Investigating the anti-cocaine effects of novel kappa opioid receptor agonists: Behavioural and cellular actions of Salvinorin A analogues

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.