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Exploring the side effect profile of 16-Ethynyl Salvinorin A
<p>Introduction: Drug addiction is a chronic and relapsing disorder that has widespread socioeconomic and health consequences. Globally, there are over 29.5 million people who are drug dependent, and New Zealand has one of the highest rates of drug use rates in the developed world. Currently, there are no Food and Drug Administration (FDA) approved pharmacotherapies that target psychostimulant addiction. Kappa opioid receptor (KOPr) agonists are being studied as a potential pharmacotherapy as it utilizes the brain’s own mechanism for controlling reward, however, KOPr agonists have unwanted side effects such as dysphoria and sedation. This thesis explores the KOPr agonists Salvinorin A (Sal A), a naturally-occurring, highly potent and short-acting non-nitrogenous KOPr agonist and a structural analogue, 16-Ethynyl Salvinorin A (16-Ethy). KOPr agonists, such as Sal A have known preclinical anti-addictive and anti-reward effects, therefore, this thesis focuses on evaluating Sal A and 16-Ethy in preclinical tests of reward and side effects. Methods: Male Sprague-Dawley rats were used in preclinical tests to evaluate common KOPr-mediated side-effects including anxiety (elevated plus maze), depression (forced swim test) sedation (locomotor activity) and aversion (conditioned place aversion). The anti-cocaine effects were also examined using self-administration, dose-response and drug-behavioural sensitisation tests. 16-Ethy was tested at 2 mg/kg in all experiments. Results: Acute pre-treatment of 16-Ethy induced sedative effects in non-habituated locomotor activity but when rats were habituated prior to administration, no sedation was observed. In contrast, Sal A (2 mg/kg) had sedative effects in habituated, but not in non-habituated locomotor activity (p = 0.0037). Compared to vehicle-treated rats, 16-Ethy and Sal A did not display pro-depressive effects in the forced swim test, show anxiogenic or aversive properties or modulate behavioural sensitisation to cocaine. Cocaine self-administration and dose-response tests were not successfully completed. Conclusion: At 2 mg/kg, 16-Ethy was found to display sedative effects in non-habituated locomotor activity but not in a habituated paradigm. Compared to vehicle-treated rats, 16-Ethy did not display pro-depressive effects in the forced swim test, or display anxiogenic or aversive properties and did not show significant cocaine sensitisation. Cocaine self-administration and dose-response tests were not successfully completed and will need to be repeated to ascertain the effects of 16-Ethy on them. However, 16-Ethy has shown glimpses of promise as a potential pharmacotherapy against addiction.</p>
<p>Introduction: Drug addiction is a chronic and relapsing disorder that has widespread socioeconomic and health consequences. Globally, there are over 29.5 million people who are drug dependent, and New Zealand has one of the highest rates of drug use rates in the developed world. Currently, there are no Food and Drug Administration (FDA) approved pharmacotherapies that target psychostimulant addiction. Kappa opioid receptor (KOPr) agonists are being studied as a potential pharmacotherapy as it utilizes the brain’s own mechanism for controlling reward, however, KOPr agonists have unwanted side effects such as dysphoria and sedation. This thesis explores the KOPr agonists Salvinorin A (Sal A), a naturally-occurring, highly potent and short-acting non-nitrogenous KOPr agonist and a structural analogue, 16-Ethynyl Salvinorin A (16-Ethy). KOPr agonists, such as Sal A have known preclinical anti-addictive and anti-reward effects, therefore, this thesis focuses on evaluating Sal A and 16-Ethy in preclinical tests of reward and side effects. Methods: Male Sprague-Dawley rats were used in preclinical tests to evaluate common KOPr-mediated side-effects including anxiety (elevated plus maze), depression (forced swim test) sedation (locomotor activity) and aversion (conditioned place aversion). The anti-cocaine effects were also examined using self-administration, dose-response and drug-behavioural sensitisation tests. 16-Ethy was tested at 2 mg/kg in all experiments. Results: Acute pre-treatment of 16-Ethy induced sedative effects in non-habituated locomotor activity but when rats were habituated prior to administration, no sedation was observed. In contrast, Sal A (2 mg/kg) had sedative effects in habituated, but not in non-habituated locomotor activity (p = 0.0037). Compared to vehicle-treated rats, 16-Ethy and Sal A did not display pro-depressive effects in the forced swim test, show anxiogenic or aversive properties or modulate behavioural sensitisation to cocaine. Cocaine self-administration and dose-response tests were not successfully completed. Conclusion: At 2 mg/kg, 16-Ethy was found to display sedative effects in non-habituated locomotor activity but not in a habituated paradigm. Compared to vehicle-treated rats, 16-Ethy did not display pro-depressive effects in the forced swim test, or display anxiogenic or aversive properties and did not show significant cocaine sensitisation. Cocaine self-administration and dose-response tests were not successfully completed and will need to be repeated to ascertain the effects of 16-Ethy on them. However, 16-Ethy has shown glimpses of promise as a potential pharmacotherapy against addiction.</p>
<p>Kappa opioid peptide receptors (KOPrs) are a class of opioid receptors which shown analgesic and anti-addictive properties. Nonaddictive analgesics would be beneficial as morphine, one of the most commonly prescribed opioids for chronic pain, activates the brain reward system and can lead to addiction. Although medical research is progressing rapidly, there is still no treatment for psychostimulant abuse. KOPr agonists show promise in this regard but display undesirable side effects and could negatively affect memory. Salvinorin A (Sal A), a structurally unusual KOPr agonist, has a reduced side effect profile compared to the more traditional KOPr agonists such as U50,488. The effect of Sal A and U50,488 on memory is controversial as they have both been shown to induce a memory impairment and also to improve memory impairments. Sal A also has a poor pharmacokinetic profile with a short duration of action. Structural analogues of Sal A have improved pharmacokinetic and side effect profiles compared to Sal A yet retain the analgesic and anti-addiction properties. This thesis will investigate whether Sal A analogues, namely Ethynyl Sal A (Ethy Sal A), Mesyl Salvinorin B (Mesyl Sal B), and Bromo Salvinorin A (Bromo Sal A), produce a memory impairment. Male Sprague-Dawley rats were evaluated in the novel object recognition (NOR) task to determine whether novel Sal A analogues impair long term recognition memory. The degree of novelty was also investigated on a cellular basis through quantifying c-Fos immunoreactive neurons within the perirhinal cortex, an area of the brain shown to respond to novelty. Acute administration of Sal A (0.3 and 1 mg/kg) and novel analogues Ethy Sal A (0.3 and 1 mg/kg), Mesyl Sal B (0.3 and 1 mg/kg), and Bromo Sal A (1 mg/kg) showed no significant differences compared to vehicle when tested in the NOR task. The prototypical KOPr agonist, U50,488 (10 mg/kg), produced a significant decrease in recognition index compared to vehicle when tested in the same task as the novel analogues. Correlating the recognition indices calculated from U50,488 in the NOR to c-Fos counts in the perirhinal cortex showed a strong positive correlation with an increase in RI relating to an increase in c-Fos activation. U50,488 (10 mg/kg) showed a non-significant trend compared to vehicle in the number of c-Fos immunoreactive cells within the perirhinal cortex. Neither Sal A nor novel analogues affected NOR, suggesting no impairment of long term recognition memory. The lack of this side-effect, among others, demonstrates that the development of potent KOPr agonists with reduced side-effect profiles is feasible. These novel analogues show improvement over the traditional KOPr agonists.</p>
<p>Kappa opioid peptide receptors (KOPrs) are a class of opioid receptors which shown analgesic and anti-addictive properties. Nonaddictive analgesics would be beneficial as morphine, one of the most commonly prescribed opioids for chronic pain, activates the brain reward system and can lead to addiction. Although medical research is progressing rapidly, there is still no treatment for psychostimulant abuse. KOPr agonists show promise in this regard but display undesirable side effects and could negatively affect memory. Salvinorin A (Sal A), a structurally unusual KOPr agonist, has a reduced side effect profile compared to the more traditional KOPr agonists such as U50,488. The effect of Sal A and U50,488 on memory is controversial as they have both been shown to induce a memory impairment and also to improve memory impairments. Sal A also has a poor pharmacokinetic profile with a short duration of action. Structural analogues of Sal A have improved pharmacokinetic and side effect profiles compared to Sal A yet retain the analgesic and anti-addiction properties. This thesis will investigate whether Sal A analogues, namely Ethynyl Sal A (Ethy Sal A), Mesyl Salvinorin B (Mesyl Sal B), and Bromo Salvinorin A (Bromo Sal A), produce a memory impairment. Male Sprague-Dawley rats were evaluated in the novel object recognition (NOR) task to determine whether novel Sal A analogues impair long term recognition memory. The degree of novelty was also investigated on a cellular basis through quantifying c-Fos immunoreactive neurons within the perirhinal cortex, an area of the brain shown to respond to novelty. Acute administration of Sal A (0.3 and 1 mg/kg) and novel analogues Ethy Sal A (0.3 and 1 mg/kg), Mesyl Sal B (0.3 and 1 mg/kg), and Bromo Sal A (1 mg/kg) showed no significant differences compared to vehicle when tested in the NOR task. The prototypical KOPr agonist, U50,488 (10 mg/kg), produced a significant decrease in recognition index compared to vehicle when tested in the same task as the novel analogues. Correlating the recognition indices calculated from U50,488 in the NOR to c-Fos counts in the perirhinal cortex showed a strong positive correlation with an increase in RI relating to an increase in c-Fos activation. U50,488 (10 mg/kg) showed a non-significant trend compared to vehicle in the number of c-Fos immunoreactive cells within the perirhinal cortex. Neither Sal A nor novel analogues affected NOR, suggesting no impairment of long term recognition memory. The lack of this side-effect, among others, demonstrates that the development of potent KOPr agonists with reduced side-effect profiles is feasible. These novel analogues show improvement over the traditional KOPr agonists.</p>
<p>Background: The mu-opioid receptor (MOPr) activating drugs such as morphine, fentanyl, etorphine and methadone are used to treat moderate to severe pain. However, their long-term use produces serious adverse effects such as respiratory depression, sedation, tolerance, nausea, dependence, and constipation and this signifies the search for an alternate pain therapeutic agent. Here we report the investigation of antinociceptive and side effect profiles of a structurally unique MOPr-activating drug, kurkinorin from Salvinorin A (Sal A) that was compared with morphine and herkinorin. Methods: Adult male B6-SJL mice (22-29 g) were used to investigate the antinociceptive effects of kurkinorin, herkinorin and morphine utilising the 50° C warm-water tail-withdrawal assay. The 2% intra-dermal formalin assay was used to evaluate acute nociceptive and inflammatory pain and paw oedema. The side effect profiles were evaluated by measuring core-body temperature and utilising behavioural tests of motor co-ordination (accelerating rotarod test). Kurkinorin’s rewarding properties were assessed using the conditioned place preference (CPP) assay in male Sprague-Dawley rats (240-350 g). Results: Kurkinorin produced significant antinociceptive effects in the tail-withdrawal assay at both 5 (p<0.01, 10 min, p<0.001, 15-60 min) and 10 mg/kg (p<0.001, 5-90 min, p<0.01, 120 min) and attenuated both nociceptive and inflammatory pain in the 2% intra-dermal formalin model in mice. The analgesic effects of kurkinorin at 10 mg/kg were similar to the analgesic effects of morphine at the same dose. The decrease in pain score in the intra-dermal formalin assay with kurkinorin and morphine produced a corresponding reduction of paw oedema. In comparison, herkinorin had reduced analgesic effects in the tail-withdrawal assay (10 mg/kg, p<0.05, 30 min) and attenuated inflammatory pain in the intra-dermal formalin assay (10 mg/kg, p<0.001) with reduced paw oedema (10 mg/kg, p<0.05). Morphine produced significant motor incoordination effects from 15-60 min post injection whereas kurkinorin produced no significant motor impairment. Kurkinorin and herkinorin (5 mg/kg, i.p) did not produce rewarding effects, whereas morphine produced a significant, rewarding effect in the CPP assay. Kurkinorin produced no change in the core body temperature while morphine significantly reduced the body temperature. Conclusions: Kurkinorin is central acting and is as potent as morphine in attenuating acute nociceptive and inflammatory pain. It produced no significant sedative and rewarding effects. Therefore, kurkinorin has been identified as a structurally new class of mu-opioid analgesic, displaying improvements compared to morphine.</p>
<p>Background: The mu-opioid receptor (MOPr) activating drugs such as morphine, fentanyl, etorphine and methadone are used to treat moderate to severe pain. However, their long-term use produces serious adverse effects such as respiratory depression, sedation, tolerance, nausea, dependence, and constipation and this signifies the search for an alternate pain therapeutic agent. Here we report the investigation of antinociceptive and side effect profiles of a structurally unique MOPr-activating drug, kurkinorin from Salvinorin A (Sal A) that was compared with morphine and herkinorin. Methods: Adult male B6-SJL mice (22-29 g) were used to investigate the antinociceptive effects of kurkinorin, herkinorin and morphine utilising the 50° C warm-water tail-withdrawal assay. The 2% intra-dermal formalin assay was used to evaluate acute nociceptive and inflammatory pain and paw oedema. The side effect profiles were evaluated by measuring core-body temperature and utilising behavioural tests of motor co-ordination (accelerating rotarod test). Kurkinorin’s rewarding properties were assessed using the conditioned place preference (CPP) assay in male Sprague-Dawley rats (240-350 g). Results: Kurkinorin produced significant antinociceptive effects in the tail-withdrawal assay at both 5 (p<0.01, 10 min, p<0.001, 15-60 min) and 10 mg/kg (p<0.001, 5-90 min, p<0.01, 120 min) and attenuated both nociceptive and inflammatory pain in the 2% intra-dermal formalin model in mice. The analgesic effects of kurkinorin at 10 mg/kg were similar to the analgesic effects of morphine at the same dose. The decrease in pain score in the intra-dermal formalin assay with kurkinorin and morphine produced a corresponding reduction of paw oedema. In comparison, herkinorin had reduced analgesic effects in the tail-withdrawal assay (10 mg/kg, p<0.05, 30 min) and attenuated inflammatory pain in the intra-dermal formalin assay (10 mg/kg, p<0.001) with reduced paw oedema (10 mg/kg, p<0.05). Morphine produced significant motor incoordination effects from 15-60 min post injection whereas kurkinorin produced no significant motor impairment. Kurkinorin and herkinorin (5 mg/kg, i.p) did not produce rewarding effects, whereas morphine produced a significant, rewarding effect in the CPP assay. Kurkinorin produced no change in the core body temperature while morphine significantly reduced the body temperature. Conclusions: Kurkinorin is central acting and is as potent as morphine in attenuating acute nociceptive and inflammatory pain. It produced no significant sedative and rewarding effects. Therefore, kurkinorin has been identified as a structurally new class of mu-opioid analgesic, displaying improvements compared to morphine.</p>
<p>Background and Purpose: Pain, although necessary for survival, can become pathological affecting an estimated 1 in 5 adults globally. It is also the most common reason people seek medical attention. Mu opioid receptor (MOPr) agonists, such as morphine, are the gold standard treatment for pain. Although these drugs have excellent analgesic properties, adverse effects such as addiction, tolerance and respiratory depression make their use problematic. An estimated 10,000 New Zealanders are addicted to prescription opiates, highlighting the need for better drugs to treat pain. Kappa opioid receptor (KOPr) agonists have analgesic properties and, unlike MOPr agonists, are also anti-addictive. Unfortunately, adverse effects such as sedation and dysphoria, have limited their therapeutic potential. The discovery of KOPr agonists that have analgesic properties without inducing adverse effects can allow for better, more efficient treatments of pain. We are the first to report the analgesic potential of novel Salvinorin A (Sal A) analogues: Tetrahydropyran Salvinorin A (THP Sal A) and Mesyl Salvinorin B (Mesyl Sal B). Experimental Approach and Compounds Tested: This study uses animal behavioural models to characterise the analgesic, anti-oedematous, sedative and hypothermic effects of a structurally new class of KOPr agonists including Sal A, THP Sal A and Mesyl Sal B. The known peripherally restricted KOPr agonist, ICI 204,448, was used to evaluate the peripherally mediated analgesic mechanisms of KOPr agonists. The tail-flick and intradermal formalin test were used to assess acute central and peripheral pain processes respectively. Sedative effects were monitored via rotarod performance test; thermoregulatory effects were also recorded. Key Results: ICI 204,448 attenuated inflammatory pain at a dose of 1 mg/kg (P<0.05, 30 min) and 2 mg/kg (P<0.001, 30-35 min). Although it showed no centrally mediated analgesic effects, it was found to be sedative at a dose of 2 mg/kg (P<0.01, 15-60 min). Sal A (2 mg/kg) attenuated inflammatory pain (P<0.01, 25-35 min) at the same dose it was sedative (P<0.01, 2-15 min). Although it treated acute thermal pain at a non-sedative dose (1 mg/kg, P<0.001, 10-15 min), it has a short duration of action (˜15 min). THP Sal A attenuated both thermal and inflammatory pain. Unfortunately, it was also sedative at both 1 mg/kg (P<0.01, 15-45 min) and 2 mg/kg (P<0.001, 15-90 min). Mesyl Sal B significantly attenuated both central (1mg/kg, P<0.01, 30-60 min) and peripheral (2 mg/kg, P<0.01, 30 min) pain processes. Although Mesyl Sal B was found to have a weak analgesic effect in all pain assays, it was not sedative. Conclusions and Implications: KOPr agonists attenuate acute nociceptive and inflammatory pain. Structural modification of Sal A at the C-2 position alters its analgesic effects in vivo. Substitution with a tetrahydropyran group greatly improves central analgesic effects; however, sedative effects were also observed. Although substitution with a mesylate group produced no sedative effects, it had reduced effects on central and peripheral pain processes. The lack of sedation by Mesyl Sal B makes it a good target for future research in pain. Its longer duration of action compared to Sal A suggests it has a better metabolic profile. The creation of more soluble KOPr compounds would allow for better dose-testing to evaluate therapeutic potential of KOPr analgesics.</p>
<p>Background and Purpose: Pain, although necessary for survival, can become pathological affecting an estimated 1 in 5 adults globally. It is also the most common reason people seek medical attention. Mu opioid receptor (MOPr) agonists, such as morphine, are the gold standard treatment for pain. Although these drugs have excellent analgesic properties, adverse effects such as addiction, tolerance and respiratory depression make their use problematic. An estimated 10,000 New Zealanders are addicted to prescription opiates, highlighting the need for better drugs to treat pain. Kappa opioid receptor (KOPr) agonists have analgesic properties and, unlike MOPr agonists, are also anti-addictive. Unfortunately, adverse effects such as sedation and dysphoria, have limited their therapeutic potential. The discovery of KOPr agonists that have analgesic properties without inducing adverse effects can allow for better, more efficient treatments of pain. We are the first to report the analgesic potential of novel Salvinorin A (Sal A) analogues: Tetrahydropyran Salvinorin A (THP Sal A) and Mesyl Salvinorin B (Mesyl Sal B). Experimental Approach and Compounds Tested: This study uses animal behavioural models to characterise the analgesic, anti-oedematous, sedative and hypothermic effects of a structurally new class of KOPr agonists including Sal A, THP Sal A and Mesyl Sal B. The known peripherally restricted KOPr agonist, ICI 204,448, was used to evaluate the peripherally mediated analgesic mechanisms of KOPr agonists. The tail-flick and intradermal formalin test were used to assess acute central and peripheral pain processes respectively. Sedative effects were monitored via rotarod performance test; thermoregulatory effects were also recorded. Key Results: ICI 204,448 attenuated inflammatory pain at a dose of 1 mg/kg (P<0.05, 30 min) and 2 mg/kg (P<0.001, 30-35 min). Although it showed no centrally mediated analgesic effects, it was found to be sedative at a dose of 2 mg/kg (P<0.01, 15-60 min). Sal A (2 mg/kg) attenuated inflammatory pain (P<0.01, 25-35 min) at the same dose it was sedative (P<0.01, 2-15 min). Although it treated acute thermal pain at a non-sedative dose (1 mg/kg, P<0.001, 10-15 min), it has a short duration of action (˜15 min). THP Sal A attenuated both thermal and inflammatory pain. Unfortunately, it was also sedative at both 1 mg/kg (P<0.01, 15-45 min) and 2 mg/kg (P<0.001, 15-90 min). Mesyl Sal B significantly attenuated both central (1mg/kg, P<0.01, 30-60 min) and peripheral (2 mg/kg, P<0.01, 30 min) pain processes. Although Mesyl Sal B was found to have a weak analgesic effect in all pain assays, it was not sedative. Conclusions and Implications: KOPr agonists attenuate acute nociceptive and inflammatory pain. Structural modification of Sal A at the C-2 position alters its analgesic effects in vivo. Substitution with a tetrahydropyran group greatly improves central analgesic effects; however, sedative effects were also observed. Although substitution with a mesylate group produced no sedative effects, it had reduced effects on central and peripheral pain processes. The lack of sedation by Mesyl Sal B makes it a good target for future research in pain. Its longer duration of action compared to Sal A suggests it has a better metabolic profile. The creation of more soluble KOPr compounds would allow for better dose-testing to evaluate therapeutic potential of KOPr analgesics.</p>
<p>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.</p>
<p>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.</p>
