PHARMACOKINETIC AND DURATION OF CYCLOOXYGENASE INHIBITION STUDIES OF PHENYLBUTAZONE, KETOPROFEN AND FLUNIXIN MEGLUMINE IN ATHLETIC THOROUGHBRED HORSES By JENNIFER NOELLE HATZEL A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2011 1 © 2011 Jennifer Noelle Hatzel 2 To the horses, without which, none of this would be possible 3 ACKNOWLEDGMENTS I thank my committee members: Dr. Patrick Colahan, for the initial concept and subsequent support through the Racing Medication Testing Consortium (RMTC) for completion of these studies, Dr. Alison Morton for her constant encouragement and Dr. Tom Vickroy for his thoughts and ideas, always in the nick of time. I thank Dr. David Hurley and Natalie Norton from the University of Georgia for their patient guidance in assisting me with learning to perform the assays. I thank Dr. Richard Sams for bestowing me with a very basic knowledge of pharmacokinetics and Marc Rumpler for his unwavering support and mentorship through every aspect of this process. I thank Dan Neal, from the department of biostatistics for not only teaching me the basics, but also assisting me when I was way over my head. This project would have never been possible without the help of the entire staff of the University of Florida Equine Performance Laboratory: Brett Rice, Allie Hreha, Amber Davidson and Megan Davidson, for whom I am very grateful. Finally, I owe a great deal of support and encouragement from Jeremiah, always. 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 8 LIST OF FIGURES .......................................................................................................... 9 LIST OF EQUATIONS ................................................................................................... 10 LIST OF ABBREVIATIONS ........................................................................................... 11 ABSTRACT ................................................................................................................... 12 CHAPTER 1 INTRODUCTION .................................................................................................... 15 The Eicosanoids ..................................................................................................... 15 History .............................................................................................................. 15 The Inflammatory Cascade .............................................................................. 16 Pharmaceutical Intervention ................................................................................... 19 The History and Mechanism of NSAIDs ........................................................... 19 Adverse Effects Affiliated with NSAID Administration ....................................... 22 Novel Classes of Selective NSAIDs ................................................................. 23 NSAID Use in Veterinary Medicine ......................................................................... 26 Equine NSAID Administration ........................................................................... 26 Drug Doping in the Performance Horse ........................................................... 29 Monitoring Drug Abuse ..................................................................................... 31 Summary and Objectives ........................................................................................ 37 2 THE PHARMACOKINETICS OF PHENYLBUTAZONE, KETOPROFEN AND FLUNIXIN MEGLUMINE FOLLOWING A SINGLE ADMINISTRATION ................. 41 Background ............................................................................................................. 41 Materials and Methods ............................................................................................ 42 Animals ............................................................................................................. 42 Standard Training Regimen for the University of Florida Equine Performance Lab ........................................................................................... 42 Incremental Exercise Test to Exhaustion ......................................................... 43 Standard Condition Test to Verify the Ability to Gallop One Mile in Two Minutes.......................................................................................................... 43 Drug Administration and Sample Collection ..................................................... 44 Drug Analysis .......................................................................................................... 44 Chemicals and Reagents ................................................................................. 44 Phenylbutazone ................................................................................................ 45 5 Sample preparation .................................................................................... 45 Instrumentation .......................................................................................... 46 Data analysis ............................................................................................. 47 Ketoprofen ........................................................................................................ 48 Sample preparation .................................................................................... 48 Instrumentation .......................................................................................... 49 Data analysis ............................................................................................. 50 Flunixin Meglumine .......................................................................................... 50 Sample preparation .................................................................................... 50 Instrumentation .......................................................................................... 51 Data analysis ............................................................................................. 53 Pharmacokinetic Modeling ............................................................................... 53 Results .................................................................................................................... 54 Phenylbutazone ................................................................................................ 54 Ketoprofen ........................................................................................................ 54 Flunixin Meglumine .......................................................................................... 55 Discussion .............................................................................................................. 55 3 THE DURATION OF CYCLOOXYGENASE INHIBITION FOR PHENYLBUTAZONE, KETOPROFEN AND FLUNIXIN MEGLUMINE FOLLOWING A SINGLE ADMINISTRATION ......................................................... 69 Background ............................................................................................................. 69 Materials and Methods ............................................................................................ 70 Animals ............................................................................................................. 70 Standard Training Regimen for the University of Florida Equine Performance Lab ........................................................................................... 71 Drug Administration and Sample Collection ..................................................... 71 Ex vivo COX-1 Assay ....................................................................................... 73 Ex vivo COX-2 Assay ....................................................................................... 73 Statistical Analysis ............................................................................................ 74 Results .................................................................................................................... 74 Ex vivo Inhibition of Phenylbutazone ................................................................ 74 Ex vivo Inhibition of Ketoprofen ........................................................................ 75 Discussion .............................................................................................................. 75 4 UTILIZING A BIOSTATISTICAL APPROACH TO MODEL COMPARISONS OF PHENYLBUTAZONE, KETOPROFEN, AND FLUNIXIN MEGLUMINE WITH SUPRESSED CONCENTRATIONS OF PGE2 AND TXB2 ..................................... 92 Background ............................................................................................................. 92 Materials and Methods ............................................................................................ 94 Animals ............................................................................................................. 94 Method of Analysis ........................................................................................... 94 Results .................................................................................................................... 95 Relating Phenylbutazone Concentrations to Prostaglandin (PGE) Concentrations