Salvinorin A (SalA), a selective κ-opioid receptor (KOR) agonist, produces dysphoria and pro-depressant like effects. These actions have been attributed to inhibition of striatal dopamine release. The dopamine transporter (DAT) regulates dopamine transmission via uptake of released neurotransmitter. KORs are apposed to DAT in dopamine nerve terminals suggesting an additional target by which SalA modulates dopamine transmission. SalA produced a concentration-dependent, nor-binaltorphimine (BNI)- and pertussis toxin-sensitive increase of ASP+ accumulation in EM4 cells coexpressing myc-KOR and YFP-DAT, using live cell imaging and the fluorescent monoamine transporter substrate, trans 4-(4-(dimethylamino)-styryl)-N-methylpyridinium) (ASP+). Other KOR agonists also increased DAT activity that was abolished by BNI pretreatment. While SalA increased DAT activity, SalA treatment decreased serotonin transporter (SERT) activity and had no effect on norepinephrine transporter (NET) activity. In striatum, SalA increased the Vmax for DAT mediated DA transport and DAT surface expression. SalA up-regulation of DAT function is mediated by KOR activation and the KOR-linked extracellular signal regulated kinase-½ (ERK1/2) pathway. Co-immunoprecipitation and BRET studies revealed that DAT and KOR exist in a complex. In live cells, DAT and KOR exhibited robust FRET signals under basal conditions. SalA exposure caused a rapid and significant increase of the FRET signal. This suggests that the formation of KOR and DAT complexes is promoted in response to KOR activation. Together, these data suggest that enhanced DA transport and decreased DA release resulting in decreased dopamine signaling may contribute to the dysphoric and pro-depressant like effects of SalA and other KOR agonists.

The neoclerodane diterpene salvinorin A, found in the leaves of Salvia divinorum, is a potent κ-opioid receptor agonist, making it an attractive scaffold for development into a treatment for substance abuse. Although several successful semisynthetic studies have been performed to elucidate structure–activity relationships, the lack of analogues with substitutions to the furan ring of salvinorin A has prevented a thorough understanding of its role in binding to the κ-opioid receptor. Herein we report the synthesis of several salvinorin A derivatives with modified furan rings. Evaluation of these compounds in a functional assay indicated that sterically less demanding substitutions are preferred, suggesting the furan ring is bound in a congested portion of the binding pocket. The most potent of the analogues successfully reduced drug-seeking behavior in an animal model of drug-relapse without producing the sedation observed with other κ-opioid agonists.

BACKGROUND AND PURPOSEAcute activation of κ opioid (KOP) receptors results in anticocaine-like effects, but adverse effects, such as dysphoria, aversion, sedation and depression, limit their clinical development. Salvinorin A, isolated from the plant Salvia divinorum, and its semi-synthetic analogues have been shown to have potent KOP receptor agonist activity and may induce a unique response with similar anticocaine addiction effects as the classic KOP receptor agonists, but with a different side effect profile. EXPERIMENTAL APPROACHWe evaluated the duration of effects of Mesyl Sal B in vivo utilizing antinociception assays and screened for cocaine-prime induced cocaine-seeking behaviour in self-administering rats to predict anti-addiction effects. Cellular transporter uptake assays and in vitro voltammetry were used to assess modulation of dopamine transporter (DAT) function and to investigate transporter trafficking and kinase signalling pathways modulated by KOP receptor agonists. KEY RESULTSMesyl Sal B had a longer duration of action than SalA, had anti-addiction properties and increased DAT function in vitro in a KOP receptor-dependent and Pertussis toxin-sensitive manner. These effects on DAT function required ERK1/2 activation. We identified differences between Mesyl Sal B and SalA, with Mesyl Sal B increasing the Vmax of dopamine uptake without altering cell-surface expression of DAT. CONCLUSIONS AND IMPLICATIONSSalA analogues, such as Mesyl Sal B, have potential for development as anticocaine agents. Further tests are warranted to elucidate the mechanisms by which the novel salvinorin-based neoclerodane diterpene KOP receptor ligands produce both anti-addiction and adverse side effects. LINKED ARTICLESThis article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx

Acute activation of κ opioid receptors produces anti-addictive effects by regulating dopamine levels in the brain. Unfortunately, classic κ opioid agonists have undesired side effects such as sedation, aversion and depression which restrict their clinical use. Salvinorin A (Sal A), a novel κ opioid receptor agonist extracted from the plant Salvia divinorum, has been identified as a potential therapy for drug abuse and addiction. Here, we review the preclinical effects of Sal A in comparison with traditional κ opioid agonists and several new analogues. Sal A retains the anti-addictive properties of traditional κ opioid receptors agonists with several improvements including reduced side effects. However, the rapid metabolism of Sal A makes it undesirable for clinical development. In an effort to improve the pharmacokinetics and tolerability of this compound, κ opioid receptor agonists based on the structure of Sal A have been synthesized. While work in this field is still in progress, several analogues with improved pharmacokinetic profiles have been shown to have anti-addiction effects. While in its infancy, it is clear that these compounds hold promise for the future development of anti-addiction therapeutics.

Rationale Kappa-opioid receptor (KOPr) agonists have pre-clinical anti-cocaine and analgesic effects. However, side-effects including sedation, dysphoria, aversion, anxiety and depression limit their therapeutic development. The unique structure of Salvinorin A has been used to develop longer-acting KOPr agonists. Objectives We evaluate two novel C-2 analogues of Salvinorin A, ethoxymethyl ether Sal B (EOM Sal B) and β-tetrahydropyran Sal B (β-THP Sal B) alongside U50,488 for their ability to modulate cocaine-induced behaviours and side-effects, pre-clinically. Methods Anti-cocaine properties of EOM Sal B were evaluated using the reinstatement model of drug-seeking in self-administering rats. EOM Sal B and β-THP Sal B were evaluated for effects on cocaine-induced hyperactivity, spontaneous locomotor activity and sucrose self-administration. EOM Sal B and β-THP Sal B were evaluated for aversive, anxiogenic and depressive-like effects using conditioned place aversion (CPA), elevated plus maze (EPM) and forced swim tests (FST) respectively. Results EOM Sal B (0.1, 0.3 mg/kg, i.p.) dose-dependently attenuated drug-seeking and EOM Sal B (0.1 mg/kg, i.p.) and β-THP Sal B (1 mg/kg, i.p.) attenuated cocaine-induced hyperactivity. No effects on locomotor activity, open arm times (EPM) or swimming behaviours (FST), were seen with EOM (0.1 or 0.3 mg/kg, i.p.) or β-THP Sal B (1 or 2 mg/kg, i.p.). However, β-THP Sal B decreased time spent in the drug-paired chamber. Conclusion EOM Sal B is more potent than Sal A and β-THP Sal B in reducing drug-seeking behaviour with fewer side-effects. EOM Sal B showed no effects on sucrose self-administration (0.1 mg/kg), locomotor, depressive-like, aversive-like or anxiolytic effects.

κ opioid receptor activation by traditional arylacetamide agonists and the novel neoclerodane diterpene κ opioid receptor agonist Salvinorin A (Sal A) results in attenuation of cocaine-seeking behavior in pre-clinical models of addiction. However, adverse effects such as sedation, depression and aversion limit their clinical utility. The Sal A analogue, 2-methoxymethyl salvinorin B (MOM Sal B) is a longer acting Sal A analogue with high affinity for κ opioid receptors. In this study, we tested MOM Sal B for its ability to modulate cocaine-seeking behavior in rats. MOM Sal B (0.3 mg/kg) successfully attenuated cocaine-seeking but also attenuated sucrose reinforcement. No change in activity was observed in either cocaine-induced hyperactivity or spontaneous open field activity tests but increased immobility and decreased swimming times in the forced swim test were observed. This study indicates that κ opioid receptor activation by more potent Sal A analogues modulates cocaine-seeking behavior non-selectively without causing sedation, suggesting an improved side effects profile. However, pro-depressive effects are seen, which may limit the therapeutic potential of this compound. Future studies with Sal A analogues having affinities at other opioid receptors are warranted as they have the potential to identify compounds having effective anti-addiction properties.

<p>Acute kappa opioid receptor (KOPr) activation by traditional agonists produces antiaddiction properties, but side effects such as sedation and depression prevent their clinical use. The novel KOPr agonist salvinorin A (Sal A), isolated from the plant Salvia divinorum, is a potent and selective KOPr agonist with a unique non-nitrogenous structure. Sal A possesses anti-addiction effects with less side effects than traditional KOPr agonists, but its short duration of action limits its therapeutic usefulness. To test the hypothesis that longer acting structural analogues of Sal A may yield a new class of therapeutics, the anti-cocaine effects of Sal A analogues such as 16-bromosalvinorin A (16-brSal A), ethoxymethyl ether salvinorin B (EOM Sal B), and methoxymethyl ether salvinorin B (MOM Sal B) were evaluated. 16-brSal A (1.0 mg/kg) displayed a longer duration of action in mice compared to Sal A, evidenced using the tail flick test (p<0.05). Both 16-brSal A and EOM Sal B produced dose-dependent decreases in cocaine-induced reinstatement of drug seeking (p<0.05). On the other hand, 16-brSal A (1.0 mg/kg) but not MOM Sal B (0.3 mg/kg) attenuated cocaine-induced hyperactivity (p<0.05), although both compounds showed no sedative effects in the locomotor activity test in rats. This indicates the superior behavioural anti-cocaine profile of 16-brSal A at its minimum effective dose. These three compounds, together with another analogue that also decreased cocaineinduced drug seeking, β-tetrahydropyran salvinorin B (β-THP Sal B), were screened for typical KOPr-mediated side effects using the minimal effective doses that attenuated drug seeking. MOM Sal B but not EOM Sal B (0.1 mg/kg), β-THP Sal B (1.0 mg/kg), or 16-brSal A produced depressive-like effects in the forced swim test (FST) in rats (p<0.05). However, EOM Sal B displayed a reduction in swimming time coupled with an increase in climbing duration in the FST (p<0.05). On the other hand, β-THP Sal B (p<0.001, between 30 – 45 min) and EOM Sal B (p<0.05, between 15 – 30 min) significantly increased sucrose intake in the rat sucrose self-administration model at different time intervals. 16-brSal A, however, produced no significant changes in natural reward intake measured by sucrose self-administration. The improved behavioural profile of 16-brSal A extended to a lack of anxiogenic effects. No significant anxiety-like behaviour was seen in the light dark or elevated plus maze, although aversion was observed in the conditioned place aversion paradigm (p<0.05). The low incidence of adverse effects of 16-brSal A compared to other iv Sal A analogues in behavioural models prompted additional cellular studies of this KOPr agonist. As the anti-cocaine effects of KOPr agonists have been attributed to their ability to modulate dopamine (DA) levels, 16-brSal A was examined for its ability to regulate dopamine transporter (DAT) function. DAT function was determined in vitro by determining uptake of a fluorescent substrate, ASP+, in HEK-293 cells expressing YFP-DAT and myc-KOPr. Ex vivo studies were also conducted by measuring DA uptake in isolated, minced rat dorsal striatum and nucleus accumbens using rotating disk electrode voltammetry. 16-brSal A significantly increased DAT function in both the in vitro (10 μM) and ex vivo (500 nM) models (p<0.05), an effect that was dependent on extracellular regulated kinase 1/2 (ERK1/2). Since late phase ERK1/2 and p38 kinase activation have been attributed to negative KOPr behavioural responses, the effects of 16-brSal A on these pathways were also examined. Western blotting studies revealed that 16-brSal A selectively activated only the early (5 – 15 min) but not late phase (120 – 180 min) ERK1/2 pathway in HEK-293 cells as well as rat dorsal striatum, prefrontal cortex, and nucleus accumbens (p<0.05). 16-brSal A also produced no significant activation of p38 kinase in the dorsal striatum or prefrontal cortex of rats, although significant phosphorylation was seen in the nucleus accumbens (p<0.05). The ability of 16-brSal A to produce desired behavioural anti-addiction effects with fewer adverse effects, matched with its regulation of KOPr signalling pathways, suggests that it may possibly be a functionally selective agonist that preferentially activates the G-protein signalling pathway at the KOPr. Since understanding the potential use of novel KOPr agonists in different phases of the addiction cycle is crucial to ensure effective administration of therapies, Sal A and 16-brSal A were tested in rats self-administering cocaine on the long access (Sal A) and progressive ratio (Sal A and 16-brSal A) schedules. Although no differences in cocaine responding were seen with KOPr agonist treatment in either paradigms, a higher dose or concurrent infusions of KOPr agonist with cocaine may improve the responses observed. Overall, the novel KOPr agonist, 16-brSal A has excellent potential as a pharmacotherapy due to its anti-cocaine effects and minimal adverse side effect profile. This is the first study to examine in detail the behavioural and cellular actions of 16-brSal A, and supports previous reports of Sal A-derived KOPr agonists as prospective therapeutics for cocaine abuse.</p>

AimTo quantify the areas of burden experienced by patients requiring repeated intravitreal injections (IVI) in the management of exudative retinal diseases.MethodsThe validated Questionnaire to Assess Life Impact of Treatment by Intravitreal Injections survey was administered to patients at four retina clinical practices across four US states. The primary outcome measure was Treatment Burden Score (TBS), a single score assessing overall burden.ResultsOf 1416 (n=657 age-related macular degeneration; n=360 diabetic macular oedema/diabetic retinopathy; n=221 retinal vein occlusion; n=178 other/uncertain) patients, 55% were women with an average age of 70 years. Patients most frequently reported receiving IVI every 4–5 weeks (40%). The mean TBS was 16.1±9.2 (range 1–48; scale of 1–54), and the TBS was higher in patients with diabetic macular oedema and/or diabetic retinopathy (DMO/DR) (17.1) compared with those with age-related macular degeneration (15.5) or retinal venous occlusive (15.3) (p=0.028). Though the mean level of discomfort was quite low (1.86) (scale 0–6), 50% of patients reported experiencing side effects more than half of the visits. Patients having received fewer than 5 IVI reported higher mean anxiety levels before (p=0.026), during (p=0.050) and after (p=0.016) treatment compared with patients having received more than 50 IVI. After the procedure, 42% of patients reported restrictions from usual activities due to discomfort. Patients reported a high mean satisfaction rating of 5.46 (scale 0–6) with the care of their diseases.ConclusionsThe mean TBS was moderate and highest among patients with DMO/DR. Patients with more total injections reported lower levels of discomfort and anxiety but higher disruption to daily life. Despite the challenges related to IVI, the overall satisfaction with treatment remained high.