Pre-clinical Anti-Addictive and Side-Effect profiles of Novel Kappa-opioid Agonists

Background: Drug addiction is a chronic, relapsing disorder with great socioeconomic and morbidity costs. An estimated 27 million people worldwide suffer from drug dependence, with over 180,000 drug abuse-related deaths reported annually (UNODC, 2015). Currently, there are no FDA-approved pharmacotherapies for psychostimulant addiction, limiting the efficacy of treatment for cocaine and amphetamine abuse. Kappa-opioid receptor (KOPr) agonists can act as inhibitors of reward, and have been investigated in pre-clinical models of drug abuse for potential anti-addictive properties, but display undesirable side-effects such as dysphoria and sedation. A naturally-occurring KOPr agonist, Salvinorin A (SalA), has been explored as a lead for new KOPr-based anti-addictive medications. SalA is a short-acting but potent non-nitrogenous KOPr agonist with known anti-cocaine effects, and chemical alterations to this structure have produced novel agonists with comparable or greater potency at the KOPr. This thesis compares two novel SalA analogues, 16-ethynyl Salvinorin A (Ethy-SalA) and 16-methyl Salvinorin A (Me-SalA), in pre-clinical models of addiction and side-effect tests.  Methods: Sprague-Dawley rats were used to model the behavioural effects of acute KOPr treatment upon cocaine self-administration and drug-seeking behaviour, natural reward-seeking, cocaine-induced and spontaneous locomotion, and pro-depressive forced-swim testing. Transiently co-transfected HEK-293 cells were used to model the influence of KOPr activation upon dopamine transporter (DAT) function in an in vitro model of dopamine uptake, using confocal microscopy to detect internalisation of the fluorescent DAT substrate ASP+.  Results: Acute pre-treatments of Ethy-SalA significantly attenuated cocaine-reinstatement of drug-seeking behaviour (at 0.1 and 0.3 mg/kg) and progressive ratio (PR) self-administration of cocaine (at 2.0 mg/kg). The less potent agonist Me-SalA did not attenuate cocaine-reinstatement or PR self-administration at the doses tested (0.3-2.0 mg/kg). Despite apparent anti-cocaine effects, Ethy-SalA (0.3 mg/kg) was not found to effectively reduce cocaine-induced locomotor hyperactivity or sensitisation in rats. Side-effect screens were carried out on the novel compounds using the doses tested in cocaine-primed reinstatement. Ethy-SalA (0.3 mg/kg) and Me-SalA (1.0 mg/kg) did not significantly affect spontaneous locomotor behaviour 0.3 mg/kg, or reduce self-administration of the natural reward sucrose at a dose of 0.3 mg/kg in rats. Depression-like effects caused by acute Ethy-SalA treatment (0.3 mg/kg) were also not detected in the Forced Swim Test. Treatment with Ethy-SalA (10 µM) significantly increased uptake of the fluorescent ASP+ in co-transfected DAT/KOPr HEK-293 cells.   Conclusions: A single treatment of the novel KOPr agonist Ethy-SalA, but not the novel agonist Me-SalA, was found to attenuate drug-seeking behaviours in models of cocaine administration with greater potency than SalA, and without detectable sedative or depression-like effects at a dose of 0.3 mg/kg. The cellular mechanism-of-action by which Ethy-SalA depresses cocaine reward is at least in part due to positive regulation of DAT, which would act to reduce extracellular dopamine within the brain. The lack of significant side-effects and the apparent improved potency of the compound support further exploration of Ethy-SalA as a lead for the development of an anti-addictive pharmacotherapy.