DISSERTATION ZUR ERLANGUNG DES DOKTORGRADES DER FAKULTÄT FÜR CHEMIE UND PHARMAZIE DER LUDWIG-MAXIMILIANS-UNIVERSITÄT MÜNCHEN Oil-Based Parenteral Depot Formulation for Veterinary Peptide Delivery YORDANKA ZHIVKOVA YORDANOVA aus Shumen, Bulgarien 2018 Erklärung Diese Dissertation wurde im Sinne von § 7 der Promotionsordnung vom 28. November 2011 von Herrn Prof. Dr. Wolfgang Frieß betreut. Eidesstattliche Versicherung Diese Dissertation wurde eigenständig und ohne unerlaubte Hilfe erarbeitet. München, den 16.09.2018 ............................................................................. Yordanka Yordanova Dissertation eingereicht am 11.10.2018 1. Gutachter: Prof. Dr. Wolfgang Frieß 2. Gutachter: Prof. Dr. Gerhard Winter Mündliche Prüfung am 30.10.2018 To my parents and my sister Thank you for always being there for me in the moments when I am most scared to make the next step. Your never ending support has inspired me to carry on moving and never stop dreaming. I feel blessed and proud to have you by my side! За моите родители и за моята сестра, които винаги са били до мен и са ми помагали да направя следващата стъпка, когато най-много ме е било страх. Вашата безкрайна подкрепа ме кара да продължавам напред и да не спирам да мечтая. Щастлива съм че ви имам! CONTENTS PREFACE ............................................................................................................................................... XI 1 GNRH [6-D-PHE] ACETATE OIL DEPOT SUSPENSION ............................................................. 3 1.1 INTRODUCTION ................................................................................................................... 3 1.2 THE ESTROUS CYCLE IN PIGS .......................................................................................... 4 1.3 CURRENT SYNCHRONIZATION TREATMENTS ............................................................ 5 1.3.1 Estrus synchronization..................................................................................................... 5 1.3.2 Stimulation of follicular development ............................................................................. 6 1.3.3 Induction of ovulation ..................................................................................................... 6 1.4 GENERAL FORMULATION REQUIREMENTS FROM A VETERINARY PERSPECTIVE ................................................................................................................................... 6 1.5 RESEARCH FOCUS, FORMULATION DEVELOPMENT REQUIREMENTS AND STUDIES ............................................................................................................................................ 8 1.5.1 Selection and Composition of the Oily Vehicle .............................................................. 9 1.5.2 Incorporated GnRH [6-D-Phe] acetate and Micronization Process .............................. 10 1.5.3 Sustained Release .......................................................................................................... 10 1.5.4 Selection of a Suitable In Vitro Release Model ............................................................ 11 1.6 AIMS ..................................................................................................................................... 11 1.7 REFERENCES ...................................................................................................................... 13 1.8 FIGURES AND TABLES ..................................................................................................... 18 2 PURE OIL MIXTURES AS A DELIVERY VEHICLE FOR GNRH [6-D-PHE] ACETATE .......... 21 2.1 INTRODUCTION ................................................................................................................. 21 2.2 RESULTS AND DISCUSSION............................................................................................ 23 2.2.1 Oil mixtures for extended release of GnRH [6-D-Phe] ................................................. 23 2.2.2 Oil Spreading at the Injection Site ................................................................................. 24 2.2.3 GnRH [6-D-Phe] Micronization and Particles Characterization ................................... 24 2.2.4 Suspension-and Cryogenic-Milled GnRH [6-D-Phe] .................................................... 26 2.2.5 GnRH [6-D-Phe] Oil Depot Suspension ....................................................................... 27 2.2.6 Rheology ....................................................................................................................... 27 2.2.7 Injection Force Determination ....................................................................................... 28 2.2.8 In vitro Release Model Selection and Studies ............................................................... 29 2.2.9 In vivo 1st Preclinical Study........................................................................................... 29 2.3 CONCLUSION ..................................................................................................................... 31 2.4 MATERIALS AND METHODS .......................................................................................... 32 2.5 REFERENCES AND ACKNOWLEDGMENTS ................................................................. 35 vii 2.6 SUPPLEMENTARY DATA ................................................................................................. 37 2.7 FIGURES AND TABLES ..................................................................................................... 41 3 GNRH [6-D-PHE] ACETATE OIL DEPOT SUSPENSION STABILITY ....................................... 45 3.1 INTRODUCTION ................................................................................................................. 45 3.2 RESULTS AND DISCUSSION............................................................................................ 46 3.2.1 Content Uniformity ....................................................................................................... 46 3.2.2 Particle Size Distribution and Characterization ............................................................. 47 3.2.3 Rheology ....................................................................................................................... 48 3.2.4 Injection Force Determination ....................................................................................... 50 3.2.5 Oil Vehicle Oxidation.................................................................................................... 51 3.3 CONCLUSION ..................................................................................................................... 51 3.4 MATERIALS AND METHODS .......................................................................................... 53 3.5 REFERENCES AND ACKNOWLEDGMENTS ................................................................. 56 3.6 SUPPLEMENTARY DATA ................................................................................................. 58 3.7 FIGURES AND TABLES ..................................................................................................... 60 4 GNRH [6-D-PHE] ACETATE OIL DEPOT SUSPENSION: THE EFFECT OF ADDITIVES ON THE RELEASE CHARACTERISTICS ................................................................................................. 63 4.1 INTRODUCTION ................................................................................................................. 63 4.2 RESULTS AND DISCUSSION............................................................................................ 65 4.2.1 Preliminary Study of Gelling Agent, Wetting Agent and Resuspendibility enhancer .. 65 4.2.2 GnRH [6-D-Phe]-Additives Oil Depot Suspension ....................................................... 66 4.2.3 Rheology ....................................................................................................................... 68 4.2.4 Injection Force Determination ....................................................................................... 71 4.2.5 Particle Size Distribution and Characterization ............................................................. 71 4.2.6 Oil-Water Interface ........................................................................................................ 73 4.2.7 Self-Emulsifying Character ........................................................................................... 74 4.2.8 In vitro Release Studies ................................................................................................. 76 4.2.9 In vivo 2nd Preclinical Study ........................................................................................ 78 4.3 CONCLUSION ..................................................................................................................... 80 4.4 MATERIALS AND METHODS .......................................................................................... 81 4.5 REFERENCES AND ACKNOWLEDGMENTS ................................................................. 84 4.6 SUPPLEMENTARY DATA ................................................................................................. 86 4.7 FIGURES AND TABLES ..................................................................................................... 91 5 GNRH [6-D-PHE] ACETATE OIL DEPOT SUSPENSION: THE EFFECT OF POLYMERS ON THE RELEASE CHARACTERISTICS ................................................................................................