1 min rather than 4 min. DAT kinetics showed increases in Vmax for all agonists investigated, and both U69,593 and DS-1-240 also showed increased Km values. This demonstrates an overall increase in function, with the possibility of increased cell surface expression. Further investigation using cell models also found an increase in uptake of the fluorescent monoamine transporter substrate ASP+ by YFP tagged human DAT (YFP-hDAT). This effect was seen with all the agonists studied after incubations of less than 5 min and was YFP-hDAT trafficking-independent. The increase in uptake seen may be due to increased active YFP-hDAT found on the cell membrane as ASP+ binding studies demonstrated an increase in binding. The acute increase in YFP-hDAT function was found to be ERK1/2 dependent for all compounds studied, and was also dependent on intact lipid rafts in the cell membrane. After a 30 min incubation, salvinorin A and U50,488H still caused increased uptake of ASP+ by YFP-hDAT, whereas DS-1-240 and DS-3-216 did not. Increases in cell surface expression of YFP-hDATwas seen at this time point with salvinorin A, U69,593, and DS-3-216. Further investigation into this found that the increase in cell surface expression of YFP-hDAT after salvinorin A treatment was ERK1/2 dependent, whereas the increase seen with U69,593 appeared to be ERK1/2 independent. Overall, this data demonstrates that KOPr rapidly regulates DAT function by a trafficking-independent, ERK1/2-, and lipid raft-dependent mechanism. The classic KOPr agonist U50,488H and salvinorin A also caused a decrease in serotonin uptake by SERT, confirming that the KOPr also regulates SERT. The data from this study provides more information on how these classic and novel KOPr agonists function to regulate DAT and SERT, which may help explain some of the anti-addictive properties displayed by these compounds.</p>

Investigating the Effects of Novel Kappa Opioid Receptor Agonists on the Dopamine Transporter

<p>Classic kappa opioid receptor (KOPr) agonists have shown anti-addictive properties in rat models of addiction (Heidbreder et al. 1998; Schenk et al. 1999; Sun et al. 2010), and this has been shown to be partially through modulation of dopamine and serotonin in the synapse (Thompson et al. 2000; Zhang et al. 2004; Zakharova et al. 2008a). However, they have side effects such as depression and dysphoria and therefore have not been moved into the clinic. The novel KOPr agonist salvinorin A has a completely different structure compared to the classic agonists, and along with its novel analogues has opened up a new family of KOPr agonists which may possess anti-addictive properties and have the potential to have decreased side effects. Salvinorin A has also demonstrated anti-addictive properties (Morani et al. 2009). In this study the novel KOPr agonist salvinorin A and its analogues DS-1-240 and DS-3-216 were investigated, along with the classic agonists U50,488H and U69,593. Their effects on the dopamine transporter (DAT) were measured using isolated rat brain tissue and cell models. The effects of U50,488H and salvinorin A on the serotonin transporter (SERT) was also measured in rat striatum using rotating disk electrode voltammetry, which was established to measure serotonin uptake in our lab during this study. We found that all of the kappa opioid receptor agonists studied in isolated rat brain tissue caused dose dependent increases in uptake of dopamine by the dopamine transporter and a decrease in uptake of serotonin by the serotonin transporter. The effect on the serotonin transporter was observed after a 15 min incubation with the agonists. Salvinorin A had a faster effect on the dopamine transporter than the other compounds investigated, with increases measured at 1 min rather than 4 min. DAT kinetics showed increases in Vmax for all agonists investigated, and both U69,593 and DS-1-240 also showed increased Km values. This demonstrates an overall increase in function, with the possibility of increased cell surface expression. Further investigation using cell models also found an increase in uptake of the fluorescent monoamine transporter substrate ASP+ by YFP tagged human DAT (YFP-hDAT). This effect was seen with all the agonists studied after incubations of less than 5 min and was YFP-hDAT trafficking-independent. The increase in uptake seen may be due to increased active YFP-hDAT found on the cell membrane as ASP+ binding studies demonstrated an increase in binding. The acute increase in YFP-hDAT function was found to be ERK1/2 dependent for all compounds studied, and was also dependent on intact lipid rafts in the cell membrane. After a 30 min incubation, salvinorin A and U50,488H still caused increased uptake of ASP+ by YFP-hDAT, whereas DS-1-240 and DS-3-216 did not. Increases in cell surface expression of YFP-hDATwas seen at this time point with salvinorin A, U69,593, and DS-3-216. Further investigation into this found that the increase in cell surface expression of YFP-hDAT after salvinorin A treatment was ERK1/2 dependent, whereas the increase seen with U69,593 appeared to be ERK1/2 independent. Overall, this data demonstrates that KOPr rapidly regulates DAT function by a trafficking-independent, ERK1/2-, and lipid raft-dependent mechanism. The classic KOPr agonist U50,488H and salvinorin A also caused a decrease in serotonin uptake by SERT, confirming that the KOPr also regulates SERT. The data from this study provides more information on how these classic and novel KOPr agonists function to regulate DAT and SERT, which may help explain some of the anti-addictive properties displayed by these compounds.</p>

Opioid Analgesia and Opioid-Induced Adverse Effects: A Review

Opioids are widely used as therapeutic agents against moderate to severe acute and chronic pain. Still, these classes of analgesic drugs have many potential limitations as they induce analgesic tolerance, addiction and numerous behavioural adverse effects that often result in patient non-compliance. As opium and opioids have been traditionally used as painkillers, the exact mechanisms of their adverse reactions over repeated use are multifactorial and not fully understood. Older adults suffer from cancer and non-cancer chronic pain more than younger adults, due to the physiological changes related to ageing and their reduced metabolic capabilities and thus show an increased number of adverse reactions to opioid drugs. All clinically used opioids are μ-opioid receptor agonists, and the major adverse effects are directly or potentially connected to this receptor. Multifunctional opioid ligands or peripherally restricted opioids may elicit fewer adverse effects, as shown in preclinical studies, but these results need reproducibility from further extensive clinical trials. The current review aims to overview various mechanisms involved in the adverse effects induced by opioids, to provide a better understanding of the underlying pathophysiology and, ultimately, to help develop an effective therapeutic strategy to better manage pain.

In Vitro Analyses of Spinach-Derived Opioid Peptides, Rubiscolins: Receptor Selectivity and Intracellular Activities through G Protein- and β-Arrestin-Mediated Pathways

Activated opioid receptors transmit internal signals through two major pathways: the G-protein-mediated pathway, which exerts analgesia, and the β-arrestin-mediated pathway, which leads to unfavorable side effects. Hence, G-protein-biased opioid agonists are preferable as opioid analgesics. Rubiscolins, the spinach-derived naturally occurring opioid peptides, are selective δ opioid receptor agonists, and their p.o. administration exhibits antinociceptive effects. Although the potency and effect of rubiscolins as G-protein-biased molecules are partially confirmed, their in vitro profiles remain unclear. We, therefore, evaluated the properties of rubiscolins, in detail, through several analyses, including the CellKeyTM assay, cADDis® cAMP assay, and PathHunter® β-arrestin recruitment assay, using cells stably expressing µ, δ, κ, or µ/δ heteromer opioid receptors. In the CellKeyTM assay, rubiscolins showed selective agonistic effects for δ opioid receptor and little agonistic or antagonistic effects for µ and κ opioid receptors. Furthermore, rubiscolins were found to be G-protein-biased δ opioid receptor agonists based on the results obtained in cADDis® cAMP and PathHunter® β-arrestin recruitment assays. Finally, we found, for the first time, that they are also partially agonistic for the µ/δ dimers. In conclusion, rubiscolins could serve as attractive seeds, as δ opioid receptor-specific agonists, for the development of novel opioid analgesics with reduced side effects.

Reversing Postcardiopulmonary Bypass Associated Cognitive Dysfunction Using k-Opioid Receptor Agonists to Regulate Microglial Polarization via the NLRP3/Caspase-1 Pathway

Cardiopulmonary bypass (CPB) is mainly used during cardiac surgeries that treat ischemic, valvular, or congenital heart disease and aortic dissections. The disorders of central nervous system (CNS) that occur after cardiopulmonary bypass are attracting considerable interest. Postoperative neurocognitive disorders (PND) have been reported as the leading cause of patients’ disability and death following CPB. The k-opioid receptor (KOR) agonists (U50488H) have been suggested to be vital in the treatment of surgically induced CNS neuroinflammatory responses. In this article, the transitions between the M1 and M2 microglial polarization state phenotypes were hypothesized to significantly affect the regulatory mechanisms of KOR agonists on postcardiopulmonary bypass (post-CPB) neuroinflammation. We investigated the effects of U50488H on neuroinflammation and microglia polarization in rats exposed to CPB and explored the method of the NLRP3/caspase-1 pathway. Thirty SD rats were randomly divided into three groups: sham operation group, cardiopulmonary bypass model group, and CPB+ k-opioid receptor agonist (U50488H) group, with ten rats in each group. The Morris water maze was used to evaluate the changes in the cognitive function of CPB rats. Hematoxylin and eosin (HE) staining and TUNEL were performed to assess the rats’ hippocampal damage. Enzyme-Linked Immunosorbent Assay (ELISA) was used to detect changes in brain injury markers and inflammatory factors. Furthermore, immunofluorescence was used to observe the expression of microglia polarization and NLRP3 followed by Western blots to detect the expression of the NLRP3/caspase-1 pathway and microglia polarization-related proteins. Rat microglia were cultured in vitro, with LPS stimulation, and treated with U50488H and a caspase-1 antagonist to evaluate the effects and mechanism of action of U50488H. KORs alleviated hippocampal damage caused by CPB and improved PND. CPB activated the NLRP3 inflammasome and upregulated pro-caspase-1 expression which promoted the expression of pro-IL-lβ and pro-IL-18 and resulted in increased inflammation. However, KORs also inhibited NLRP3 and transformed microglia from the M1 to the M2 state. Caspase-1 inhibitor treatment reduced the microglial polarization induced by KORs. The κ-opioid receptor agonists inhibited the inflammation mediated by microglia and improved PND through the NLRP3/caspase-1 signaling pathway.