PHARMACOKINETIC CHARACTERIZATION OF ANGIOTENSIN IV ANALOGS WITH THERAPEUTIC POTENTIAL FOR CANCER AND DEMENTIA. By ALENE MCCOY A dissertation submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY WASHINGTON STATE UNIVERSITY Graduate Program in Pharmacology and Toxicology MAY 2010 To the Faculty of Washington State University: The members of the Committee appointed to examine the dissertation of ALENE MCCOY find it satisfactory and recommend that it be accepted. Joseph W. Harding, Ph.D., Chair John W. Wright, Ph.D. Neal Davies, Ph.D. Raymond Quock, Ph.D. Heiko Jansen, Ph.D. ii ACKNOWLEDGEMENTS There are many people without whose support the research presented in this dissertation would not have been possible. First and foremost, I would like to thank my thesis advisor Joseph Harding for his professional and personal guidance and support throughout my graduate career. Your great generosity and wisdom will forever be an inspiration to me. I would also like to thank my thesis committee and the faculty in the Program in Pharmacology and Toxicology. Thank you for always being available and for your professional guidance. Special thanks to Neal Davies for your generous assistance with all of my pharmacokinetic queries. Thanks to all of the past employees of Pacific Northwest Biotechnology and to present members of the Harding laboratory. You made PNB and the Harding lab a wonderful place to work and taught me a great deal. I would especially like to acknowledge Brent Yamamoto who was a mentor to me during my first three years in graduate school, and my good friend Caroline Benoist. Your support and friendship have enriched both my personal and laboratory life. iii PHARMACOKINETIC CHARACTERIZATION OF ANGIOTENSIN IV ANALOGS WITH THERAPEUTIC POTENTIAL FOR CANCER AND DEMENTIA Abstract By Alene McCoy, Ph.D. Washington State University May 2010 Chair: Joseph W. Harding Angiotensin IV is a peptide hormone that acts via the AT4 receptor with physiological functions that have not yet been fully elucidated but appear to include important roles in the processes of cognition, fluid balance, blood flow and vascular repair and remodeling. Angiotensin IV shares a partial homology with hepatocyte growth factor (HGF) and our laboratory has recently demonstrated that angiotensin IV analogs are able to effect changes in intracellular signaling cascades via the HGF receptor (c-Met). HGF/c-Met signaling is essential for embryonic development and is responsible for direction of cellular processes including differentiation, proliferation and migration. As the product of an oncogene, the c-Met receptor is also implicated to play a critical role cancer. Our laboratory has developed a library of angiotensin IV analogs with a number of these exhibiting strong potential as therapeutic agents for the treatment of cancer or dementia. Given that the failure of many therapeutic candidates in clinical trials can be attributed to unsuitable pharmacokinetics, our laboratory wished to establish a basic understanding of the iv pharmacokinetic characteristics of these molecules. We, therefore, set out to establish the basic pharmacokinetic profiles of three molecules, which serve as representative molecules for classes of chemical modifications to the angiotensin IV peptide made in our laboratory. This dissertation presents results from in-vitro and in-vivo studies describing pharmacokinetic characteristics for three angiotensin IV analogs in rats. These characteristics include measures of metabolic stability, clearance mechanisms and rates, and kinetic parameters, including half life, volume of distribution, and area under the concentration/time curve. These studies indicate strong potential as pharmaceutical candidates for some of our angiotensin IV analogs; and a need for further chemical modification of others in order to overcome impractical pharmacokinetic characteristics. We additionally describe the development and validation of assays for quantification of these molecules in biological fluids. v TABLE OF CONTENTS Page ACKNOWLEDGEMENTS .........................................................................................................iii ABSTRACT .................................................................................................................................iv LIST OF TABLES .......................................................................................................................viii LIST OF FIGURES .....................................................................................................................ix CHAPTER 1. INTRODUCTION, BACKGROUND AND SIGNIFICANCE .................................1 1.1. Cancer and Alzheimer’s Disease – Two Pathologies, One Receptor .................1 1.2. Angiotensin IV and the AT4 Receptor ................................................................4 1.3. The c-Met Receptor ............................................................................................8 1.4. Angiotensin IV Analogs .....................................................................................9 1.5. Overview of Experimentation .............................................................................11 2. HIGH-PERFORMANCE LIQUID CHROMATOGRAPHIC ANALYSIS OF NORLEUAL IN BLOOD ..........................................................................................13 2.1. Introduction .........................................................................................................13 2.2. Methods...............................................................................................................16 2.3. Results and Discussion .......................................................................................21 2.4. Conclusion ..........................................................................................................26 3. PHARMACOKINETICS AND METABOLISM OF NORLEUAL IN RATS ........27 3.1. Introduction .........................................................................................................27 3.2. Methods...............................................................................................................29 vi 3.3. Results and Interpretation ...................................................................................36 3.4. Discussion ...........................................................................................................43 4. HIGH-PERFORMANCE LIQUID CHROMATOGRAPHIC ANALYSIS, PHARMACOKINETICS AND METABOLISM OF PNB-0405 IN RATS .............46 4.1. Introduction .........................................................................................................46 4.2. Methods...............................................................................................................49 4.3. Results and Interpretation ...................................................................................57 4.4. Discussion ...........................................................................................................68 5. HIGH-PERFORMANCE LIQUID CHROMATOGRAPHIC ANALYSIS, PHARMACOKINETICS AND METABOLISM OF DIHEXA IN RATS ...............70 5.1. Introduction .........................................................................................................70 5.2. Methods...............................................................................................................73 5.3. Results and Interpretation ...................................................................................84 5.4. Discussion ...........................................................................................................107 6. DISCUSSION ............................................................................................................110 BIBLIOGRAPHY ........................................................................................................................121 vii LIST OF TABLES 1. Structure of Angiotensin IV analogs........................................................................................11 2.1 Structure of Angiotensin IV, Norleual and the Internal Standard ........................................15 2.2 Recovery of Norleual and PNB-0719 from blood compared to water .................................23 2.3 Within- and between-run precision and accuracy of Norleual determination in blood .........24 3.1. Predicted Physicochemical Properties of Norleual ..............................................................36 3.2. Intrinsic clearance values for Norleual and reference compounds ......................................38 3.3. WinNonlin estimated pharmacokinetic paramters for Norleual after intravenous administration to adult male Sprague-Dawley rats .....................................................................41 4.1. Within- and between-run precision and accuracy of PNB-0405 determination in serum ....59 4.2. Predicted Physicochemical Properties of PNB-0405 ...........................................................62 4.3. PK parameters of PNB-0405 following single-dose IV administration in rats ...................64 4.4. PK parameters of PNB-0405 following single-dose SC administration in rats ...................64 5.1. Structural modifications of angiotensin IV to produce Dihexa and the internal standards .....................................................................................................................................71 5.2. Recovery of Dihexa from urine ............................................................................................88