Thesis (Ph.D.) - University of Queensland, 2003. / Includes bibliography.

Thesis (M.Phil.) - University of Queensland, 2005. / Includes bibliography.

Abstract Ligand biased signaling of opioid agonists for phosphorylation andregulation of μ -opioid receptors Student: Vida Basti. Supervisor: Prof Macdonald Christie. Departmement of Neuropharmacology, The University of Sydney. Examiner: Prof Ingrid Nylander. Departement of Phamacology, University of Uppsala. Opioid drugs are of great use in the medical practise. The drugs are commonly prescribed formany types of illnesses, mostly in cases of pain management. Although opioids come withmany benefits they are causing a lot of problems as well. The side effects are many andamongst these is tolerance development which may lead to abuse and addiction. Because ofthe fast tolerance development in patients, higher doses up to 10 times the therapeutic doseare being prescribed. This is a major issue in today’s society and must be addressed.Scientists are trying to figure out the mechanism behind tolerance by comparing differenttypes opioid drugs. Some opioids causes tolerance in a much faster rate than others but it isstill uncertain why and what is causing this. Two of the most commonly prescribed opioidsare oxycodone and morphine and so in this rapport these opioids are compared with respectto their capability to cause internalization in neurons. In the experiments a positive control,DAMGO, is being used as well as a negative control. The method being used is an indirectmethod of immunohistochemistry on AtT20 transfected cell culture. The results show thatOxycodone seems to cause no internalization at all in comparison to the control.

Class of 2005 Abstract / Objectives: To standardize six tablets that share a statistically insignificant in vitro dissolution profile consisting of an experimental mixture of oxycodone HCl paired with dextromethorphan. We wanted to see if the release dynamics were not statistically different in an aqueous environment utilizing testing via USP Apparatus II (rotating paddles) in order to establish a drug release profile. Methods: Six experimental formulation tablets of oxycodone/DM were placed in separate dissolution vessels. The medium contained 900ml of water (standard media per USP) at 37°C (standard temperature per USP). Samples were taken at the 1, 2, 4, 6, 8, and 24 hour time periods and quantified using HPLC. The aim of this experiment was not meant to simulate an in vivo environment but simply to gain preliminary data for future research. Results: A one-sample t-test was used to calculate significant differences between the release profiles of oxycodone and dextromethorphan. We found that the release of all 6 tablets were not significantly statistically different for active ingredients, oxycodone and dextromethorphan. This data validated our hypothesis that the six experimental tablets would release the active ingredients over a 24-hour period at very similar and statistically insignificant rates. Implications: We now have a tablet formulation that can be replicated and used for further research including animal studies, and possibly human clinical trials, in order to develop a new pharmacotherapeutic approach for pain management.

The aim of this study was to investigate the influence of soft tissue decomposition on the stability of drugs incorporated in hair antemortem. Two burial trials were conducted: in the first trial, rats were administered oxycodone over five days; in the second trial, rats were administered heroin over nine days then drug abstinent for another nine days. After each respective treatment the rats were sacrificed and buried in controlled burial microcosms. Concentrations of oxycodone and selected metabolites or the metabolites of heroin; 6-monoacetylmorphine and morphine, incorporated within rat hair and liver were measured before and during the decomposition process. Oxycodone was analysed in hair and liver samples, while morphine and 6-monoacetylmorphine were analysed in hair samples by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Oxycodone concentrations in both hair and liver decreased as the interment period and decomposition of the carcasses progressed. 6-Monoacetylmorphine was not detected in any hair samples collected from the exhumed carcasses. / UOIT

Oxycodone is a semi-synthetic opioid originally developed as a safer alternative to morphine. It is commonly prescribed for its pain-relieving effects, but has recently been implicated as a major underlying cause of the current opioid epidemic due to its clinical limitations that include tolerance, dependence and a high abuse liability. Simultaneous consumption of opioids and ethanol has been shown to increase the risk of overdose and death from opioids in opioid-tolerant individuals. We hypothesized that ethanol reversed opioid tolerance and previous studies showed that ethanol reversed morphine tolerance. This dissertation investigated whether ethanol reversed tolerance to other opioids in mice, primarily oxycodone. We found that tolerance developed to the antinociceptive effects of both oxycodone and hydrocodone, and that the same dose of ethanol (1 g/kg i.p.) reversed that tolerance. Oral ethanol (2 g/kg) also effectively reversed oxycodone tolerance. Ethanol did not significantly alter either acute or chronic oxycodone brain concentrations, suggesting that the reversal effect was mediated by neuronal mechanisms. DRG neurons were isolated from adult mice and the effects of oxycodone were assessed using whole-cell patch clamp electrophysiology experiments. Oxycodone [3µM] acutely reduced neuronal excitability as measured by a shift in threshold potentials to a more positive value. DRG neurons incubated overnight with 10µM oxycodone did not respond to the 3µM oxycodone challenge, indicating tolerance developed within these neurons. To test if ethanol was reversing tolerance through neuronal mechanisms, we incubated DRG neurons overnight with 10µM oxycodone and applied 20mM ethanol to the media prior to recording. Tolerance was robustly reversed in these neurons, as indicated by a response to 3µM oxycodone. The PKC inhibitor, Bis XI, also reversed oxycodone tolerance. In these studies we have clearly shown that tolerance develops to oxycodone in both the whole animal in an isolated neuronal preparation. In addition we have shown that the tolerance produced in these two preparations was reversed by ethanol at blood levels similar to those seen in humans. Further we have also included preliminary data that suggest that this reversal of oxycodone tolerance by ethanol may well be due to its actions on PKC.

Disputats.

Opioid Use Disorder is known to have a heritable component, but the genetics underlying this heritability are not well understood. Inbred laboratory mice provide a stable genetic platform useful for interrogating the genetic component of specific opioid related behaviors, with the goal of identifying novel biology underlying opioid phenotypes. In this work we used inbred mice, leveraging both the near isogenic nature of substrains and the strain-specific inheritance of ancestral haplotypes, to explore the genetics of opioid phenotypes. Through these studies we found 1) Robust, female-specific increases in oxycodone state-dependent reward learning and whole brain concentrations of the oxycodone metabolite oxymorphone in BALB/cJ vs BALB/cByJ substrains. Oxymorphone is a full agonist at the mu opioid receptor, with a higher potency and efficacy than oxycodone, and thus, increased brain oxymorphone could enhance state-dependent oxycodone reward learning. Quantitative trait locus (QTL) mapping in a BALB/c F2 reduced complexity cross revealed one major QTL on chromosome 15 underlying brain oxymorphone concentration that explained 32% of the female variance. Cis-expression QTL analysis (eQTL), exon-level eQTL analysis, liver and brain proteomics identified Zhx2 as candidate underlying these differences. 2) In a study of ancillary phenotypes, BALB/cByJ mice showed enhanced sensitivity to thermal nociception and mechanical stimulation and decreased brain weight versus BALB/cJ. We identified a QTL on chromosome 13 for hot plate sensitivity and a QTL on chromosome 5 for brain weight. Cis-eQTL mapping identified H2afy and spliceome analysis revealed differential H2afy exon usage. Whole brain proteomics further supported H2afy as a candidate gene for thermal nociception and identified Acads as a candidate for reduced brain weight. 3) Using a panel of 29 inbred mice we assessed behavioral variation and associations within opioid sensitivity, reward, antinociception, and dependence. We identified very little correlation between measures, suggesting distinct genetic components regulating theses facets of OUD. This study is preliminary to GWAS analysis and identification of loci underlying phenotypic variation. In summary, inbred mice, combined with a multi-omic approach, provide a powerful tool to identify genetic loci and candidate genes underlying complex behaviors. Future studies will validate Zhx2 and H2afy through reciprocal gene editing and tissue-specific viral manipulations in BALB/c substrains and involve GWAS analysis using oxycodone strain survey data.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 150-161).

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