Byron C. Yoburn - US grants

Chronic treatment with the opioid antagonist drug, naltrexone, increases opioid binding sites in the central nervous system of rats and produces a 50% increase in the antinociceptive (analgesic) action of morphine (supersensitivity). In the mouse, naltrexone treatment produces a 3-fold greater increase in the analgesic potency of morphine than in the rat. While the increase in binding and morphine analgesia is a regularly observed phenomenon, little is known about the pharmacodynamics of opioid antagonist-induced upregulation and supersensitivity. The purpose of this application is to examine the consequences of chronic naltrexone-treatment upon opioid and nonopioid pharmacodynamics and brain opioid binding. We will determine the optimal dose of naltrexone to produce supersensitivity in the rat and mouse, and use this information to examine the development of tolerance and dependence to opioid agonists. The effect of naltrexone treatment on the development of supersensitivity and brain opioid binding in morphine-sensitive and insensitive strains of mice will be determined. In order to examine the role of particular opioid receptors in supersensitivity, the effect of selective opioid agonists will be determined. Since it is not known if supersensitivity is mediated by populations of neurons in the brain or spinal cord, or both, the contribution of these sites will be studied. The potential alterations in psychopharmacologic and toxic response to opioid and nonopioid drugs following opioid antagonist treatment will also be examined as will the possible pharmacokinetic changes in opioid agonists in brain and biofluids following naltrexone treatment. The effects of naltrexone-treatment on stress-induced (cold water swim) analgesia in the rat will be evaluated to assess the contribution of opioid and nonopioid systems in stress. Given that naltrexone has been recently approved for chronic use in the treatment of opioid dependence, these questions are of particular interest, since the possible consequences of upregulation in humans are unknown. Studies outlined in this proposal will systematically examine the functional consequences of opioid antagonist treatment on a variety of opioid and nonopioid effects. These studies will allow us to evaluate the significance, generality and specificity of opioid antagonist-induced upregulation and supersensitivity.

The objective of this project is to integrate minority students into a comprehensive research program aimed at addressing the relationship between opioid binding sites and pharmacodynamic potency of opioid agonists. Opioid receptor density and opioid agonist potency can be reliably altered by opioid antagonists, opioid agonists and CNS stimulants (e.g., cocaine and amphetamine). It is important to examine the pharmacologic and biochemical consequences of these drug treatments since they include drugs of abuse and significant tools in the clinical management of pain and drug abuse. The proposed studies that minority students will be involved in will explore the factors that mediate increases (upregulation) in opioid binding sites in the CNS. The effect of CNS stimulants and endocrine factors on opioid antagonist-induced upregulation will be studied. CNS stimulants and opioid agonists can also produce receptor upregulation. Studies will investigate the common mechanisms that converge on opioid receptor upregulation. Parallel pharmacodynamic studies using receptor selective agonists, and standard opioid analgesics, will assess the functional consequences of receptor changes. The relationship between receptors and pharmacodynamic response will also be examined in different species. These studies will build on the differences between the rat and mouse in terms of opioid potency and receptor characteristics. The differences in CNS stimulant potentiation of opioid agonist potency at mu and delta spinal cord and brain opioid receptors will be examined. We will assess in a simple in vitro system the mechanism by which CNS stimulants reliably increase opioid receptor density. The in vivo effects of CNS stimulants on opioid binding in the CNS will also be evaluated. We will examine alterations in opioid receptor-effector coupling using pertussis toxin, a bacterial toxin which interferes with opioid mediated effects. Using binding and pharmacodynamic assays, we will explore the effects of the toxin on opioid actions including analgesia, tolerance and toxicity mediated by specific opioid receptors. The overall goal is to provide an opportunity for minority students to be exposed to the excitement of basic science research in the context of probing the nature of opioid pharmacodynamics and receptor regulation. The scientific importance of these studies includes a more complete understanding of how opioid effects (analgesia, tolerance, dependence, toxicity), as well as the actions of cocaine and amphetamine, might be mediated. The proposed studies may increase understanding of the basic mechanisms of drug abuse and pain and may provide important practical information for developing new strategies for treatment.

The overall objective of this project is to examine the pathways that mediate mu-opioid receptor regulation and opioid tolerance in the intact animal. It has been established that opioid agonists differ in their ability to regulate CNS mu-opioid receptors in vivo. Chronic treatment with high intrinsic efficacy opioid agonists (e.g., etorphine) downregulates mu opioid receptors, while at the same time producing tolerance and regulation of mu-opioid receptor mRNA levels. Chronic treatment with lower intrinsic efficacy opioid agonists (e.g., morphine) can induce tolerance without altering mu-receptor density or mu-receptor mRNA. At present, the basis for the different effects of treatment with high and low intrinsic efficacy opioid agonists in vivo are not known. However, compelling data raise the possibility that opioid tolerance and mu-opioid receptor regulation require the activation of different intracellular signaling systems. This project will test the hypothesis that opioid agonist-induced downregulation of mu-opioid receptors and regulation of mu-opioid receptor mRNA levels in vivo involves three signaling proteins: G-protein receptor kinase 2,beta arrestin 2 and dynamin. It is further hypothesized that morphine-induced tolerance is independent of this signaling pathway in vivo. Finally, it is hypothesized that the magnitude of opioid tolerance is increased by mu-opioid receptor downregulation. The experiments proposed in this application will use behavioral, biochemical and molecular pharmacological methods to determine the role of these proteins in mediating opioid agonist-induced tolerance, downregulation of mu-opioid receptors and regulation of mu- receptor mRNA in vivo. The results of these studies will significantly enhance our knowledge of the pathways that regulate chronic opioid effects in the intact, behaving animal. As such, these results may provide important insights for developing strategies to treat opioid drug abuse and pain.

[unreadable] DESCRIPTION (provided by applicant): This project is in response to a request from NIDA for studies on opioid agonists that are found in commonly abused prescription pain medications. Recent data suggest a substantial increase in abuse of prescription opioid analgesics that are used to treat several clinical concerns, most notably pain. The overall goal of this project is to examine the pharmacodynamics of these opioid agonists. To address this goal, the project has 2 Specific Aims. Specific Aim 1 is to examine the relative analgesic potency of and production of tolerance to opioid agonists commonly found in prescription pain medications. Specific Aim 2 is to determine the relative efficacy of these opioid agonists and evaluate the role of efficacy in the development of analgesic tolerance and the regulation of mu-opioid receptors. The opioids to be studied were chosen based on the most recent SAMHSA data in the DAWN survey and the National Survey on Drug Use. The project will test 3 hypotheses. Hypothesis 1 is that the efficacy of opioid agonists determines the development of tolerance. Specifically, high efficacy agonists will produce less tolerance than equi-effective doses of low efficacy agonists in the intact mouse. Hypothesis 2 is that the efficacy of opioid agonists determines the regulation of mu-opioid receptor density in spinal cord of the intact mouse. Specifically, high efficacy agonists will reduce mu-opioid receptor density in the mouse spinal cord, whereas low efficacy agonists will either not affect, or increase mu-opioid receptor density. Hypothesis 3 is that directly changing the efficacy of an opioid ligand will alter the magnitude of tolerance and regulation of mu-opioid receptor density in spinal cord of the intact mouse. It is anticipated that irreversible alkylation of mu-opioid receptors will reduce the efficacy of opioid agonists and will increase opioid tolerance at equi-effective doses and reduce regulation of mu- opioid receptor density. Overall, the results of this project will provide new and important information on the pharmacodynamics and mechanism of action of a group of opioid analgesics that are potential drugs of abuse. [unreadable] [unreadable] [unreadable]