In situ forming biodegradable microparticles for sustained parenteral delivery of protein drugs

Transcription

1 In situ forming biodegradable microparticles for sustained parenteral delivery of protein drugs Dissertation zur Erlangung des akademischen Grades des Doktors der Naturwissenschaften (Dr. rer. nat.) eingereicht im Fachbereich Biologie, Chemie, Pharmazie der Freien Universität Berlin vorgelegt von MARTIN KÖRBER aus Berlin November, 2007

2 1. Gutachter: Prof. Dr. R. Bodmeier 2. Gutachter: Prof. Dr. P. Maincent Tag der mündlichen Prüfung: II

3 To my parents III

4 Acknowledgements I am very thankful to Prof. Dr. R. Bodmeier for providing this interesting and challenging research topic and for his support throughout the dissertation. Additionally, I want to thank him for the opportunity to gain a broad spectrum of experiences in other scientific areas while participating in industrial collaborations under his supervision. I want to thank Prof. Maincent for co-evaluating this thesis and Prof. Gust, Prof. Maincent, Prof. Pertz and Dr. Schwabe for serving as members of my thesis advisory committee. My deepest appreciation goes to Ms. Eva Ewest for her support during my thesis. Thanks to Burkhard Dickenhorst and Mirko Voigt for evaluating parts of the thesis and to Katrin Möbus and Dr. Xiaosong Luan for their help with a number of poster and presentation manuscripts. Special thanks also to Oliver Bley and Burkhard Dickenhorst for helping with complex hardware or software problems and Angelika Schwarz for her support with administrative issues. Thanks to Andreas Krause, Dr. Mesut Ciper, Dr. Wenyu Dong, Dr. Valerie Hoffart, Dr. Andrey Dashevsky, Dr. Nantharat Pearnchob, Dr. Abid Ahmed Riaz, Dr. Mark Skupin, Dr. Nattawut Charoenthai for contributing to a pleasant and professional team atmosphere in industrial collaborations. I would like to thank present workgroup members and alumni Christine Curbach, Araya Raiwa, Katrin Steiner, Duangrutana Shuwisitkul, Julia Herrmann, Soravoot Rujivipat, Nutsawadee Apichatwatana, Joana Mota, Martin Schulz, Samar El-Samaligy, Diana Klose, Dirk Sticha, Frauke Kreye, Lin-Phan Thong, Dr. Ildiko Terebesi, Dr. Oliver Bley, Dr. Till Bussemer, Dr. Heike Friedrich, Dr. Vivian Voigt, Dr. Khaled El-Karraz, Dr. Ulrike Bertram, Dr. Achmad Mohammad, Dr. Jan Möschwitzer, Dr. Saowanee Jaraswekin, Dr. Florence Siepmann, Prof. Dr. Jürgen Siepmann, Dr. Alexander Streubel, Dr. Heiko Kranz, Dr. Srisagul Sungthongjeen Dr. Erol Yilmaz and Dr. Wandee Im-Emsap for a friendly atmosphere in our workgroup. My deepest thank is dedicated to my mother, who we lost in 2004, as well as to my courageous father and my beloved Annett for their patience and their inestimable support. IV

5 TABLE OF CONTENTS Chapters Pages 1. INTRODUCTION...1 _ 1.1. Protein delivery Proteins as pharmaceuticals Challenges of protein delivery Routes of administration - Bioavailability Protein stability Protein structure Reversible inactivation Irreversible inactivation Stabilization approaches Sustained and controlled protein delivery Biodegradable materials Biodegradable polyesters based on lactic and glycolic acid Synthesis of PLGA Degradation of PLGA PLGA-based delivery systems for controlled protein delivery Biodegradable implants Biodegradable microparticles Microencapsulation of proteins Storage Protein release Biodegradable in situ forming systems In situ forming implants In situ forming microparticles Objective...55 V

6 2. MATERIALS AND METHODS...56 _ 2.1. Materials Proteins Polymers Solvents Fatty acid ester Surfactants Other compounds Methods Lysozyme characterization in aqueous medium Lysozyme aggregation and adsorption release media screening Long-term stability of lysozyme in acetate buffer Coomassie assay HPLC Biological activity of lysozyme Electrophoresis Preparation of in situ formulations In-situ implants (ISI) Lysozyme dispersions Lysozyme solutions / in situ precipitated lysozyme dispersions In-situ microparticle systems (ISM) Lysozyme extraction from nonaqueous systems Protein separation with ethyl acetate Alkaline extraction Lysozyme extraction of in situ implants wetted with release medium Lysozyme in PLGA solvents Lysozyme amount dissolved in in situ implants Phase diagrams of lysozyme and PLGA in ternary solvent blends Storage stability of lysozyme in DMSO / triacetin blends Drug release Study of flow behavior and viscosity Milling of lysozyme powder Particle size determination of precipitated lysozyme in PLGA solutions...67 VI

7 Optical light microscopy Photon correlation spectroscopy Solvent miscibilities Degradation study of PLGA (RG 502H) Injection force measurements Thermogravimetric analysis (TGA) RESULTS AND DISCUSSION.70 _ 3.1. Protein delivery with in situ forming microparticles - methodical aspects Evaluation of dissolution conditions for the release of lysozyme Agitation-induced lysozyme aggregation Lysozyme adsorption to surfaces and interfaces Long term stability of aqueous lysozyme solutions Polymer degradation Influence of buffer (ion species, ionic strength and ph) Medium exchange Protein separation method Alkaline drug extraction Recovery with organic solvents Lysozyme solubility in organic solvents Miscibility of solvents Lysozyme separation with ethyl acetate Formulation parameters of in situ forming drug delivery systems In situ systems based on water-immiscible polymer solvent Macroscopic appearance In vitro drug release of lysozyme from in situ formulations based on triacetin Effect of drug particle size Effect of polymer concentration Comparison of uncapped and capped PLGA Comparison of in situ microparticle with in situ implant systems Qualitative characterization of released and unreleased lysozyme In situ systems based on water-miscible polymer solvents VII

8 In situ systems based on N-methyl-2-pyrrolidone Lysozyme stability in NMP Drug release from blends of DMSO and NMP In situ systems based on dimethyl sulfoxide Macroscopic appearance In vitro drug release of lysozyme from in situ formulations based on DMSO Lysozyme incorporation into PLGA solutions in DMSO Effect of polymer concentration Comparison of uncapped and capped PLGA Effect of drug loading Comparison of in situ microparticle with in situ implant systems Mathematical modeling of lysozyme release from in situ implants Addition of release modifying agents Blends of water-miscible and water-immiscible polymer solvents The ternary solvent system DMSO / ethyl acetate / water DMSO / ethyl acetate / water blends Lysozyme in DMSO / ethyl acetate / water blends PLGA in DMSO / ethyl acetate / water blends Lysozyme incorporation into PLGA solutions Particle size of precipitated lysozyme Effect of the DMSO / ethyl acetate / water ratio on lysozyme release Effect of the lysozyme supplier on drug release The ternary solvent system DMSO / triacetin / water Lysozyme stability in DMSO / triacetin / water blends PLGA in DMSO / triacetin / water blends Lysozyme stability in PLGA solutions in DMSO / triacetin blends External phase composition of ISM Oil-in-oil vs. water-in-oil in situ microparticles Internal to external phase ratio Mixing rate during emulsification Injectability Stability of oil-in-oil emulsions _ VIII

9 4. SUMMARY ZUSAMMENFASSUNG REFERENCES PUBLICATIONS & PRESENTATIONS CURRICULUM VITAE IX