Opioid-induced constipation (OIC) is one of the major adverse effects of opioid analgesics used by millions of patients each year. While progress has been made, there remains a significant unmet medical need in the treatment of OIC. Major gaps remain in our understanding of the role of the gastrointestinal tract and central nervous system (CNS) in precipitating OIC. For the last four decades, numerous investigations to study the sites of action of opioid analgesics have utilized peripherally acting mu-opioid receptor antagonists (PAMORAs), which have been incorrectly believed to have limited penetration across the blood-brain barrier (BBB). Several preclinical and clinical reports indicate that significant amounts of PAMORAs penetrate the BBB quite readily. As a result, the usage of current PAMORAs have resulted in misunderstandings of the role of the CNS and gastrointestinal tract in causing side effects such as opioid-induced constipation (OIC). We have developed a transthyretin-based novel drug delivery approach for restricting the passage of small molecules across the BBB. Our approach involves endowing the opioid agonist/antagonist with the selective transthyretin ligand, AG10. The newly synthesized naloxone- and oxycodone-based conjugates have demonstrated superior peripheral selectivity, improved pharmacokinetics, and efficacy in rats compared to other clinically used PAMORAs. Here we present chemical synthesis, in vitro binding and stability studies, as well as pharmacokinetic and pharmacodynamic evaluations of the AG10-opioid conjugates in rats. Our AG10-based PAMORA allowed us to obtain new insights into the important role of mu-opioid receptors in the central nervous system (CNS) in causing constipation. Additionally, our results demonstrate for the first time that synergy between mu-opioid receptors in the central nervous system and the gastrointestinal tract is crucial to the understanding of OIC and the development of effective treatment regimens. These findings contradict prior ideas that OIC was caused by a mechanism that involves primarily the gastrointestinal mu-opioid receptors. Moreover, we confirmed our findings by a AG10-oxycodone conjugate, a peripherally restricted opioid agonist. This molecule demonstrated the predominant role of CNS in OIC precipitation. The newly synthesized AG10-opioid conjugates represent a novel class of pharmacological probes that will aid in our understanding of OIC and other undesirable adverse effects of opioids. In addition, these conjugates have been evaluated for their potential therapeutic value in the preclinical studies. Collectively our approach to limit the BBB penetration of opioids will contribute to develop safer and more effective opioid medications.

Increases in basolateral amygdala (BLA) activity drive avoidance-seeking behavior that may be associated with stress induced drug seeking. Activity of BLA pyramidal neurons is regulated by local and paracapsular gamma aminobutyric acid (GABA) interneurons. The lateral paracapsular interneurons (LPCs) border the external capsule, receive dense cortical/thalamic input and provide feed-forward inhibition onto BLA principle neurons. The GABAergic LPCs also express high concentrations of g-protein coupled µ-opioid receptors (MORs). Therefore, the effects of opiates on LPC activity and local GABA release were examined. Fluorescently double labeled LPCs were observed in glutamate decarboxylase (GAD) 65-mcherry/GAD67-green fluorescent protein (GFP) transgenic mice. Whole-cell electrophysiology experiments demonstrated that acute exposure to [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO; a synthetic selective MOR agonist), reduced LPC firing and spontaneous inhibitory postsynaptic current (sIPSC) frequency in LPCs, with no apparent effect on spontaneous excitatory currents (sEPSCs). Current injection induced firing in LPC neurons, but less effectively than in saline controls. Morphine-exposed mice (10mg/kg/day, across 5 days, 1-2 days off) had increased sIPSCs compared to saline-injected controls, as well as enhanced adenylyl cyclase (AC) activity. Together these data show that LPC neurons are a highly sensitive targets for opiate-induced inhibition, and that long-term opiate exposure results in impaired LPC excitability, possibly contributing to anxiety observed during opiate withdrawal.

The purpose of this thesis project was to examine the effects of chronic alcohol on the excitability and molecular adaptation of GABA neurons of the ventral tegmental area (VTA). GABA neurons are of interest with regards to ethanol intoxication, reinforcement, and dependence due to their widespread distribution and connectivity to mesocorticolimbic dopamine (DA) neurons implicated in alcohol reward and addiction. Since we have previously shown adaptation of VTA GABA neuron firing rate to chronic ethanol (Gallegos, Criado et al. 1999) and suppression of gap-junction (GJ) mediated coupling between these neurons by acute ethanol (Stobbs, Ohran et al. 2004), we wanted to further characterize the effects of chronic ethanol on VTA GABA neuron excitability, electrical coupling and molecular adaptation. In particular, we analyzed the GJ mediated coupling and protein regulation of VTA GABA neurons following a three week period of continuous ethanol exposure via liquid diet. Although some animals showed tolerance, there was no significant tolerance to ethanol inhibition of GJ-mediated electrical coupling. In addition, we were able to characterize differences in mRNA expression levels for the DA synthesizing enzyme tyrosine hydroxylase (TH), the DA D2 receptor and the NMDAR2B receptor subunit in DA versus GABA neurons, all three of which were expressed at higher levels in DA neurons. We also determined the effects of chronic ethanol on mRNA levels of these same proteins as well as μ-opioid receptors (μORs) and connexin-36 (Cx36) GJs. Most significantly, we found a down-regulation of the DA D2 receptor, confirming that molecular modification occurs in these VTA GABA neurons with chronic alcohol. While we reject our hypothesis that acute ethanol inhibition of VTA GABA neuron electrical coupling would undergo tolerance to chronic ethanol in these non-dependent rats, which was the focus of this thesis, it remains to be determined if tolerance to chronic ethanol might be obtained in ethanol-dependent rats.

Opioid analgesics have been a major contribution to pain therapy with opioids being used as an effective treatment for various recalcitrant pain conditions. The drug class has come under increased scrutiny due to the raising concerns about the public health crisis of opioid misuse and addiction, thereby increasing the need for alternative and safer analgesics. The exploration of alternative pharmacotherapy for pain management has led to an increasing paradigm shift towards the development of a single-drug-multiple-target approach that takes inspiration from numerous naturally occurring drugs. The mu-opioid receptor has been the primary target for the management of pain; however, the voltage-gated sodium channel Nav1.7 is gaining attention as a putative antinociceptive target based on human genetic evidence. The proposed research aims to develop multi-target directed ligands (MTDL) that modulates two key targets for pain perception, the MOR, and Nav1.7 to generate analgesics with reduced side effects and enhanced analgesia. This will be achieved by exploiting polypharmacology to develop hybrid analgesia in two ways: (i) performing structure-activity relationship (SAR) studies to design a single drug with two pharmacophores that specifically interacts with both the targets (ii) exploiting in silico techniques by performing structure-based virtual ligand screening (VLS) of a chemical library. In our work, we report that through SAR studies and molecular docking studies that the designed compounds having in combination the pharmacophore of PZM21 and aryl sulfonamide demonstrate significant interactions between the active compounds and both the MOR and Nav1.7 proteins. This study also reports the first ever bifunctional virtual ligand screening where a library consisting of over a million compounds was screened for bifunctional activity at the MOR and the Nav1.7 ion channel. We also report the development of a novel mechanism-specific membrane potential assay to that can be used to screen for subtype selective Nav1.7 inhibitors. The research performed in this thesis will serve as a platform to explore the possibility of MTDL as potential therapeutic solutions to diseases of complex etiologies such as chronic pain. It will also serve as a starting point to exploring bifunctional VLS as a way to screen large chemical libraries for MTDLs.

Les opioïdes sont les analgésiques les plus efficaces dans le traitement des douleurs sévères. Ils produisent leurs effets en ciblant spécifiquement les récepteurs opioïdes localisés tout le long de la voie de perception de la douleur où ils modulent la transmission de l'information douloureuse. La plupart des études dans ce domaine essaient de caractériser les récepteurs opioïdes à l'état isolé de tout partenaire de signalisation. Cette thèse, par contre, montre que le récepteur opioïde delta (DOR) peut former un complexe avec sa protéine G et l'un de ses effecteurs impliqués dans la production de l'effet analgésique, le canal potassique à rectification entrante activée par les protéines G (Kir3 ou GIRK). Après avoir établi la présence de ce complexe constitutif, on a ensuite caractérisé sa stabilité, modulation et régulation suite à une stimulation avec des agonistes opioïdes. En premier lieu, on a caractérisé la transmission de l'information du récepteur DOR, suite à son activation par un agoniste, vers le canal Kir3. On a remarqué que cette transmission ne suit pas le modèle de collision, généralement accepté, mais nécessite plutôt un simple changement dans la conformation du complexe préformé. Ensuite, on a déterminé que même suite à l'activation prolongée du récepteur DOR par un agoniste complet, le complexe DOR/Kir3 maintenait son intégrité et a été reconnu par la βarrestine (βarr) comme une seule unité signalétique provoquant ainsi l'internalisation de DOR et Kir3 par un mécanisme clathrine et dynamine-dépendant. Ainsi, prises ensemble, ces données montrent que l'activation du récepteur DOR déclenche non seulement l'activation de l'effecteur Kir3 mais également un mécanisme de régulation qui élimine cet effecteur de la membrane plasmique. / Opioids are the most effective analgesics in the treatment of severe pain. They produce their effects by specifically targeting opioid receptors located all along the pain perception pathway where they modulate the transmission of pain information. Most studies in this area try to characterize the opioid receptor in isolation from any signaling partner. This thesis, on the other hand, shows that the delta opioid receptor (DOR) can form a complex with its G protein and one of its effectors involved in the production of the analgesic effect, the G protein coupled inward rectifying potassium channel (Kir3 or GIRK). Having established the presence of this constitutive complex, we then characterized its stability, modulation and regulation following stimulation with opioid agonists. First, we characterized the transmission of information from DOR, following its activation by an agonist, to the Kir3 channel. We have noticed that this transmission does not follow the collision model, generally accepted, but rather requires a simple change in the conformation within the preformed complex. Then, we have determined that even following prolonged DOR activation by a full agonist, the DOR/Kir3 complex maintained its integrity and was recognized by βarrestin (βarr) as a single signaling unit producing the internalization of DOR and Kir3 by a clathrin and dynamin-dependent mechanism. Thus, taken together, these data show that DOR activation triggers not only activation of the Kir3 effector but also a regulatory mechanism that removes this effector from the plasma membrane.

Bases: A antinocicepção induzida por substâncias doces tem sido largamente estudada. Contudo, a investigação dos neurotransmissores envolvidos nesse processo antinociceptivo ainda carece de mais estudos, pois é de extrema importância entender o envolvimento desses neurotransmissores no sistema neural que controla este tipo de antinocicepção. Objetivo: O objetivo deste estudo é clarificar o envolvimento dos sistemas opióide e serotoninérgico na antinocicepção induzida por substância doce. Método: O presente trabalho foi realizado em modelo animal (Rattus norvegicus, Rodentia, Muridae), objetivando investigar se a ingestão crônica de solução de sacarose é seguida de antinocicepção. A latência de retirada de cauda após a aplicação de estímulo nocivo térmico foi medida antes e após esse tratamento no teste de retirada de cauda (provavelmente um reflexo espinal). Não houve diferenças estatisticamente significantes entre os valores de linha basal dos diferentes grupos e foi calculado um índice de analgesia da latência de retirada de cauda antes e depois do tratamento. O envolvimento de opióides endógenos e de serotonina neste processo antinociceptivo foi pesquisado com fármacos antagonistas específicos e não-específicos dos receptores opióides e serotoninérgicos. Resultados: O efeito analgésico da ingestão de sacarose depende da concentração da solução de sacarose e do tempo de duração do consumo da mesma. Naltrexona e metisergida diminuíram a antinocicepção induzida por substâncias doce (após 14 dias de ingestão da sacarose). Estes efeitos foram corroborados pela administração periférica de naloxonazina e cetanserina. Conclusões: Os resultados sugerem o envolvimento de opióides endógenos e serotonina no processo antinociceptivo atualmente estudado. Tudo apontando para a participação de receptores opióides µ1 e serotoninérgicos 5-HT2 na regulação central da antinocicepção induzida por substâncias doces. / Rationale: Sweet substance-induced antinociception has been widely studied, and the investigation of the neurotransmitters involved in the antinociceptive process is an important way for understanding the involvement of neural system controlling this kind of antinociception. Objective: The aim of this study is to investigate the involvement of opioid and serotonergic system in the sweet substance-induces antinociception. Methods: the present work was made in animal model (Rattus norvegicus, Rodentia, Muridae); with the aim of investigating if the chronic intake of sweet substance, such as sucrose, is followed by antinociception. Their tail withdrawal latencies in the tail-flick test (probably a spinal reflex) were measured before and immediately after this treatment. As there was not statistic significant differences between baseline values of different groups, an analgesia index was calculated from the withdrawal latencies before and after treatment. The involvement of endogenous opioid and serotonin in the antinociceptive process was investigated with specific and non-specific pharmacological antagonism on opioid and serotonergic receptors. Results: The analgesic effect of sucrose intake depends on the concentration of sucrose solution and on the time during which the solution is consumed. Naltrexone and methysergide decreased the sweet substance-induced antinociception (post 14 days of sucrose intake). These effects were corroborated by peripheral administration of naloxonazina and ketanserin. Conclusions: The present results suggest the involvement of endogenous opioids and serotonin in the antinociceptive process presently studied. µ1-opioid and 5-HT2 serotonergic receptors may be involved in the central regulation of the sweet substance-produced antinociception.

<p>In this study, we have addressed the serotonergic and the opioid system within the CNS. Both systems are of outmost importance in the etiology of disease states, especially mental disorders. </p><p>In our investigation of the serotonergic system, we have synthesized novel enantiomerically pure 6-aryl-3-amino- and 8-aryl-3-aminochromans as ligands for the 5-HT₇ receptor. One reason for the lack of understanding of the physiological functionality of the serotonin 5-HT₇ receptor, the most recently discovered member of the serotonin receptor family, is the absence of partial agonists and agonists. In this series, we have identified partial agonists with more than189 fold selectivity over the 5-HT_1Areceptor and one agonist with 29 fold greater selectivity over the serotonin 5-HT_1Areceptor. Thus the present series constitutes a starting point for developing highly selective ligands for the 5-HT₇ receptor. </p><p>In our investigation of the opioid system, our focus has been on the natural product salvinorin A, which is a highly selective kappa opioid receptor agonist. In the total synthesis of salvinorin A, we have accomplished the synthesis of a key intermediate, 6-(3-furyl)-4-methyl-5,6-dihydro-pyran-2-one via ring closing metathesis. Furthermore, synthetic methodologies have been developed as a part of the total synthesis. Several lipases have been screeened for their ability to generate enantiomerically pure 1-(3-Furyl)-3-buten-1-ol via bio-catalyzed hydrolysis of the corresponding acetate. The lipase from <i>Pseudomonas fluorescens</i> was identified as having stereoselectivity high enough to generate a % <i>ee </i>value above 98%. We have also developed a route for the introduction of a hydroxyl functionality in the γ position of α,β-unsaturated cyclic ketones by the regioselective oxidation of 1-silyloxy-1,3-dienes using dimethyldioxirane. We have initiated the investigation of the pharmacophore responsible for the kappa opioid activity by synthesizing simplified analogues of salvinorin A. A synthetic route providing easy access to simplified analogues of salvinorin A have been established.</p>

In this study, we have addressed the serotonergic and the opioid system within the CNS. Both systems are of outmost importance in the etiology of disease states, especially mental disorders. In our investigation of the serotonergic system, we have synthesized novel enantiomerically pure 6-aryl-3-amino- and 8-aryl-3-aminochromans as ligands for the 5-HT7 receptor. One reason for the lack of understanding of the physiological functionality of the serotonin 5-HT7 receptor, the most recently discovered member of the serotonin receptor family, is the absence of partial agonists and agonists. In this series, we have identified partial agonists with more than189 fold selectivity over the 5-HT1A receptor and one agonist with 29 fold greater selectivity over the serotonin 5-HT1A receptor. Thus the present series constitutes a starting point for developing highly selective ligands for the 5-HT7 receptor. In our investigation of the opioid system, our focus has been on the natural product salvinorin A, which is a highly selective kappa opioid receptor agonist. In the total synthesis of salvinorin A, we have accomplished the synthesis of a key intermediate, 6-(3-furyl)-4-methyl-5,6-dihydro-pyran-2-one via ring closing metathesis. Furthermore, synthetic methodologies have been developed as a part of the total synthesis. Several lipases have been screeened for their ability to generate enantiomerically pure 1-(3-Furyl)-3-buten-1-ol via bio-catalyzed hydrolysis of the corresponding acetate. The lipase from Pseudomonas fluorescens was identified as having stereoselectivity high enough to generate a % ee value above 98%. We have also developed a route for the introduction of a hydroxyl functionality in the γ position of α,β-unsaturated cyclic ketones by the regioselective oxidation of 1-silyloxy-1,3-dienes using dimethyldioxirane. We have initiated the investigation of the pharmacophore responsible for the kappa opioid activity by synthesizing simplified analogues of salvinorin A. A synthetic route providing easy access to simplified analogues of salvinorin A have been established.

憂鬱症是盛行的精神疾病之一。慢性疼痛或是處在長期壓力情境的患者常與憂鬱症產生共病。在動物研究中，類鴉片delta受體制效劑能產生抗憂鬱效果，並且在發炎性疼痛的研究也指出類鴉片delta受體制效劑能展現抗痛覺過敏的效果。本研究主要利用大白鼠腦室內給予類鴉片delta受體制效劑SNC80以及三環抗憂鬱劑amitriptyline，來探討並比較其所產生的抗憂鬱效果在發炎性疼痛或長期壓力情境下與正常情境下的異同。大白鼠強迫游泳試驗被用來比較測試藥物的抗憂鬱效果；佛氏完全佐劑經由皮下注射至大白鼠右後腳掌底板來產生發炎性疼痛；腎上腺皮質酮經由皮下注射且持續21天來產生長期性壓力；西方墨點法用來檢驗在發炎性疼痛或長期壓力下，類鴉片delta受體蛋白質在大白鼠海馬迴的細胞膜上的改變。另外，拮抗劑實驗則用來確認類鴉片delta受體所產生的抗憂鬱效果。實驗結果顯示，大白鼠在正常情境下，SNC80及amitriptyline皆能產生抗憂鬱效果；然而在發炎性疼痛下，SNC80所產生的抗憂鬱效果有提高的表現，並且類鴉片delta受體蛋白質的數量在海馬迴的細胞膜上也隨著疼痛的時間增長而增加，amitriptyline則跟正常情境下的效果相似。另外，大白鼠在長期性壓力下，SNC80的抗憂鬱效果則沒有提高的表現，並且類鴉片delta受體蛋白質的數量在海馬迴的細胞膜上也未受到改變。本研究透過行為實驗提出類鴉片delta受體制效劑的藥理特性，並用分子生物學的方法來對應行為實驗的結果。本研究可做為未來類鴉片delta受體制效劑在治療慢性疼痛的憂鬱症患者上，可能發展為抗憂鬱藥的一個證據。 / Depression is one of the most prevalent mental illnesses all over the world. Patients with chronic pain or stress often have depression. Previous studies have shown that delta opioid receptor (DOR) agonists produced antidepressant-like effects in animal models and that antihyperalgesic effects of DOR agonists can be enhanced in rats under inflammatory pain. The aim of the study was to investigate and compare the antidepressant-like effects of a DOR agonist, SNC80, and a tricyclic antidepressant, amitriptyline, following intracerebroventricular (i.c.v.) administration in rats under different states. The forced swim test was used to determine the antidepressant-like effects of test compounds. Complete Freund’s adjuvant was injected subcutaneously into the right hind paw of rats to elicit inflammatory pain. Corticosterone was injected subcutaneously once per day for 21 days to induce chronic stress. The western blot was used to quantify the levels of DOR protein on plasma membrane in the hippocampus of rats under inflammatory pain or chronic stress. In addition, antagonist experiment was conducted to verify the receptor mechanism underlying the antidepressant-like effects of DOR agonist. Results indicated that i.c.v. SNC80 and amitriptyline dose-dependently produced antidepressant-like effects in rats under normal state. More importantly, the potency of SNC80-induced antidepressant-like effects, but not amitriptyline, was enhanced in rats under inflammatory pain. In addition, up-regulation of supraspinal DORs was time-dependently associated with enhanced antidepressant-like effects of SNC80 in rats under inflammatory pain. On the other hand, SNC80 did not produce enhanced antidepressant-like effects, and DOR density was not changed in rats under chronic stress. This study provides evidence of the DOR agonist’s state-dependent effects and suggests that DOR agonists may be more effective as potential antidepressants for patients with depression comorbid with chronic pain.

5 Summary: Currently, the outcomes from traumatic exsanguination cardiac arrest (CA) show that over 50% of deaths due to trauma occur at the scene, where medical care is limited. Less than 10% of patients who become pulseless from trauma survive. However, in an appropriate setting, some of those traumatic injuries could be surgically repairable. Emergency preservation and resuscitation (EPR) is a novel approach for resuscitation of exsanguination CA victims. EPR uses deep hypothermic preservation for prolonged CA to buy time for transport, damage control surgery, and delayed resuscitation with cardiopulmonary bypass (CPB). Initially, we used a dog model to maximize clinical relevance. We showed that the efficacy of EPR is related to the depth of hypothermia and duration of CA. Pharmacologic adjuncts tested to augment hypothermia generally failed. Extended hemorrhagic shock did not prevent the success of EPR vs. conventional resuscitation if extended post-resuscitative hypothermia was provided. Oxygenation of the flush allowed extending of survivable duration of deep hypothermic CA. Because of the lack of molecular tools available for use in dogs, we developed a rat EPR model to study the cellular and molecular mechanisms underlying deep hypothermic neuroprotection to allow us to define specific targets for...