Improved procedures for the glycosylation of serine and threonine utilizing Schiff base activation are reported. The procedures are less expensive and more efficient alternatives to previously published methods. The Schiff bases exhibited ring-chain tautomerism in CDCl₃ as shown by ¹H NMR. Acting as glycosyl acceptors, the Schiff bases reacted at RT with simple sugar peracetate donors with BF₃•OEt₂ promotion to provide the corresponding protected amino acid glycosides in good yields. With microwave irradiation, the reactions were complete in 2-5 minutes. Glycosylation with the dipeptide Schiff base shows the potential of this method in the preparation of peptide building blocks. To investigate this reaction further, direct glycosylation of sugar acetates with FMOC-Ser-OH/OBZl under BF₃•OEt₂ promotion in a microwave provided glycosides in high yield. In addition to the expected glycoside products acetylated side products resulting from acetate migration were isolated, suggesting that activation of the anomeric sugar acetates with a Lewis acid such BF₃•OEt₂ led to an oxocarbenium ion, which rearranged to a 1,2-dioxocarbenium ion because of the acetate participating group at C-2. Solvent participation was also illustrated with acetate migration being more pronounced when CH₃CN was used as a solvent and resulted in less product yield and higher amounts of the acetylated product. The acyl transfer products in these reactions where sugar acetates serve as glycosyl donors is reported for the first time, which also implies that ortho-ester like intermediates are important in the reaction mechanism. Keeping the message segment constant in the sequence H-Tyr-DThr-Gly-Phe-Leu- Ser-CO-NH₂ and modification of the address segment with different carbohydrate moieties had little effect on selectivity for binding at the μ, δ, or κ-opiod receptors. However, substitution of D-threonine with D-serine or the less polar D-alanine in the message segment resulted in a loss of κ-receptor affinity. Further replacement of D-threonine with the more hydrophobic D-valine resulted in complete loss of κ-binding affinity generating pure μ-δ agonists. These data suggests that changes in the message segment of the pharmacophore results in the glycopeptide adopting a conformation that is less favorable for 􀀁-binding receptor activity. Finally, the peripheral administration and i.c.v. tests of the drugs suggest that modifications in the message segment of the pharmacophore influences the potency of these compounds.

Pain is the most common and debilitating sign of a medical problem, with nearly 15 million patients suffering from chronic pain, including neuropathic pain. Widely used therapies for treating neuropathic pain include tri-cyclic antidepressants, opioids, anticonvulsants, non-steroidal anti-inflammatory agents and combinations thereof. Despite the abundance of treatments, the management of chronic pain remains difficult due to an inability for many patients to achieve appropriate pain relief at doses which are tolerable over long periods of time.Opiates (natural products), or opioids (synthetic derivatives), are considered the gold standard of analgesic care, though with little efficacy for neuropathic pain. Opioids are associated with unwanted side effects, including paradoxical pain and abuse liability that may result from several nervous system adaptations within the pain modulating neural network. These dose related side effects become more prevalent as clinicians try to overcome analgesic tolerance.Molecular mechanisms underlying these unwanted side effects have been studied extensively, and the literature purports a variety of contributing factors and neurobiological adaptations. The studies herein describe additional molecular adaptations and novel pharmacological approaches to counteract these changes. First, the contributions of neurobiological remodeling within a single receptor system (the opioid system) were investigated in the spinal dorsal horn after peripheral nerve ligation and chronic exposure to an opioid agonist in combination with an ultra-low-dose of opioid antagonist. The effects of the ultra-low-dose opioid antagonist naltrexone on the efficacy of oxycodone for neuropathic pain were investigated after both central and systemic administration.Secondly, molecular remodeling occurs across different receptor systems in the pain network, including altered regulation of pronociceptive molecules (e.g. substance P; SP). Previous studies have reported that opioid-induced hyperalgesia, tolerance and reward can be prevented by a blockade or ablation of SP activity at the neurokinin 1 receptor (NK1). We have characterized single compounds, rationally designed to act as opioid agonists and an NK1 antagonist using in vitro assays and the efficacy in vivo using rodent models of pain, antinociceptive tolerance and reward. Collectively, these studies validate the concept of targeting multiple neurobiological adaptations as a therapeutic option for neuropathic pain and reducing opioid- mediated side effects.

Metastatic bone cancer is the most common cause of pain in patients with malignant tumors. Prolonged opioid treatment remains the primary method to treat pain in these patients. Sustained morphine exposure enhances both bone cancer-induced pain and bone loss in mice implanted with sarcoma cells. Sustained treatment of bone marrow cultures with morphine results in COX-2 dependent upregulation of RANKL and PGE2, and suppression of OPG. This results in increased osteoclastogenesis which was dependent on COX-2 and OPG/RANKL regulatory axis. Treatment with morphine does not induce any direct changes in osteoclasts or sarcoma cells. The in vitro data was validated in the animals where morphine induces an increase in the osteoclastogenesis and RANKL, and suppresses OPG. These data indicate that morphine enhances osteoclastogenesis by modulating the OPG/RANKL regulatory axis in osteoblasts through a COX-2 dependent mechanism.Prolonged opioid exposure induces an opioid-receptor dependent hyperalgesia in humans and in animals. Studying the direct effect of opioids on primary sensory neurons we demonstrate a modest increase in CGRP cellular content that was not opioid-receptor dependent. Although dynorphin A (2-13) and PGE2 enhanced the release of the neuropeptide, pretreatment with opioids does not influence the capsaicin or KCl evoked CGRP release. These date indicate that the neurochemical changes seen in vivo may be dependent on factors upregulated in the periphery and/or the CNS.It has been demonstrated that sensory neurons innervating the femur express markers of neuronal injury and the intramedullary region of the femur becomes devoid of nerve fibers as the tumor expands. In this study we demonstrate that the sarcoma cells generate high levels of ROS and release hydrogen peroxide into the surrounding space, which induces death and injury to both sensory neurons and glia. This death was prevented by the anti-oxidants BHA and catalase. The present study provides evidence that ROS released by cancer cells can directly lead to injury and death of sensory neurons. ROS induced injury may be one of the mechanism through which sensory neurons are injured in the murine bone cancer pain model.

Regulation of the human delta opioid receptor (hDOR) is implicated in the development of tolerance to chronic morphine (Zhu et al., 1999). In addition, DORs are promising analgesic targets for the management of chronic pain states such as inflammatory or neuropathic pain (Cahill et al., 2007). Therefore, in this study, we investigated multiple aspects of hDOR regulation, including receptor phosphorylation, beta-arrestin binding, receptor internalization, down-regulation and desensitization, using recombinant Chinese hamster ovary (CHO) cells expressing the wild-type or various mutant hDOR constructs. We found that structurally diverse delta opioid agonists regulate the hDOR by different mechanisms. We demonstrate that morphine is able to activate the initial step of the regulatory events, phosphorylation of S363, but due to requirements for simultaneous activation of multiple sites, morphine fails to promote beta-arrestin binding, receptor internalization and down-regulation. We also report that peptide delta opioid receptor agonists and a non-peptide agonist SNC80 differ in their ability to down-regulate the hDOR. Further differences in receptor phosphorylation, desensitization and beta-arrestin translocation between these two classes of full DOR agonists are reveled by truncation of the receptor's C-terminus or by mutation of the primary phosphorylation site, S363. Studies using the mutant receptors identify the C-terminus as the important domain for hDOR phosphorylation, beta-arrestin binding and down-regulation by both peptide and non-peptide agonists. S363 within the C-terminus is critically involved in receptor phosphorylation, desensitization and down-regulation, but not in beta-arrestin binding and receptor internalization. In contrast to peptide agonists, SNC80 is able to phosphorylate and activate secondary intracellular domain(s), in addition to the C-terminus, which participate in beta-arrestin recruitment and receptor desensitization and down-regulation. Therefore, agonist-specific differences were detected for multiple regulatory events between morphine, peptide agonists and SNC80. Differential agonist-mediated regulation of the human delta opioid receptor may be used to design pain therapy drugs with improved analgesic properties and minimal side effects.

The work presented in this thesis concerns the opioid peptide dynorphin A (DynA). DynA functions primarily as a neurotransmitter and belongs to the family of typical opioid peptides. These peptides are a part of the opioid system, together with the opioid receptors, a family of GPCR membrane proteins. The opioid system system is involved or implicated in several physiological processes such as analgesia, addiction, depression and other types of neurological disorders. In this thesis, two biologically relevant aspects of DynA have been investigated with biophysical methods. First, interactions between DynA and an opioid receptor, and second, the direct membrane interactions of DynA. The DynA–receptor studies were focused on the selectivity-modulating second extracellular loop (EL2) of the kappa-opioid receptor (KOR). A protein engineering approach was used in which the EL2 was grafted onto a soluble protein scaffold. The results show that DynA binds with low affinity but high specificity to EL2 in the construct protein environment. The strength of the interaction is in the micromolar range, and we argue that this interaction is part of the receptor recognition event. With bicelles as a mimetic, membrane interactions were probed for wild-type DynA and for two DynA peptide variants linked to a neurological disorder. R6W–DynA and L5S–DynA were shown to be very different in terms of bicelle association, penetration and structure induction. In these experiments, as well as in investigations of DynA dynamics in bicelles, the lipid environment was shown to have much larger effects on peptide dynamics than on structure; and both these properties depend on lipid charge. Additionally, in a methodological project, DHPC/DMPC bicelle morphology as a function of total PC concentration was characterised by diffusion NMR in combination with two-way decomposition. The results may contribute to providing guidelines for the appropriate use of bicelles as a membrane mimetic. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 5: Manuscript.</p>

The experiments reported in this thesis studied the contributions of opioid and NMDA receptors to predictive fear learning, as measured by freezing in the rat. The first series of experiments (Chapter 2) used a within-subject one-trial blocking design to study whether opioid receptors mediate a direct action of predictive error on Pavlovian association formation. Systemic administrations of the opioid receptor antagonist naloxone or intra-vlPAG administrations of the selective μ-opioid receptor antagonist CTAP prior to Stage II training prevented one-trial blocking. These results show for the first time that opioid receptors mediate the direct actions of predictive error on Pavlovian association formation. The second series of experiments (Chapter 3) then studied temporal-difference prediction errors during Pavlovian fear conditioning. In Stage I rats received CSA ?? shock pairings. In Stage II they received CSA/CSB ?? shock pairings that blocked learning to CSB. In Stage III, a serial overlapping compound, CSB → CSA, was followed by shock. The change in intra-trial durations supported fear learning to CSB but reduced fear of CSA, revealing the selective operation of temporal-difference prediction errors. This bi-directional change in responding was prevented by systemic NMDA receptor antagonism prior to Stage III training. In contrast opioid receptor antagonism differentially affected the learning taking place during Stage III, enhancing learning to CSB while impairing the loss of fear to CSA. The final series of experiments (Chapter 4) then examined potential neuroanatomical loci for the systemic effects reported in Chapter 3. It was observed that intra-BLA infusion of ifenprodil, an antagonist of NMDA receptors containing the NR2B subunit, prevented all learning during Stage III, whereas intra-vlPAG infusion of the μ-opioid receptor antagonist CTAP facilitated learning to CSB but impaired learning to CSA. These results are consistent with the suggestion that opioid receptors in the vlPAG provide an important contribution to learning. Importantly, this contribution of the vlPAG is over and above its role in producing the freezing conditioned response. Furthermore, the findings of this thesis identify complementary but dissociable roles for amygdala NMDA receptors and vlPAG μ-opioid receptors in predictive fear learning.

Opioid dependence is a complex and persistent disorder with a high mortality rate and severe impact on health and social situation. It is associated with much harm, including the transmission of blood-borne bacterial and viral infections, self-harm, traumatic injury and drug overdose. All of these harms carry a risk of death, and accordingly, mortality rates in opioid-dependent people are many times higher than those in the general population of the same age and sex. One of the more commonly used strategies for reducing the risks of opioid dependence is the provision of maintenance treatment. In Australia, available maintenance treatments include methadone, buprenorphine, oral naltrexone, and the unregistered sustained-release formulation of naltrexone, naltrexone implants. This thesis reports on a range of data collections and study designs to investigate the levels, predictors and causes of mortality in opioid-dependent persons entering methadone, buprenorphine and naltrexone maintenance treatment in Australia. The studies used data linkage to examine mortality rates and causes of death in a longitudinal cohort of the early entrants to the NSW methadone program, examined the predictors of mortality (particularly the impact of methadone and buprenorphine treatment) using survival analysis in a longitudinal cohort study, compared national mortality rates between methadone, buprenorphine and naltrexone maintenance treatments in a cross-sectional analytic study, and used a small case series of coronial cases to examine whether death from opioid overdose was possible in a recipient of a naltrexone implant. This thesis demonstrates that mortality rates as a whole and from particular causes of death are many times higher in Australian opioid-dependent subjects than the general population, exposure to methadone or buprenorphine maintenance treatment significantly reduced mortality in a sample of opioid-dependent subjects, naltrexone treatment appears to have higher mortality than both methadone and buprenorphine maintenance treatments, and fatal opioid overdose while in receipt of sustained-release naltrexone treatment is possible. These results support longer retention in and repeated access to methadone and buprenorphine maintenance treatments in order to reduce mortality in opioid-dependent people, and greater regulation of the access to and more rigorous monitoring of the mortality associated with oral and sustained-release naltrexone maintenance treatments.

Opioid dependence is a complex and persistent disorder with a high mortality rate and severe impact on health and social situation. It is associated with much harm, including the transmission of blood-borne bacterial and viral infections, self-harm, traumatic injury and drug overdose. All of these harms carry a risk of death, and accordingly, mortality rates in opioid-dependent people are many times higher than those in the general population of the same age and sex. One of the more commonly used strategies for reducing the risks of opioid dependence is the provision of maintenance treatment. In Australia, available maintenance treatments include methadone, buprenorphine, oral naltrexone, and the unregistered sustained-release formulation of naltrexone, naltrexone implants. This thesis reports on a range of data collections and study designs to investigate the levels, predictors and causes of mortality in opioid-dependent persons entering methadone, buprenorphine and naltrexone maintenance treatment in Australia. The studies used data linkage to examine mortality rates and causes of death in a longitudinal cohort of the early entrants to the NSW methadone program, examined the predictors of mortality (particularly the impact of methadone and buprenorphine treatment) using survival analysis in a longitudinal cohort study, compared national mortality rates between methadone, buprenorphine and naltrexone maintenance treatments in a cross-sectional analytic study, and used a small case series of coronial cases to examine whether death from opioid overdose was possible in a recipient of a naltrexone implant. This thesis demonstrates that mortality rates as a whole and from particular causes of death are many times higher in Australian opioid-dependent subjects than the general population, exposure to methadone or buprenorphine maintenance treatment significantly reduced mortality in a sample of opioid-dependent subjects, naltrexone treatment appears to have higher mortality than both methadone and buprenorphine maintenance treatments, and fatal opioid overdose while in receipt of sustained-release naltrexone treatment is possible. These results support longer retention in and repeated access to methadone and buprenorphine maintenance treatments in order to reduce mortality in opioid-dependent people, and greater regulation of the access to and more rigorous monitoring of the mortality associated with oral and sustained-release naltrexone maintenance treatments.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2005. / Härtill 4 uppsatser.

Diss. (sammanfattning) Uppsala : Univ., 2003. / Härtill 5 uppsatser.