Topical delivery of α1-Antichymotrypsin for wound healing Dissertation zur Erlangung des Doktorgrades der Fakultät für Chemie und Pharmazie der Ludwig-Maximilians-Universität München vorgelegt von Roland Schmidt aus Treuchtlingen München 2005 Erklärung Diese Dissertation wurde im Sinne von § 13 Abs. 3 und 4 der Promotionsordnung vom 29. Januar 1998 von Herrn Prof. Dr. G. Winter betreut. Ehrenwörtliche Versicherung Diese Dissertation wurde selbständig, ohne unerlaubte Hilfe erarbeitet. München, 01. Januar 2005 (Roland Schmidt) Dissertation eingereicht am: 10. Januar 2005 1. Berichterstatter: Prof. Dr. G. Winter 2. Berichterstatter: Prof. Dr. W. Frieß Tag der mündlichen Prüfung: 1. Februar 2005 ACKNOWLEDGMENTS Foremost, I wish to express my deepest appreciation to my supervisor, Prof. Dr. Gerhard Winter. I am much obliged to him for his professional guidance and his scientific support. On a personal note, I especially want to thank him for inspiring my interest in protein pharmaceuticals, for teaching me so much, and for creation of an outstanding working climate. I am also grateful to the Switch Biotech AG, Neuried, Germany for financial support. I would like to acknowledge Dr. Uwe Goßlar for rendering every assistance and the always professional and personally warm contact. Moreover, I would like to thank Annette, Björn, and especially Olivia for performing the Bioassays. Thanks are also extended to Prof. Dr. Bracher, Prof. Dr. Frieß, PD Dr. Paintner, Prof. Dr. Schlitzer, and Prof. Dr. Wagner for serving as members of my thesis advisor committee. I very much enjoyed working at the Department for Pharmaceutical Technology and Biopharmaceutics of the Munich Ludwig-Maximilians-University, what was mainly due to the cooperative and most convenient atmosphere. Wolfgang, Silke, Sandra, Iris, Steffi, Fritz, Ingo and all the others, it was a pleasure to work with you. To my parents Table of contents 1 Introduction 1 1.1 Wound healing 3 1.1.1 Physiology of wound healing 3 1.1.1.1 Wound healing process 3 1.1.1.2 Growth factors in physiological wounds 8 1.1.1.3 Proteases in physiological wounds 12 1.1.1.4 Protease inhibitors in physiological wounds 15 1.1.2 Pathophysiology of chronic wounds 18 1.1.2.1 Cellular and biochemical imbalance in chronic wounds 19 1.1.2.2 Clinics of chronic wounds 20 1.1.2.3 Infection of wounds 21 1.1.3 Treatment of chronic wounds 21 1.1.3.1 Debridement 22 1.1.3.2 Moist wound treatment 23 1.1.3.2.1 History of moist wound treatment 23 1.1.3.2.2 Effects of moist wound treatment 23 1.1.3.2.3 Products for moist wound treatment 24 1.1.3.3 Infection control in wounds 25 1.1.3.4 Skin substitutes for wound healing 26 1.1.3.5 Growth factors control in chronic wounds 26 1.1.3.6 Protease control in chronic wounds 27 1.1.3.6.1 Active dressings for chronic wounds 28 1.1.3.6.2 Delivery of ACT in chronic wounds 28 1.2 Protein delivery from hydrogel formulations 30 1.2.1 Suitability of hydrogels for protein delivery 31 1.2.2 Protein delivery from hydrogels 32 1.2.2.1 Application in wounds 32 1.2.2.2 Transdermal delivery 33 1.2.2.3 Oral delivery 34 1.2.2.4 Ophthalmic delivery 35 1.2.2.5 Delivery by injection and general approaches 35 1.2.3 Summary 37 1.3 Aim of the thesis 39 2 Materials and Methods 41 2.1 Materials 41 2.1.1 α1-Antichymotrypsin (ACT) 41 2.1.2 Excipients and chemicals 42 2.1.3 Polymers 43 2.1.3.1 Cellulose ethers 43 2.1.3.2 Gellan gum 43 2.1.3.3 Other polymers 44 2.2 Methods 45 2.2.1 Characterisation of ACT 45 2.2.1.1 ACT activity assay 45 2.2.1.2 ACT ELISA 45 2.2.1.3 Gel electrophoresis 46 2.2.2 Manufacture of matrices 46 2.2.2.1 Wet film manufacture with the scraper 46 2.2.2.2 Freeze-drying 46 2.2.2.3 Warm air drying 47 2.2.3 Characterisation of matrices 47 2.2.3.1 Viscometry 47 2.2.3.2 Mechanical tests 47 2.2.3.3 In vitro Release tests 48 2.2.3.4 Karl Fischer Titration 48 2.2.3.5 Differential scanning calorimetry (DSC) 48 2.2.3.6 X-ray diffraction 49 3 Results and Discussion 50 3.1 Analytical tools for the characterisation of ACT 51 3.2 Stabilisation of ACT in solution 52 3.2.1 Effects of pH, buffers, and electrolytes on ACT solution stability 52 3.2.1.1 Effect of pH on ACT solution stability 52 3.2.1.2 Effect of buffer species on ACT solution stability 56 3.2.1.3 Effect of salts on ACT solution stability 57 3.2.1.4 Effect of buffer content on ACT solution stability 58 3.2.1.5 Summary of the effects of electrolytes on ACT solution stability 60 3.2.2 Effects of stabilisers and excipients on ACT solution stability 61 3.2.2.1 Surfactants for the stabilisation of ACT in solution 61 3.2.2.2 Sugars and polyols for the stabilisation of ACT in solution 63 3.2.2.3 Cyclodextrins for the stabilisation of ACT in solution 65 3.2.2.4 Amino acids for the stabilisation of ACT in solution 68 3.2.2.5 Preservatives for ACT containing solutions 70 3.2.3 Summary of ACT solution stability studies 71 3.3 Hydrogels as delivery system for ACT into wounds 72 3.3.1 Development as delivery system for wound healing 72 3.3.1.1 Sterilisation of hydrogels 73 3.3.1.2 Viscosity of hydrogels 73 3.3.1.3 Viscosity of gellan gum/hydroxyethyl cellulose hydrogels 76 3.3.2 Stability of ACT in hydrogel formulations 80 3.3.2.1 Effects of polymers on ACT stability in hydrated formulations 80 3.3.2.2 Aseptic manufacture of ACT loaded hydrogels 83 3.3.2.3 Analysis of ACT loaded hydrogels 85 3.3.2.4 Mid term stability of ACT in hydrogel formulations 85 3.3.2.4.1 Principles of data interpretation 86 3.3.2.4.2 Experimental results and discussion 90 3.3.2.4.3 Summary 93 3.3.2.5 Freeze/thaw stability of ACT in hydrogel formulations 93 3.3.3 Summary of hydrogels as ACT delivery systems 94 3.4 Dry delivery systems 95 3.4.1 Xerogels as drug delivery systems for wound healing 96 3.4.1.1 Lyophilisation process 96 3.4.1.1.1 DSC studies 97 3.4.1.1.2 Lyophilisation program 97 3.4.1.2 Gel composition for xerogel formation 100 3.4.1.2.1 Hydroxyethyl cellulose qualities for xerogel formation 101 3.4.1.2.2 Excipients in hydroxyethyl cellulose xerogels 103 3.4.1.2.3 Hydroxyethyl cellulose/gellan gum mixtures for xerogels 108 3.4.1.2.4 Other polymers for xerogel formation 109 3.4.2 Stability of ACT in xerogel formulations 112 3.4.2.1 Stability of ACT during the lyophilisation process 112 3.4.2.2 Mid term stability of ACT in xerogel formulations 115 3.4.2.2.1 Principles of data interpretation 116 3.4.2.2.2 Experimental results and discussion 118 3.4.2.2.3 Summary 121 3.4.3 Polymer films as drug delivery systems for wound healing 122 3.4.3.1 Production process 122 3.4.3.2 Gel composition for polymer film formation 124 3.4.3.2.1 Gelling agents for film formation 124 3.4.3.2.2 Polymers as additives to hydroxyethyl cellulose films 125 3.4.3.2.3 Hydroxyethyl cellulose / gellan gum mixtures for film formation 132 3.4.3.2.4 Protein stabilisers in polymer films 134 3.4.4 Stability of ACT in film formulations 135 3.4.4.1 Stability of ACT during the film manufacturing process 136 3.4.4.2 Mid term stability 138 3.4.4.3 Summary 141 3.4.5 Summary for dry matrices as ACT delivery systems 141 3.5 Release of ACT from dry delivery systems 143 3.5.3 Experimental setup 143 3.5.3.1 Membrane 143 3.5.3.2 Acceptor medium 144 3.5.3.3 Chamber model 145 3.5.4 Theoretical background and data interpretation 146 3.5.5 Release of model substances from gel based matrices 147 3.5.6 Release of ACT from formulations 151 3.5.6.1 Dynamic model 151 3.5.6.2 Static model 153 3.5.6.2.1 Evaluation of the model 153 3.5.6.2.2 Release of ACT from xerogel formulations 157 3.5.6.2.3 Release of ACT from film formulations 161 3.5.6.2.4 Summary 166 4 General summary 167 5 References 171 Curriculum vitae 183 Chapter 1 - Introduction 1 Introduction The World Health Organisation of the United Nations prognoses the development of world-wide diabetes cases over the next decades in actual studies. Accordingly, the number of type II patients will more than double until 20301. Next to the disease itself, moreover, 25% of diabetes patients frequently develop chronic wounds with about half of them requiring elaborate inpatient treatment. Therefore, the diabetic foot causes more hospitalisation than does any other complication associated with diabetes and represents approximately 2.5% of all hospital admissions2.