Neuropathic (NP) pain is a debilitating form of chronic pain that can result from a variety of disease states that target the sensory nervous system. NP pain can manifest as burning or shooting pain, and is commonly refractory to traditional analgesics including opioids. Clinically used opioids target the mu opioid receptors (MORs); preclinical reports, however, suggest that the delta opioid receptor (DOR) may be a valid target. It is now accepted that MORs and DORs oligomerize in heterologous expression systems and exhibit novel pharmacology distinct from their monomers. This research aimed to utilize both functional and molecular evidence to identify if a heteromeric mu-delta opioid receptor oligomer (M/DOR) forms in vivo and whether it is upregulated in NP pain. In an animal model of NP pain, animals displayed characteristic behaviours including protecting of the ipsilateral hindpaw from environmental stimuli, and mechanical allodynia in this paw. Behavioural studies reported that acute injection of DOR- selective agonists that bind M/DOR produced enhanced thermal antinociception and reversed mechanical allodynia in NP rats. DOR agonists that have low binding affinity for M/DOR did not produce enhanced thermal antinociception but did reverse mechanical allodynia in NP rats. Molecular studies were employed to characterize the molecular species of ORs in the lumbar spinal cord. Isolated spinal cord membranes were subjected to coimmunoprecipitation with a M/DOR antibody. Co-immunoprecipitation was unable to conclusively identify changes in M/DOR levels in the dorsal horn but did confirm that such a species exists in vivo. Furthermore, antibody characterization was completed to determine if the commercial antibodies used were labeling the appropriate OR proteins. HEK293T cells transfected with MOR and/or DOR plasmids were used in Western blotting and immunocytochemistry protocols to test commercially available antibodies. These studies determined that MOR and DOR antibodies do label their respective OR type, but also recognize additional proteins (non-specifically) in Western blotting protocols. In conclusion, behavioural studies revealed a putative role for M/DOR agonists in the treatment of NP pain; however, more sensitive tools and protocols must be developed before molecular experiments are able to identify quantifiable changes with endogenous M/DORs. / Thesis (Master, Pharmacology & Toxicology) -- Queen's University, 2009-09-24 12:43:58.472

Angiogenesis is a fundamental process in reproduction and wound healing. Angiogenesis is also indispensable for solid tumor growth and metastasis, and also associated with angiogenic diseases. Beta-endorphin (£]-EP), derived from its precursor pro-opiomelancortin (POMC), is well known for its role in nociception and immune regulation. However, the function of morphine and £]-EP during angiogenesis remains characterization. One previous study indicated that morphine inhibited the proliferation and hypoxia-induced vascular endothelial growth factor (VEGF) release of endothelial cells. Contrastingly, another report found that morphine via Ras/PI3k/MAPK/ERK signaling promotes the survival and angiogenesis in endothelial cells. Besides, endogenous opioid peptides stimulated angiogenesis in chicken allantoic membrane assay through opioid receptors. Thus, the function and mechanism of £]-EP and opioid receptors in angiogenesis are controversial. This study evaluated the culture effects of £]-EP and morphine on angiogenesis . It was found that £]-EP stimulated the proliferation, migration, and tube formation of endothelial cells in a dose-dependent manner. Morphine at a high dose inhibited the proliferation, migration, and tube formation of endothelial cells. In the ex vivo rat aortic ring assay, £]-EP enhanced, whereas morphine perturbed, the microvessel sprouting. We also confirmed the expression of MOR¡ADOR¡AKOR opioid receptor in endothelial cells. Application of naloxone, a selective opioid antagonist, and neutralizing antibodies of MOR abolished the angiogenic effect of £]-EP and morphine. Thus £]-EP and morphine exert the pro- and anti-angiogenic effect via MOR, respectively .Besides, £]-EP can be regarded as a novel angiogenic factor.

Recently we have begun to investigate a novel role of EphB receptors in opiate-dependant analgesia. EphB2-β-galactosidase knockins demonstrate that EphB2 is persistently expressed within a number of neural pathways involved in MOR-mediated nociception in vivo and that EphB2 colocalizes with markers of the MOR at the cellular level in the spinal cord and dorsal root ganglia. Despite demonstrating wild-type levels of sensory and motor activity, EphB2 null mice exhibit a significantly altered analgesic response to repeated (but not naive) opiate exposure compared to controls. Investigation of EphB2 null mice and wild type animals revealed no differences in MOR protein levels or affinity. Analysis of this opiate-mediated tolerance suggests that associative phenomena play a substantial role in mediating the analgesic effects observed, possibly due to defeciencies in CA1-mediated learning. Therefore, loss of EphB2 may diminish context-dependent learning and that such learning plays a substantial role in regulating morphine-dependent tolerance.

Recently we have begun to investigate a novel role of EphB receptors in opiate-dependant analgesia. EphB2-β-galactosidase knockins demonstrate that EphB2 is persistently expressed within a number of neural pathways involved in MOR-mediated nociception in vivo and that EphB2 colocalizes with markers of the MOR at the cellular level in the spinal cord and dorsal root ganglia. Despite demonstrating wild-type levels of sensory and motor activity, EphB2 null mice exhibit a significantly altered analgesic response to repeated (but not naive) opiate exposure compared to controls. Investigation of EphB2 null mice and wild type animals revealed no differences in MOR protein levels or affinity. Analysis of this opiate-mediated tolerance suggests that associative phenomena play a substantial role in mediating the analgesic effects observed, possibly due to defeciencies in CA1-mediated learning. Therefore, loss of EphB2 may diminish context-dependent learning and that such learning plays a substantial role in regulating morphine-dependent tolerance.

Accumulating evidence suggests a prominent role of the arrestin-dependent signaling pathway in triggering most of the deleterious side effects observed using δ-OR targeting drugs. Numerous small molecules targeting the δ-OR receptors have been developed but their pharmacological properties, including their functional selectivity, have been poorly characterized. The absence of functionally selective opioid drugs, and the lack of knowledge of the pharmacological profile and signaling properties of the δ-OR receptor, limits its therapeutic exploitation. The development of functionally selective modulator toward the canonical G protein pathway could importantly increase the therapeutic potential of this receptor while decreasing its deleterious effects. An approach to fine-tune the functional selectivity of a GPCR is by using allosteric modulators. These allosteric modulators would reduce problems associated with drugs targeting the orthosteric site by not chronically activating the receptor. The overall goal of the proposed research is to study the molecular mechanism by which sodium-channel inhibitors allosterically regulates the delta opioid receptor (δ-OR) signaling and functional selectivity. Additionally, the signaling features of the δ-OR signal transduction triggered by biased receptor activation have been investigated. A combination of approaches, including functional studies, molecular modeling and mutagenesis, were used to study the general mechanism underlying the activation and tuning of the δ-OR signal transduction behavior. Thus, this work suggests the druggability of the allosteric sodium pocket by using sodium channel inhibitors. The current research represent discovery of two different allosteric profiles for the β-arrestin recruitment and one allosteric profile for the G-protein pathway at activated DOR and would serve as scaffold for further refinement of modulators with the desired pharmacological profile.

Most clinical opioids produce analgesia through the Mu Opioid Receptor (MOR) providing the only effective treatment for chronic pain patients. These studies explore three pre-clinical strategies to improve MOR analgesia and minimize side effects: 1) compounds that target G-protein Coupled Receptors (GPCRs) heterodimers, such as heterodimerization between the Delta Opioid Receptor (DOR) and MOR (MDOR); 2) multi-functional compounds that target multiple receptor systems for synergistic effects, such as a MOR agonist and a the serotonin reuptake transporter (SERT) inhibitor; or 3) biased agonists that preferentially activate one signaling pathway associated with analgesia over another associated with side effects at the same receptor. First, several indirect lines of evidence indicate the MOR-DOR heterodimer (MDOR) can regulate MOR opioid tolerance and withdrawal. However, studying MDOR remains difficult because no selective MDOR antagonists are available. To address this need, we created a novel series of bivalent MDOR antagonists by connecting a low affinity MOR antagonist (H-Tyr-Pro-Phe-D1Nal-NH2) to a moderate affinity DOR (H- Tyr-Tic-OH) antagonist with variable length polyamide spacers (15-41 atoms). In vitro radioligand binding and [35S]-GTPγS coupling assays in MOR, DOR, and MDOR expressing cell lines show bivalent ligands produce a clear length dependence in MDOR but not MOR or DOR cell lines. The lead compound – D24M with a 24-atom spacer – displayed high potency (IC50MDOR = 0.84 nM) with 91-fold selectivity for MDOR:DOR and 1,000-fold MDOR:MOR selectivity. Second, clinicians have long appreciated subtle but distinct differences in analgesia and side effects of MOR opioids. A variety of non-MOR targets including DOR, Kappa Opioid Receptor (KOR), the Cannabinoid Receptor-1 (CB1), the Sigma-1 Receptor (σ1R), the Dopamine- (DAT), Serotonin- (SERT) and Norepinephrine- Reuptake Transporters (NET) induce analgesia and/or modulate MOR mediated side effects. To determine if different opioid profiles arise from non-MOR interactions, we evaluated the binding and function of nine clinical analgesics at the nine aforementioned targets revealing several clinical opioids contain previously unidentified affinity’s or activity’s. Hydrocodone displayed low affinity at the MOR (KI = 1800 nM) and only ~2 fold less affinity at the σ1R (KI = 4000 nM). Second buprenorphine promoted monoamine influx at DAT, SERT and NET with EC50 > 1,000 nM. These novel interactions suggest the nuanced differences of clinical opioids may arise from previously unappreciated off-target effects. Future studies will assess whether these in vitro results predict hydrocodone and buprenorphine activity in vivo. Finally, the unique function of the numerous endogenous opioid peptides at a given receptor remains unclear. How endogenous ligands interact with ORs produces obvious drug design consequences. These studies show two endogenous Dynorphin analogues – Dynorphin A and Dynorphin B – differentially regulate two ubiquitous signaling modules – βarrestin2 and Gαi/o– at the DOR. Dynorphin A and Dynorphin B swap potency rank orders for β-arrestin2 recruitment and [35S]-GTPγS signaling, indicating two distinct signaling platforms are formed. Dynorphin A but not Dynorphin B treatment simulated AC super activation, while Dynoprhin B internalized DOR better than Dynorphin A. These in vitro assays suggest endogenous Dynorphin analogues differentially regulate signals at the DOR in vitro. Future work includes further characterizing signaling differences in vitro and testing these changes in vivo.

L’addiction ou dépendance aux substances psychoactives est une affection chronique, fréquente et grave, émaillée de rechutes et de périodes d’abstinence. Les études épidémiologiques montrent que l’abstinence aux opiacés est fortement associée à une prévalence accrue de la dépression. Nous résumons ici les principaux aspects cliniques de la dépendance aux opiacés et de la dépression, en détaillant leurs mécanismes physiopathologiques. Puis, nous présentons notre modèle d’abstinence aux opiacés chez la souris. Suite à un traitement morphinique chronique et au cours de l’abstinence apparaissent progressivement des comportements apparentés à la dépression. Ce traitement morphinique modifie profondément le fonctionnement du système sérotoninergique, notamment dans le noyau du raphé dorsal. De plus, les déficits comportementaux observés peuvent être prévenus par un traitement chronique par la fluoxétine, un antidépresseur ciblant ce système. Nous avons généralisé ce modèle à l’héroïne, un autre opiacé illicite. Nous avons révélé par des approches génétiques de délétion constitutive et conditionnelle les rôles distincts des 3 récepteurs opioïdes (mu, delta et kappa) lors de l’abstinence à l’héroïne. Enfin, nous avons initié une étude de caractérisation, à l’échelle de l’ensemble du génome, des adaptations transcriptomiques (ARN messagers et micro-ARN) dans le noyau du raphé dorsal au cours de l’abstinence à l’héroïne et du traitement antidépresseur. Ce travail devrait permettre d’améliorer notre compréhension des mécanismes neurobiologiques à l’œuvre dans la comorbidité entre dépendance aux opiacés et dépression et pourrait suggérer de nouvelles pistes thérapeutiques. / Addiction is a chronic, frequent and serious brain disease, with relapse alternating with abstinence periods. Epidemiological studies show that abstinence, notably from opiates, is strongly associated with depression.Here we present the main clinical aspects of opiate addiction and depression, and most recent advances in molecular pathophysiology of both disorders. Then, we present our mouse model of opiate abstinence. Following chronic morphine exposure, depressive-like behaviours progressively emerge. Morphine treatment profoundly disrupts serotonergic signalling, notably in the dorsal raphe nucleus. In addition, behavioural deficits can be prevented by chronic treatment with fluoxetine, an antidepressant targeting serotonergic neurons. We then generalized our mouse model to heroin, another major illicit opiate. Using constitutive and conditional knockout strategies, we documented distinct roles for all 3 opioid receptors (mu, delta and kappa) in heroin abstinence. Finally, we initiated a large-scale analysis of transcriptomic regulations (mRNA and micro-RNA) occurring in our model as a function of heroin abstinence and fluoxetine treatment.These studies should reveal an unforeseen contribution of the dorsal raphe nucleus to addiction. They should uncover new molecular mechanisms underlying depressive-like behaviors in mice during opiate abstinence and thus put forward new therapeutic targets in humans.

The goal of this dissertation was two fold: 1) To relate dopamine responses in the ventral striatum to ethanol preference and consumption, and 2) to investigate the role of the mu opioid receptors in this ethanol induced dopamine release in the ventral striatum. First a two bottle choice experiment established that a substrain of C57BL/6 mice (C57BL/6NCrl) had significantly less preference for and consumption of ethanol than a second substrain of mouse based on the same background (C57BL6/J). The C57BL/6 strain has been extensively used in alcohol drinking studies and is well known for it’s propensity to consume alcohol over water. To determine if differences in ventral striatal dopamine response vii could contribute to this variability in drinking behavior, we characterized the dopamine response in both substrains of mice after intraperitoneal injections of 1.0, 2.0 or 3.0 g/kg ethanol or saline. We found that the acute intraperitoneal ethanol injections in naïve mice caused a significant elevation in dopamine in both substrains at all three doses with a significant difference between substrains at the two highest alcohol doses. Therefore, ethanol induced dopamine release in the ventral striatum may contribute to ethanol preference and consumption. Next, we investigated the effect of acute intraperitoneal ethanol injections on naïve mu opioid receptor knockout mice and in mice pretreated with a mu opioid receptor antagonist. The mice used were all established on the C57BL/6J background. We found that ventral striatal dopamine response was similar in these mice after 1.0, 2.0 and 3.0 g/kg intraperitoneal ethanol injections compared to appropriate controls. As both gene deletion and pharmacological blockade of the mu opioid receptor did not affect ethanol stimulated dopamine release, it points to the conclusion that this receptor may not play a significant role in ethanol induced ventral striatal dopamine release. / text

Thesis advisor: Alexa H. Veemena / Across species, the juvenile period is characterized by increased social interaction with peers and heightened novelty-seeking behavior, as compared to any other life stage. These behaviors are likely to be highly adaptive during this developmental phase. Still, an excessive novelty-seeking phenotype may predispose individuals to risk-taking and substance abuse, while too little social engagement and low novelty-seeking are characteristics of neuropsychiatry disorders such as autism. The over-arching aim of this dissertation research has been to elucidate the neural mechanisms underlying juvenile social novelty-seeking behavior. Central activation of oxytocin, vasopressin V1a, and µ-opioid receptors (OTR, V1aR, and MOR, respectively) have been implicated in the regulation of adult social behavior, but our understanding of the expression and function of OTR, V1aR, and MORs in the juvenile brain is incomplete. Therefore, in Studies 1 and 2, age differences in binding density of OTR, V1aR, and MOR throughout the rat brain were identified using receptor autoradiography. Next, in Study 3, I established the social novelty preference test, a new paradigm designed to assess the preference of juvenile rats to interact with either a novel or a familiar (cage mate) conspecific. Using this social novelty preference test, in Studies 3, 4, and 5, the functional involvement of OTR, V1aR, and MOR in the regulation of juvenile social novelty preference was characterized using both intracerebroventricular and local in-vivo pharmacological manipulations. The results of these experiments demonstrate that both OTR and MOR activation in the brain are involved in the regulation of juvenile social novelty preference, particularly acting within the nucleus accumbens. Finally, in Study 5, I investigated the impact of social isolation on juvenile social novelty preference. My findings show that social isolation potently reduces social novelty preference, which, in turn, can be restored by MOR activation in the nucleus accumbens. Taken together, this body of work significantly advances our understanding of the neural systems underlying juvenile social novelty preference, and suggests that both oxytocin and opioid systems in the brain may be potential clinical targets for restoring social novelty-seeking behavior in neurodevelopmental disorders, such as autism.

Living organisms are capable of producing novel terpenoids with both remarkable ease and great selectivity. Many of these natural products exhibit significant biological activity useful for treatment of human diseases, but isolation of highly sought chemicals often results in only minute quantities. Consequently, extraction of these potential therapeutics from natural sources becomes an unrealistic method for obtaining enough material for a thorough biological evaluation, and so synthesizing these compounds becomes essential. Synthesis of terpenoids as potential therapeutics requires exceptional selectivity, especially when corresponding isomers elicit a contrasting biological response. The necessity for such selective syntheses along with the inherent structural complexities of terpenoids, often presents a number of significant challenges for the synthetic chemist. Isolation of the terpenoids pawhuskins A–C and petalostemumol from Dalea purpurea was reported by Belofsky in 2004, and of the collected compounds pawhuskin A was found to exhibit the most significant activity in an opioid receptor assay in vitro. Natural pawhuskin A was extracted from “Purple Prairie Clover” in only a 39 mg quantity and therefore syntheses of the natural product along with several analogues were pursued. Two of the synthesized analogues demonstrated greater potency than pawhuskin A, and interestingly these two isomeric derivatives were found to be selective for two different opioid receptors. However, the synthetic route utilized to form these two derivatives was not very selective for either isomer, and thorough purification proved challenging. Ergo, an alternative approach was sought to ensure the purity of these potential therapeutics. Parallel syntheses affording high selectivity for the key isomeric intermediates as well as a third regioisomer have been developed. The new isomeric intermediate also allowed the synthesis of two new analogues. This work is described in this report along with the formation of additional pawhuskin derivatives. The activity of these analogues as opioid receptor modulators also will be discussed.