GASTROINTESTINAL ABSORPTION OF HEPARINS A Dissertation Submitted to the College of Graduate Studies and Research in Partial Fulfillments of the Requirements for the Degree of Doctor of Philosophy in the Graduate Program of Veterinary Biomedical Sciences University of Saskatchewan Saskatoon Bita Moazed Copyright Bita Moazed, November 2009. All Rights Reserved PERMISSION TO USE In presenting this dissertation in partial fulfillment of the requirements for a Doctor of Philosophy degree from the University of Saskatchewan, I agree that the Libraries of this University may make it freely available for inspection. I further agree that permission for copying of this thesis/dissertation in any manner, in whole or in part, for scholarly purposes may be granted by Dr. Linda M. Hiebert who supervised my dissertation work or, in her absence, by the chair of the Graduate Program of Veterinary Biomedical Sciences. It is understood that any copying or publication or use of this dissertation or parts thereof for financial gain shall not be allowed without my written permission. It is also understood that due recognition shall be given to me and the University of Saskatchewan in any scholarly use which may be made of any material in this dissertation. Requests for permission to copy or to make other use of other material in this dissertation in whole or part should be addressed to: Chair of the Graduate Program of Veterinary Biomedical Sciences Western College of Veterinary Medicine University of Saskatchewan 52 Campus Drive Saskatoon, Saskatchewan S7N 5B4 Canada i PREFACE This dissertation has been organized as a series of manuscripts that was or will be submitted for publication in scientific journals. Some repetition of introductory and methodological material is unavoidable. ii ABSTRACT Heparin, a highly sulfated and acidic glycosaminoglycan, has been clinically used in parenteral formulations to prevent and/or treat thromboembolic disorders for more than 90 years. Actions of heparin are not limited to anticoagulation and antithrombosis. Rather heparin has several other important actions which include fat clearing, antitumor and anti-inflammatory effects. However, use of heparin for such applications has been limited by its route of administration. Historically, it has been believed that heparin is not absorbed following oral administration and therefore is only available for clinical use by parenteral administration. This belief has been challenged several times by our laboratory and other researchers showing heparin binding to endothelium following oral administration as well as prevention of thrombosis and lowering blood pressure, etc. However, the site of oral heparin absorption and the mechanism responsible for absorption have not been investigated. This in vitro study was designed to address these important questions. We mounted pieces of rat gastrointestinal mucosa in a vertical diffusion Ussing chamber with both sides of the mucosal membrane exposed to Kreb’s bicarbonate buffer solution containing mannitol on the mucosal side (lumen) and glucose on the serosal side. Electrical properties across the membrane including potential difference (PD), resistance (R), and short circuit current (Isc) were recorded following heparin addition to the mucosal buffer under different mucosal buffer pH conditions. Mucosal and serosal buffer and tissue were collected and extracted for heparin and heparin recovery was performed by gel electrophoresis and anticoagulation tests. iii The first chapter (chapter 4) was designed to investigate if stomach mucosal tissue is a site for unfractionated heparin (UFH) absorption when mounted in the Ussing chamber. We found that stomach mucosa is able to transport UFH in an intact form when both mucosal and serosal buffers are at neutral pH of 7.4. When the mucosal buffer pH is made more acidic, at pH 4, transport is facilitated. The second study (chapter 5) was designed to investigate if stomach mucosal tissue is also capable of transporting low molecular weight heparins (LMWHs). We showed that LMWHS were transported across stomach mucosa. However, the rate of transport was faster at mucosal buffer pH of 7.4 compared to pH 4. The third study (chapter 6) investigated the effect of molecular weight on rate of heparin transport across stomach mucosal tissue since pH dependency of this transport was evident from both previous studies. This study suggested that decreasing the molecular weight increases the rate of heparin transport across stomach tissue under neutral pH but not acidic pH conditions. The fourth study (chapter 7) investigated how UFH is transported across the ileal mucosa and if Peyer’s patches contribute to this transport. It was shown that UFH is transported across ileal mucosa containing Peyer’s patches at a rate faster than ileal mucosa without Peyer’s patches. Making the mucosal buffer pH acidic facilitated UFH transport in the absence of Peyer’s patches but not when ileal mucosa contained Peyer’s patches. The final study (chapter 8) investigated the mechanism of UFH transport across stomach mucosa mounted in the Ussing chamber using pharmacological inhibitors sodium fluoride, colchicine, and amiloride. Results showed that UFH is transported across the iv stomach mucosa at physiological acidic pH by an active transport mechanism using metabolic energy, cytoplasmic tubule formation, and sodium-coupled systems. From this, we conclude that oral heparins are absorbed across the gastrointestinal tract. The acidic environment of the stomach is a better absorption site for UFH. On the other hand, the more basic environment of the intestine is a better site for absorption of LMWHs. UFH is mainly absorbed across the stomach mucosa by an active transport mechanism using metabolic energy, cytoplasmic tubule formation, and sodium-coupled systems. Considering the very much larger surface area of the intestine than the stomach and that intestine, especially the ileum, contains many Peyer’s patches where UFH transport is not pH-dependent, larger amounts of UFH may be transported across the intestinal tissue compared to the stomach. v Acknowledgements I wish to express my sincere gratitude and appreciation to my supervisor, Dr. Linda M. Hiebert, for her invaluable guidance, endless support and encouragement throughout the course of my Ph.D. program. Her persistent critical appraisal and suggestions have always been quite helpful at all stages of this research. I would like to especially thank her for being a patient listener to me and teaching me a great deal in the science of pharmacology. It was a complete joy working in her lab. I am indebted to her for the many enjoyable and instructive discussions we've had that gave me a new perspective on available opportunities. I am greatly thankful to my advisory committee members, Dr. Lily Wu, Dr. George Forsyth, Dr. Anthony Carr, and my advisory chairs Dr. Baljit Singh and Dr. Gilian Muir for their valuable suggestions and feedback. I take this opportunity to thank Mrs. Sandra Wice and Mrs. Tilly Ping for their assistance and help throughout the laboratory program of my research. Financial support through a grant from the Heart and Stroke Foundation of Saskatchewan and several teaching fellowships from the University of Saskatchewan are also acknowledged. I would like to thank my husband, Hossein Azinfar, for his unwavering support during the many long days of my Ph.D. program. I would like to thank God for bringing an enjoyment to my life with my son, Armin, who was born during my Ph.D. program. I would like to especially thank my brother, Reza Moazed, for his encouragement and scientific conversations throughout my Ph.D. program. Reza, you have always been there for me to share my life, thoughts, fears, joys, laughter and tears. You are a star shining brightly in my life. I would like to thank my sisters, Dr. Banafsheh Moazed and Rosa Moazed, for their never fading support and loving care. My special gratitude to my parents, Dr. Hadi Moazed and Malikeh Jafarnejadi, whose love and affection is always the source of inspiration and encouragement in all aspects of my life. vi I dedicate this work to my loving husband, my spiritual master Hossein Azinfar, and to my precious son, Armin My darling husband, Hossein, you direct my understanding of life. You supported and encouraged me through all of my desires and goals even when my dreams seemed totally out of reach. I am so very blessed to have you as my husband. Without you in my life I would have been a lost human being not knowing your sincere and secure love. My dear son, Armin, you hold a place in my heart where no one’s ever been. You are the most precious son a mother could ask for. I love you more than my life. You have brought me more pleasure in a single moment of your life, than all other moments combined. Hossein and Armin, you both are my link to life and the continuity of life, and of love. I love you both very much vii Table of Contents Permission to use…….…………………………………………………………………….i Preface…………………………………………………………………………………….ii Abstract……………………………………………………………………………… .….iii Acknowledgments…………………………….……………………………..…..……….vi Table of contents.……………………..…………………….…………………………..viii List of Tables…………………………………………………………………………...xvii List of Figures…………………………………………………………………….…..…xix Abbreviations……………………………………………..………….…………….…...xxii Chapter 1 Introduction…………………………………………………………….……1 1.1 Hemostasis…………………………………………………………………………….1 1.1.1 Primary Hemostasis…………………………………………………………2 1.1.1.1 Vascular Spasm……………………………………………………2