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Opioid peptide permeation across the blood- brain and blood-cerebrospinal fluid barriers Item Type text; Dissertation-Reproduction (electronic) Authors Abbruscato, Thomas John, 1970- Publisher The University of Arizona. Rights Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. Download date 04/10/2021 05:24:05 Link to Item http://hdl.handle.net/10150/282429 INFORMATION TO USERS This manuscript has been reproduced from the microfihn master. UMI fihns the text direct^ from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter &ce, while others may be from any type of computer printer. The quality of this reproductioii is dependent upon the quality of the copy submitted. 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Contact UMI directly to order. UMI A BeU A Howell Infonnation Compai^ 300 North Zeeb Road, Ann Aibor MI 48106-1346 USA 313/761-4700 800/521-0600 OPIOID PEPTIDE PERMEATION ACROSS THE BLOOD-BRAIN AND BLOOD- CEREBROSPINAL FLUID BARRIERS by Thomas John Abbruscato A Dissertatioa Submitted to the Faculty of the COMMITTEE ON PHARMACOLOGY AND TOXICOLOGY (Graduate) In Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY In the Graduate College THE UNIVERSITY OF ARIZONA 1997 UMX Number: 9806814 UMI Microform 9806814 Copyright 1997, by UMI Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. UMI 300 North Zeeb Road Ann Arbor, MI 48103 9 THE UNIVERSITY OF ARIZONA ® GRADUATE COLLEGE As members of the Final Examination Committee, we certify that we have read the dissertation prepared by Thomas John Abbruscato entitled OPIOID PEPTIDE PERMEATION ACROSS THE BLOOD- BRAIN AND BLOOD-CEREBROSPINAL FLUID BARRIERS and recommend that it be accepted as fulfilling the dissertation requirement for the Degree of Doctor of Philosophy Final approval and acceptance of this dissertation is contingent upon the candidate's submission of the final copy of the dissertation to the Graduate College. I hereby certify that I have read this dissertation prepared under my direction and recommend that it be accepted as fulfilling the dissertation requirement. W Dissertation Director Date 3 STATEMENT BY AUTHOR This dissertation has been submitted in partial flilfiUment of requirements for an advanced degree at The University of Arizona and is deposited in the University of Library to be made available to borrowers under rules of the Library. Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his or her judgement the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author.. SIGNED: 4 Acknowledgements I would first like to thank my advisor Dr. Thomas P. Davis for his support and encouragement throughout my undergraduate and graduate studies. He is an excellent role model and has influenced me both scientifically and personally. I thank him for his consistent drive to develop all aspects of my scientific method. I would also like to thank the members of my committee, namely Dr. Henry Yamamura and Dr. Robert Dorr for their pharmacological expertise, and Dr. A. Jay Gandolfi and Dr. Klaus Brendel for their chemical and toxicological assistance with my project. I have also been fortunate enough to work with many people in the Davis lab. Dr. Steve Weber played a major role in teaching me what it takes to be a research scientist. Dr. Elizabeth Brownson exposed me to many aspects of neuroscience and helped to apply them to my project. As well, she introduced me to my wife Robyn. Dr. Sarah Thomas nee' Williams trained me to proficient in the in situ brain perfusion technique and helped me to develop my scientific writing. I will always be gratefiil for her attention Dr. Richard Egleton was a key influence in helping me finished my dissertation with his expertise in the area of the blood-brain barrier. Dr. Chris Konkoy helped me immensely with his extensive pharmacology background. Terry Gillespie provided assistance with the analytical and enzymatic aspects of my project. His expertise in peptide research extended the scope of my project. Dr. Matthew Rounseville also helped me also with his experience in molecular biology. I would also like to thank a long time fiiend. Brad Merrill, who was instrumental in getting me started in biomedical research. I would also like to thank the other members of the lab that I have worked with over the years; Dr. Steve Waters, Dr. Barbara Mania-Famell, Dr. Dave Clark, Dr. Mary Oakes, Dr. Pierre Konings, Craig Mayr, Sharon Hom and Vincent Hau. The graduate students also deserve great thanks for making the past four years enjoyable, especially C.J. Kovelowski, Art Riegal, Marc Oshiro, Mark Bowers, Sandi Wegert, Kim Mitchell and Jack Adams. I would especially like to thank my family for their love and support: mom (Jackie), dad (Joseph), Joe and Tim. Lastly, I would like to thank my wife, Robyn, who has supported me throughout my graduate studies and understands my love for science. She brings out the best characteristics in myself and I love her for that. She is my life long best fiiend. 5 Dedication This work is dedicated to the woman I love, my wife Robyn, who has always encouraged and supported me. 6 TABLE OF CONTENTS LIST OF FIGURES 9 LIST OF TABLES 11 ABSTRACT. 13 1- General Introduction 15 Anatomy and physiology of blood-brain and blood-CSF barriers IS History of blood-brain barrier. 15 Site of the blood-brain barrier. 15 Anatomy and physiology of the blood-brain barrier. 16 Types of transport across the blood-brain barrier. 21 Periendothelial structures. .......... ............ ....... .......... ..22 Cerebral spinal fluid. 24 Anatomy and physiology of the blood-CSF barrier. 26 Techniques to study blood-CNS permeability and stability of drugs 31 Brain uptake index................... 31 Intravenous bolus injection 32 Brain perfusion studies. 33 Positron emission tomography. 34 Octanol/saline partition coefficient. 35 HPLC capacity factor. 35 Isolated cultures of cerebral capillary endothelial cells. 36 Endogenous opioid peptides and opioid pharmacology ....37 History ofopioids. 37 Endogenous opioid peptides. 38 Midtiple opioid receptor types. 39 Regional CNS distribution of opioid receptors. 41 Mechanism of opioid inhibitory action on neurotransmission 42 Peptide drug delivery to the brain 44 Peptides as neuropharmaceuticals. 44 Present study 47 Hypothesis. 47 Specific aims. 47 2- Functional and Enzymatic Characterization of In Vitro Bovine Brain Microvessel Endothelial Cell Model of the In Vivo Blood-Brain Barrier. 49 Introduction 49 Methods 55 In vitro BMEC method. 55 Specific enzymatic assays...... 61 In vivo brain uptake 61 HPLC analysis. 62 Data and statistics. 63 Results 64 I. Functional.................................................. 64 In vitro BMEC studies. 64 In vivo BBB studies. 64 H. Enzymatic aspects of BMECs 68 In vitro enzymatic activity of AP, APM, NEP and ACE. 68 In vitro BBB permeability. 68 Discussion 74 3- Defining the Mechanism of DPDPE Central Nervous System Entry. 78 Introduction 78 Methods 81 In situ brain perfiision studies. 81 Calcidation of kinetic constants. 84 Capillary depletion analysis. 86 Protein binding. 87 Octanol/saline partition coefficient. 88 HPLC analysis of ^HJDPDPE brain and perfusate extraction 88 Results 91 Multiple time uptake analysis of [^HJDPDPE compared to ['*C]sucrose 91 Self inhibition studies. 91 Contribution of vasadar component to ^HJDPDPE brain uptake 92 Octanol /saline partition coefficient. 92 Discussion 101 4- Effects of Chloro-Halogenation on CNS Entry Biphalin 106 Introduction 106 Methods 110 In situ brain perfiision IIO In vitro BMEC. 110 Brain extraction after in situ vascular brain perfiision 114 Results 119 Midtiple time uptake analysis of radiolabeled biphalin, chloro-biphalin and sucrose 119 In vitro permeability coefficient, octanol /saline partition coefficient, and R litsue percent for radiolabeled biphalin, chloro-biphalin and sucrose..Ill 8 Brain extraction of radiolabeled peptide......................,......................A22

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