Mary Jeanne Kreek - US grants

Drug and alcohol abuse and dependency, along with all of the medical and behavioral complications of drug abuse, ranging from hepatitis B and delta, cirrhosis and AIDS to psychiatric disorders and sociopathic behaviors, are the major medical problems confronting our nation and the world today. Effective treatments, based on fundamental knowledge about the drugs of abuse, pharmacologic agents used in the treatment of drug abuse, and the biological bases of addictive disease, as well as information about and approaches to the management of medical and behavioral problems which may complicate treatment, are needed urgently. Heroin abuse continues to afflict over 2 million persons at this time, with over one-half million "hard-core" heroin addicts in the United States, constituting the greatest numbers of patients in the second highest risk group for AIDS. Cocaine abuse continues to escalate, with around 6 million persons now reported to be regular users of cocaine and with increasing numbers using the more potent free-base form, "crack". The central theme of the proposed Center will be to identify and study the biological correlates of treatment of addictions, and as a major part of this effort, to study in depth several factors which may negatively, and also positively, affect treatment outcome. Problems such as medical and behavioral including pharmacological, metabolic and behavioral aspects of treatments, will be studied. In addition, some studies will attempt to elucidate further the biological bases of addictive diseases, with the goal of developing in the future novel, improved or more specific pharmacological as well as behavioral approaches for the treatment of various addictions. To the same end, with the goal of developing new or modifying existing treatments to improve both short- and long-term treatment outcome, studies are proposed to delineate the effects of existing treatments on those aspects of physiology which may by intrinsically involved with the addictive disease process, or which may, by inadvertent alterations of physiology, contribute to treatment failure. In the proposed Center, studies of basic science, basic clinical research, and applied clinical research types will all be focused on the central theme of studying the biological correlates of the treatment of addiction. These various scientific approaches will be integrated through the Core Resources and other interactions which only a Center can appropriately provide.

This is an application for renewal of the Research Scientist Award by Mary Jeanne Kreek, MD. Narcotic addiction continues to be a major problem in the United States, with heroin abuse at some time identified in around 2 million persons, and regular use of heroin in over one-half million persons. The second largest risk group for the development of AIDS and for presence of infection with the human immunodeficiency virus (HIV) are parenteral drug abusers, primarily heroin addicts. This group may be the primary source of spread of HIV to the heterosexual population and to infants. At this time approximately 75,000 former heroin addicts are in chronic methadone maintenance treatment; however waiting lists for treatment continue to grow. Cocaine abuse including abuse by smoking the new free-base "crack" form, the most rapidly increasing drug abuse problem, with around 6 million people in the U.S. using cocaine regularly. Although several studies on treatment of cocaine abuse are in progress, to date there is no effective treatment generally available. Knowledge as to the biological basis of narcotic addition, although increasing, remains incomplete; even less is known about the biological basis of cocaine dependency in humans. The overall objectives of our proposed studies, which will involve both clinical and experimental research, as well as new technique development, will continue to focus on: 1) studies of disposition, metabolism, and drug interactions of addictive drugs and drugs used in the management of addictions; 2) studies of the physiological and pharmacological effects of opioids, opioid antagonists, and cocaine on neuroendocrine, gastroenterological, hepatic and immunological function, with emphasis on elucidating the biological correlates of treatment of addictions, the roles of endogenous opioids in normal physiology and in pathological states, and the possible relationships of defined physiological effects of the metabolic basis of addictive disease; and 3) surveillance of patients in chronic pharmacological treatment of narcotic addiction, and also cocaine dependency, coupled with studies of the natural history, during treatment, of co-existing major medical problems, especially alcoholic liver disease, hepatitis-B, hepatitis delta, and HIV infections, all of which are major public health problems.

Heroin, cocaine and alcohol addiction with their frequent complications including hepatitis B, C, non-A, non-B and Delta, cirrhosis, and AIDS to psychiatric and sociopathic disorders, remain the major medical problems confronting our nation. Effective treatments based on a fundamental understanding of the biological basis of addictive diseases, the effects of drugs of abuse, and of pharmacological agents used or potentially useful in the treatment of drug abuse, as well as information about approaches to the management of the medical and behavioral problems which may complicate treatment, are still urgently needed. Our Treatment Research Center will continue to identify and study the biological correlates of addictions, and as a major part of this effort, to study factors which affect treatment outcome. Pharmacological, metabolic, medical, and behavioral problems which complicate treatment will be studied. In this proposal for continuation of our Center, all laboratory and clinical research studies are developed on an interactive basis, relying upon the Center for their integration. Six continuing projects are: 1) to study further the disposition, metabolism, processing, and interactions of exogenous and endogenous opioids and their antagonists, developing novel techniques of laser desorption mass spectrometry to study opioids and neuropeptides; 2) to define the effects of selected drugs of abuse and treatment agents on neuroendocrine function and to examine the relationship of atypical responsivity to induced stress which we have previously observed in preliminary studies, demonstrated in heroin and cocaine addicts to the clinical phenomenon of craving and relapse; 3) to further study the endogenous opioid system of the gastrointestinal tract and its relationship to that of the brain, and to extend clinical studies of an opioid antagonist with limited systemic bioavailability for the management of opiate induced gastrointestinal disorders; 4) to extend studies on the molecular biology of the viroid-like Delta agent, and to explore the use of custom constructed oligonucleotide ribozymes to destroy the Delta agent and also to cleave selected mRNAs: 5) to study further mechanisms involved in control of enkephalin gene expression in the hamster adrenal medulla. Three are new projects: 6) to study the effects of cocaine, morphine, alcohol and potential treatment agents on opioid receptors and peptides; 7) in parallel, to study the effects of drugs of abuse and treatment agents on opioid gene expression using the sensitive technique of solution hybridization protection assays for quantitation of mRNAs. These scientific projects will be integrated through the Core Laboratory and Clinical Resources, strengthening interactions which only a Research Center can provide, an advantage which has been documented during the first four years of this Research Center.

This subproject examines neuroendocrine function during the early and late phases of abstinence in heavy cocaine users. We are also interested in studying the effects of serotonin uptake inhibition on depressive symptoms in this group. An additional interest is in characterizing the pharmacokinetics of serotonin uptake inhibition in cocaine abusers. It should be emphasized that the study concerns neuroendocrine and neurotransmitter mechanisms, and is not a study of treatment efficacy.

The most effective treatments for each of the addictive diseases probably will be based on a fundamental understanding of the biological bases of addictive diseases; the physiological as well as pharmacological effects of drugs of abuse and of agents used for the treatment of drug abuse or addiction; as well as on information about approaches to the management of the other medical and behavioral problems which may complicate treatment. Research activities will continue to identify and study the biological correlates of addictions, factors which affect treatment outcome and also the neurobiological bases of addiction. Pharmacological, metabolic, neuroendocrine, immunological, medical and behavioral problems which are present in addicts at time of entry to treatment, and which may complicate treatment, will be studied. Specific projects include: 1) further studies using conventional techniques of the disposition, metabolism, processing, and interaction of exogenous and endogenous opioids and their antagonists, as well as their related neuropeptides, along with development of novel techniques using laser desorption extended range matrix assisted mass spectrometry for qualitative and quantitative measurements of opioids, other neuropeptides, opiates, and opioid antagonists; 2) studies of the effects of selected drugs of abuse and treatment agents as well as selected neuropeptides, including dynorphin peptides, on neuroendocrine function, immune function, and on mood and behavior, and to study the relationship of atypical responsivity to induced stress, which we have previously observed in preliminary studies in both abstinent heroin addicts and cocaine addicts, to the clinical phenomena of craving and relapse; 3) further studies of the effects of cocaine, morphine, alcohol, and potential treatment agents using rodent models (rat, guinea pig and mouse), a) on opioid receptors, using techniques of quantitative autoradiography and other in vivo techniques, b) on opioid peptide gene expression by use of a modified sensitive technique of solution hybridization protection assay for quantitation of specific mRNA's, and c) measurement of neuropeptides and the processing of active neuropeptides using conventional and novel techniques under development; 4) use of the technique of microdialysis to study neurotransmitter and potential neuropeptide release as well as possibly levels of administered drugs or drug metabolites in specific brain regions in the setting of administration of drugs of abuse and/or potential treatment agents; 5) further studies of the endogenous opioid system of the gastrointestinal tract and its relationship to the brain in animal models, and basic clinical research studies of the use of an opioid antagonist with limited systemic bioavailability for the management of opioid induced gastrointestinal disorders; 6) Studies of the possible utility of the effects of the opioid antagonist nalmefene in the management of chronic alcoholism; and 7) continued prospective surveillance of the medical status of patients in methadone maintenance treatment, determining the changing patterns of hepatitis Beta, delta, and HIV-1 infection in this group, and also determination of the response to hepatitis Beta vaccination in former heroin addicts in methadone maintenance treatment, and studies of the events of early HIV-1 infection.

Heroin, cocaine and alcohol addiction with their frequent complications including hepatitis B, C, non-A, non-B and Delta, cirrhosis, and AIDS to psychiatric and sociopathic disorders, remain the major medical problems confronting our nation. Effective treatments based on a fundamental understanding of the biological basis of addictive diseases, the effects of drugs of abuse, and of pharmacological agents used or potentially useful in the treatment of drug abuse, as well as information about approaches to the management of the medical and behavioral problems which may complicate treatment, are still urgently needed. Our Treatment Research Center will continue to identify and study the biological correlates of addictions, and as a major part of this effort, to study factors which affect treatment outcome. Pharmacological, metabolic, medical, and behavioral problems which complicate treatment will be studied. In this proposal for continuation of our Center, all laboratory and clinical research studies are developed on an interactive basis, relying upon the Center for their integration. Six continuing projects are: 1) to study further the disposition, metabolism, processing, and interactions of exogenous and endogenous opioids and their antagonists, developing novel techniques of laser desorption mass spectrometry to study opioids and neuropeptides; 2) to define the effects of selected drugs of abuse and treatment agents on neuroendocrine function and to examine the relationship of atypical responsivity to induced stress which we have previously observed in preliminary studies, demonstrated in heroin and cocaine addicts to the clinical phenomenon of craving and relapse; 3) to further study the endogenous opioid system of the gastrointestinal tract and its relationship to that of the brain, and to extend clinical studies of an opioid antagonist with limited systemic bioavailability for the management of opiate induced gastrointestinal disorders; 4) to extend studies on the molecular biology of the viroid-like Delta agent, and to explore the use of custom constructed oligonucleotide ribozymes to destroy the Delta agent and also to cleave selected mRNAs: 5) to study further mechanisms involved in control of enkephalin gene expression in the hamster adrenal medulla. Three are new projects: 6) to study the effects of cocaine, morphine, alcohol and potential treatment agents on opioid receptors and peptides; 7) in parallel, to study the effects of drugs of abuse and treatment agents on opioid gene expression using the sensitive technique of solution hybridization protection assays for quantitation of mRNAs. These scientific projects will be integrated through the Core Laboratory and Clinical Resources, strengthening interactions which only a Research Center can provide, an advantage which has been documented during the first four years of this Research Center.

The objectives of this subproject are (1) examine possible genetic factors underlying individual differences in susceptibility to drug addiction and chemical dependency (2) establish a bank of both DNA samples and cell lines from individuals actively or formerly addicted to heroin, cocaine, alcohol, and other abused drugs as well as from a group of normal control volunteers and (3) learn whether genetic or inherited differences in the endogenous opioid system, as well as other related systems, might cause an individual to be predisposed to addictive diseases.

The objectives of this subproject are (1) to study the distribution, metabolism, excretion, persistence, and drug interactions of long-acting exogenous opioids (narcotics) using methadone and LAAM as prototypic agents in methadone maintained patients; (2) to extend studies of the physiological effects of methadone and LAAM, as well as the effects of other pharmacotherapeutic agents in chemical dependency, and drug free treatment in chronic maintenance patients during treatment and following detoxification from methadone; and (3) to determine the natural history of diseases common in addicts during treatment.

This pilot, open-label, single-center trial is designed to evaluate the safety, tolerance, CD4+ cell response, viral load (in plasma, cerebrospinal fluid and intestinal tissue) reduction, and durability of viral load reduction of long-term quadruple antiretroviral therapy, using zidovudine (ZDV), epivir (3TC), 1592U89 and 141W94. Additionally, the study will enable determination of rates of decay of HIV in gut-associated lymphoid tissue (GALT) and cerebrospinal fluid, providing an understanding of the decay characteristics of HIV replicates in viral reservoirs other than peripheral blood. Twenty-four HIV-1 subjects will be studied: 12 with acute (within 90 days) infection and 12 with chronic infection. The initial duration of the study is 24 weeks. Based on review of ongoing toxicology studies, agreement with the sponsor and FDA, and contingent upon IRB approval, the treatment duration will extended to 48 weeks. If the safety profile of the four-drug combination remains acceptable, and contingent upon agreement with the sponsor and the FDA and approval of the IRB, the treatment duration may be extended indefinitely.

This study will examine the effect of acute administration of alcohol in non-alcoholic human volunteers following different modes of administration. In addition, this study will examine the effect of opioid antagonist administration in non-alcoholic human volunteers and abstinent alcoholics. Most studies so far have either given alcohol intravenously, or per os. Only a few studies have, to our knowledge, been done to compare these modes of administration of alcohol in humans.

Using 18-F cyclofoxy,which binds to both mu and kappa subtype opioid receptors, it will be possible through positron emission tomography scanning (performed at the NIH campus) to determine the localization of opioid receptors in specific brain responses of both volunteer subjects with addictive diseases and healthy subjects.

We aim to investigate the mechanisms of reinforcement of co-existing heavy alcohol and nicotine abuse. Specifically, we will determine the neuroendocrine activity, mood effects, and any elicited symptomatology during and after cigarette smoking in heavy smokers with different pre- existing alcohol drinking patterns. We will also study the effects of a pre-administered dose of an opioid antagonist on smoking response in the same smokers of differing drinking patterns.

This subproject examines neuroendocrine function during the early and late phases of abstinence in heavy cocaine users. We are also interested in studying the effects of serotonin uptake inhibition on depressive symptoms in this group. An additional interest is in characterizing the pharmacokinetics of serotonin uptake inhibition in cocaine abusers. It should be emphasized that the study concerns neuroendocrine and neurotransmitter mechanisms, and is not a study of treatment efficacy.

alcoholism /alcohol abuse; corticotropin releasing factor; drug addiction antagonist; hypothalamic pituitary adrenal axis

This subproject utilizes the opioid antagonist oral naloxone (or oral nalmefene glucuronide) to treat opioid-induced constipation in former heroin addicts currently in methadone maintenance treatment. Constipation is a common side effect of methadone maintenance treatment.

Comparison studies in both normal healthy volunteers and also on defined contrast groups of patients with defined addictive diseases will be studied to determine the effects of typical and atypical response to stress on neuroendocrine indices.

The purpose of this study is to test the hypotheses that endogenous opioids may play an important role in maintaining normal physiological control of intestinal function and they may also play a major role in the pathophysiology of some defined (as well as idiopathic) gastrointestinal motility disorders.

We have recently made a novel discovery which supported a much earlier hypothesis, that activation of the hypothalamic-pituitary-adrenal axis may precede, rather than simply follow, opioid antagonist (eg. naloxoneprecipitated withdrawal in opioid dependent persons, whether they are persons receiving opioids on a chronic basis for the treatment of opiate addiction or for the management of chronic pain. In this study, which will be reported in Metabolism (Culpepper-Morgan, J and Kreek, MJ, Volume 46, February, 1997) we were able to document hypothalamic- pituitary-adrenal axis hypersensitivity in a patient with chronic pain management with chronic opioid agonist and thus, with opioid dependence. As part of a larger study on attempts to manage opioid-induced gastrointestinal motility disorders in patients receiving opioids on a chronic basis, a titration study was conducted in which small amounts of the opioid antagonist naloxone, which has extremely limited systemic bioavailability after oral administration, was administered in incremental doses on separate days. Both clinical symptoms and signs of opiate withdrawal, including both adrenergic and opioidergic signs of withdrawal, as well as objective measurements of hormones of the hypothalamic- pituitary-adrenal axis were assessed. It was found that objective changes in the hypothalamic-pituitary-adrenal axis preceded the onset or appearance of clinical signs and symptoms of withdrawal and in fact, at some low doses, occurred in the absence of any clinical signs or symptoms of withdrawal. Also, plasma levels of naloxone were strongly correlated with plasma cortisol levels. Neuroendocrine changes reflecting activation of the hypothalamic- pituitary-adrenal axis persisted long after the adrenergic changes in clinical symptoms had ameliorated, either spontaneously or by administration of a short-acting opioid antagonist. This study suggests that the hypothalamic- pituitary-adrenal axis is a more sensitive indicator of opioid withdrawal than the adrenergic system. Moreover, it suggests that activation of the stress- responsive hypothalamic-pituitary-adrenal axis may contribute to or cause many of the changes seen in both acute opiate withdrawal and possibly contribute to the protracted abstinence syndrome. These findings have potentially enormous implications both in the management of pain and also in the management of opiate addiction. In the management of chronic pain, especially in the setting of the potential for eliminating opioid agonist treatment, an understanding of the role of the activation of the hypothalamic- pituitary-adrenal axis in the signs and symptoms of opiate withdrawal may allow new approaches to be targeted to preventing or attenuating such activation. Similarly, in the management of short-term opiate addiction, where so-called "detoxification" treatments of either the pharmacological or nonpharmacological type may be indicated, again, increased information about the role of activation of the stress-responsive hypothalamic-pituitary- adrenal axis may be of value in developing new therapeutic innovations. Of possibly greatest importance are the implications of these findings for the understanding of both drug craving or drug hunger, and also the high propensity to relapse in medication-free former opiate-dependent persons. These are novel findings which may ultimately lead to improvement of therapeutic approaches both for the management of short-term and long-term addiction and the management of patients with chronic pain requiring opioid agonist treatment.

Drug addiction continues to be a major medical and social problem. It is estimated that one million or more persons in the United States are currently addicted to heroin, with millions more worldwide. Cocaine addiction and alcohol dependence are frequent comorbid conditions in heroin addicts in addition to being major primary addictions. Many studies over the past thirty years have shown that these drugs disrupt physiologic systems, and that these disruptions may contribute to drug addiction and alcohol dependence and to relapse to drug or alcohol abuse following withdrawal and abstinence. Clinical observations suggest that individuals differ in their response to heroin, cocaine, and alcohol; however, little is known about specific underlying hereditary genetic factors which might influence individual susceptibility to the addictive properties of these substances. Studies also suggest that both common and distinct heritable factors account for the genetic variance in the susceptibility to the separate addictive diseases. We hypothesize that there is a heritable as well as environmental basis for the acquisition and persistence of, and relapse to, specific addictive diseases. In this R01 application we propose to expand and increase our existing collection of DNA and immortalized cell lines from individuals without and with opioid and related drug dependencies and psychiatric comorbidities. Two separate types of genetic analyses will be used to determine association and linkage. All study subjects will be extensively characterized with respect to the addictive diseases, medical history, family medical addictive disease history; psychiatric comorbidity, and psychological profile, as well as ethnic background. A hypothesis driven approach will be utilized for the discovery of unknown polymorphisms in genes known to be involved in the responses to drugs of abuse, using traditional and novel methods. Functional studies of variant gene products caused by polymorphisms we identify will be performed, since altered function of these proteins may be important in individual responses to drugs of abuse, individual differences in the development and persistence of addiction, or in relapse to addiction, as well as in many aspects of normal physiology in which that gene product is involved. A better understanding of the consequences of genetic contributions with respect to protection from, or susceptibility to, heroin addiction and related codependencies and comorbid conditions, could have enormous importance in both prevention and treatment of this problem.

The overall goal of this project is to understand the biological mechanisms underlying the addictive diseases, focusing on three addictions that pose enormous public health problems and carry devastating morbidity and mortality: addictions to heroin and other related opiates, cocaine, and alcohol. Pursuing this goal has led not only to the development of safe and effective treatments, but in parallel, has led to the elucidation of fundamental aspects of normal human physiology, and also the neurobiological mechanisms underlying addictive diseases which continues to guide the development of new treatments.1 A related goal is to study the natural history of each of these three addictive diseases and how other diseases which are very prevalent in the addict population, including hepatitis B, delta, C, HIV-1 infection, and AIDS can alter this natural history. Studies of the disposition, metabolism, excretion and pharmacokinetics of both drugs of abuse and actual or potential treatment agents are also an essential component of the ongoing clinical research of this project. We propose to define the neuroendocrine disturbances associated with cocaine addiction (alone, complicated with, and in contrast to alcohol, nicotine, and/or opioid exposure), and to evaluate existing and potential pharmacological treatments for addictions from the perspective of their ability to protect against, and/or correct these disturbances.

DESCRIPTION (provided by applicant): This is a competitive renewal to provide the major source of salary support for the applicant, Mary-Jeanne Kreek, M.D., as a senior Research Scientist Awardee (and also as a Principal Investigator and Scientific Director of an NIH-NIDA Treatment-Related Research Center.) This award will allow the applicant to continue to spend the majority of her time in basic laboratory-based and basic clinical research, as well as to continue to spend a significant amount of time in scientific training and mentoring in the graduate, postgraduate, and mid-career levels, as well as science education at the undergraduate, graduate and high school levels. The applicant will also continue to be active in science education, particularly as pertains to the biological basis of the addictive disease, to a more general public. The most effective treatments for the addictive diseases, including opiate addiction, cocaine addiction, alcoholism, and nicotine addiction, probably will continue to be based on a fundamental understanding of the biological and molecular bases of addictive diseases; the physiologic and pharmacologic effects of drugs of abuse, and of agents used for the treatment of specific addictions, and also of medical and behavioral problems which frequently coexist with specific addictions and may complicate treatment, such as HIV-1 (AIDS), hepatitis B, and especially hepatitis C. Research activities will continue to identify and study the biological correlates of addictive diseases, factors which affect treatment outcome, and primarily the molecular neurobiological basis of addiction, and the human molecular genetics of addictions. There will be four domains of Specific Aims: 1) Laboratory-based research (Projects 1, 2 and 3); 2) Basic clinical research (Project 4); 3) Human molecular genetics (Project 5); and, 4) Applied clinical research (Project 6), each related to four specific addictions: opiate (primarily heroin) addiction, cocaine addiction, alcoholism, and nicotine addiction. Three specific projects within these four Specific Aims are, e.g.: 1) Effects of chronic opiates and cocaine, withdrawal, and challenge on receptors, and gene expression of the endogenous opioid and related neurotransmitter systems; 2) Effects of opiates and cocaine on the molecular biology and expression of the stress responsive hypothalamic-pituitary-adrenal axis; 5) Human genetics research of addictions with emphasis on studies of the human molecular genetics.

hypothalamic pituitary adrenal axis; stress; neuroendocrine system; hormone regulation /control mechanism; glucocorticoids; liver disorder; prolactin; drug addiction; biofeedback; neuropharmacology; opiate alkaloid; brain electrical activity; patient oriented research; human subject; clinical research;

cocaine; opioid receptor; neurophysiology; dynorphins; drug addiction; neuropharmacology; hypothalamic pituitary adrenal axis; pituitary gonadal axis; hormone regulation /control mechanism; cortisol; drug abuse chemotherapy; adrenocorticotropic hormone; methadone; prolactin; patient oriented research; clinical research; human subject;

genetic susceptibility; endogenous opioid; family genetics; genetic polymorphism; heroin; alcoholism /alcohol abuse; disease /disorder proneness /risk; drug addiction; tissue resource /registry; single nucleotide polymorphism; disease /disorder etiology; pharmacogenetics; genetic screening; clinical research; cell line; human subject; human genetic material tag;

endogenous opioid; metabolism; inhibitor /antagonist; stimulant /agonist; biomedical resource; opiate alkaloid; macromolecule; clinical research; mass spectrometry;

[unreadable] DESCRIPTION (provided by applicant): The overall goal of the proposed research is to determine the relationship of both specific gene variants and epigenetic modification of genes of the opioidergic, stress response, serotonergic, and dopaminergic systems in the brain of HIV-1 infected subjects with the progression of HIV-1 disease. Many individuals infected with HIV-1 suffer from comorbid psychiatric illnesses, including depression, anxiety, and impulsive behaviors. Host genetic factors-can modify HIV-1 infection and have been shown to affect the vulnerability to develop psychiatric illnesses. Opioids promote HIV-1 replication in immune and glial cells in vitro, presumably by increasing levels of the chemokine CCR5 (one of the coreceptors for HIV-1 entry into the cell) and by the suppression of host HIV-protective factors. The relationship between the genotype and expression patterns of the mu-opioid receptor gene, OPRM1, and CCR5 in brain samples obtained from the National NeuroAIDS Tissue Consortium will be investigated. Comorbid depression, anxiety, atypical stress responsivity, impulsivity, and risk-taking behaviors, of which drug abuse/addiction may be a manifestation, will be examined. To identify peripheral markers for the progression of HIV-1 infection, the role of epigenetic factors in both brain and lymphocytes from individuals characterized as to psychiatric comorbidity and abuse/addiction, which may modulate key genes related to infectivity and risk-taking, will be examined. In a large study using samples from the Women's Interagency HIV Study repository, we will investigate the relationship between polymorphisms in genes of the serotonergic, dopaminergic and opioidergic systems with HIV-1 infection progression. The relationship of these variants with impulsivity and risk-taking to HIV-1 disease progression will be examined. Clarifying the functional relevance of polymorphisms associated with susceptibility to complex disorders such as psychiatric illnesses and abuse/addiction may provide the foundation for future clinical studies. Also, these studies may identify the role of genetic variants and epigenetic mechanisms in the progression of HIV- 1 infection and may point to targets for pharmacotherapy in HIV-1 disease with psychiatric comorbidity. The information gained from this research should help to alleviate the suffering of HIV-1 infection with comorbid psychiatric diseases. [unreadable] [unreadable] [unreadable]

Animal Experimental Use; Animal Experimentation; Animal Research; Clinical; Clinical Research; Clinical Study; Development; Environment; Goals; Information Dissemination; Mentors; R01 Mechanism; R01 Program; RPG; Research; Research Grants; Research Project Grants; Research Projects; Research Projects, R-Series; Training; Universities; career; experience; novel; outreach

[unreadable] DESCRIPTION (provided by applicant): Heroin, cocaine, and alcohol addictions, alone and in combination, along with their multiple and frequent medical complications (including hepatitis B and C, AIDS, and psychiatric comorbidity) remain the major medical problems confronting our nation and much of the world. Effective treatments must be based on a fundamental understanding of the biological bases of each specific addictive disease, including the effects of chronic exposure to specific drugs of abuse and the interaction with environment and genetic factors. Our NIH-NIDA P60 Treatment Research Center, "Treatment of Addictions: Biological Correlates," will continue to identify and study the dynamic molecular, neurobiological, and behavioral changes caused by heroin and cocaine. Research will focus on the endogenous opioid system, to elucidate its importance in the biological basis of addictive diseases, and interactions with related neurotransmitters and neuropeptides, emphasis on dopaminergic and glutamatergic systems, and stress-responsive systems. All laboratory and clinical research studies, and the Research Cores, have been developed on an interactive basis with thematic integration for the entire Center. Bidirectional translational research is a hallmark of the work of this Center. Findings from basic and applied clinical research are used to formulate specific hypotheses and develop novel animal models. Findings from laboratory research (such as vasopressin alterations) are applied into innovative clinical research. The specific research projects include: 1) studies of the effect of exposure, withdrawal, and re-exposure to heroin or cocaine, dynamic neurobiological adaptations in the opioid system underlying addictive-like behavioral changes; 2) effects of drugs of abuse on stress-responsive brain systems and hypothalamic-pituitary-adrenal axis function; 3) cocaine-induced synaptic plasticity in limbic brain regions; and 4) alterations in neuroendocrine stress responsivity in specific addictive diseases without or with codependency or comorbidity. Increasing effectiveness of existing treatments and developing novel treatments depends on expanding our insights into the molecular neurobiology of addictions, which is the primary goal of these projects, each utilizing five core resources, including a Research Training Core. Thematic integration and scientific interaction fosters synergism, which only a multidisciplinary center can provide. [unreadable] [unreadable] CENTER CHARACTERISTICS [unreadable] [unreadable] [unreadable]

The Clinical Research Core is a fundamental and essential core of our NIH-NIDA funded Center. The goals of the core are: 1) To provide highly trained staff (physicians, nurse practitioners, nurses, and assistants for research) at all levels to perform the rigorous inpatient molecular neurobiology studies as detailed in Project 4, as well as an outpatient site for performance of related genetic studies, as well as for subject screening, evaluation, and follow-up. 2) The second goal is to develop or maintain strong on-site relationships with a limited number of large, successful programs delivering primarily pharmacotherapy for heroin addiction using methadone maintenance or buprenorphine maintenance for: a. Recruitment of subjects for studies at The Rockefeller University Hospital b. In some cases, conducting onsite human molecular genetics ascertainment studies with local clinic IRBs' Informed Consent, as well as Rockefeller University IRB Informed Consent, both signed c. To identify new problems, including psychiatric and medical problems, new drug abuse problems confronting these groups, as well as any unexpected positive or adverse responses to specific treatments. A broad spectrum of subjects in the greater New York area are used for recruitment. Our related clinic in Las Vegas, NV, is used primarily for identification of problems and confirming or refuting the generalizability of observations made in the greater New York area in another part of our nation. 3) The third goal is to provide a nurturing and rigorous environment for the training of clinical investigators, including physicians, nurse practitioners, research nurses, counselors, psychologists, and a wide variety of healthcare personnel, including social workers and counselors interested in becoming directly involved in addictive diseases research or for the purpose of general education and training in research medicine and/or addictive diseases treatment.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Brief anesthesia, such as following exposure to high levels of carbon dioxide, prior to decapitation is considered a more humane alternative for the euthanasia of rodents, compared to use of the decapitation alone. Studies of the levels of certain stress hormones in plasma such as corticosterone and adrenocorticotropic hormone (ACTH) have supported the use of this method of euthanasia in endocrinological and molecular studies. In the current study, rats were briefly exposed to a chamber filled with carbon dioxide until recumbent (20-25 s), and immediately sacrificed via decapitation, and trunk blood collected;findings were compared to rats sacrificed via decapitation with no exposure to carbon dioxide. Radioimmunassays were used to measure arginine vasopressin (AVP) and ACTH immunoreactivity (ir) in plasma. While ACTH-ir levels remained steady following brief exposure to carbon dioxide (in accordance with results of other investigators), AVP-ir levels were increased by more than an order of magnitude. These results were confirmed by quantitative capillary-liquid chromatography-mass spectrometry, indicating this observation of rapid increase in plasma AVP-ir levels is not due to nonspecific recognition by the antibody used in the radioimmunoassay. Likewise, using capillary-liquid chromatography-mass spectrometry, we observed a rapid increase in plasma oxytocin levels following carbon dioxide exposure. These surprising findings have important implications for the design and interpretation of studies involving brief carbon dioxide exposure prior to decapitation, as well as those with euthanasia resulting from carbon dioxide-induced asphyxiation. A manuscript describing this work is currently under review.