INTRA-ARTICULAR DEPOT FORMING DRUG DELIVERY SYSTEM FOR OSTEOARTHRITIS Matthew Freddi, BSc (Hons) Thesis submitted to the University of Nottingham for the degree of Doctor of Philosophy May 2012 Abstract Osteoarthritis (OA) is a chronic degenerative disease of the joint. Current treatments for this disease (such as glucocorticoid steroids) aim to reduce pain and increase mobility. Intra-articular injection is used in OA as treatment can be targeted to affected joints only. There is currently a lack of sustained release formulations for intra-articular injection. The aim of this thesis was to produce and characterise an injectable intra- articular drug delivery system capable of providing delivery of the steroid dexamethasone phosphate (DXMP) over 3 months. This would be an injectable hydrogel that contains drug loaded nanoparticles. Initially two systems Pluronic F127 gels and polyelectrolyte complexes between hyaluronic acid (HA) and chitosan were investigated. The complexes between HA and chitosan were selected for the hydrogel portion of this system as they showed the greatest stability and promise in initial studies. To improve the polyelectrolyte complex properties a modified HA was synthesised. This modified polymer caused faster complex formation and produced stronger, more resilient complexes. DXMP was incorporated into poly(glycerol-adipate) (PGA) nanoparticles. A low but sufficient drug loading was achieved and particles were found to give a sustained drug release over 28 days. i Nanoparticles were found to be efficiently incorporated and well retained within complexes. Nanoparticles slightly improved complex formation and properties. Composites were able to be formulated into an injectable form. Drug release from directly loaded complexes was rapid. A full release profile was not determined from composites of nanoparticle loaded complexes; however, over 60% of the loaded drug was recovered after 56 days of release study. Dexamethasone crystals were also incorporated directly into complexes to investigate the necessity of the use of nanoparticles. This gave a sustained drug release over 90 days making this system worthy of further investigation. These results highlight the different responses of these systems using drugs with different hydrophobicities. ii Acknowledgments Firstly and most importantly I would like to thank my supervisors Dr. Martin Garnett and Dr. Snow Stolnik whose guidance and support were invaluable. I would also like to thank my AstraZeneca supervisors Simon Cruyws, Terry Woolley and Paul Gellert for their useful input. My thanks are also due to the EPSRC and AstraZeneca for funding and presenting me with this opportunity. I would like to thank the people who helped with my work during this project: Sanya Puri, Ahmed Abushrida and Mieke Heyde for help with the polymers; Bettina Wolf and Lloyd Hamilton for support with rheology. I would also like to thank the technical staff for their amazing work and dedication: Christy Grainger-Boultby, Paul Cooling, Teresa Marshall and Lee Hibbett. I would also like to acknowledge other students who have contributed to the results in this thesis: Fabrice Bayard who carried out the work on the PAA hydrogels in Chapter 3; Salome Gamble and Shy Lih Teo who both made contributions to some initial studies. My thanks are also due to Chris Grindon and Claudia Matz for emotional and administrative support as DTC project officer. Finally I would like to thank the other members of the Targeted Therapeutics DTC for their friendship and support, particularly Vicky Hutter, Klara Lovrics, Adnan Khan, Helen Angell and Gavin Hackett. Lastly I would like to thank all my friends and family for their constant support and love; especially my grandmother Sylvia Hope Butters who passed away in 2010 aged 94 and is greatly missed. iii Table of Contents Abstract ............................................................................................................ i Acknowledgments ......................................................................................... iii Table of Contents ........................................................................................... iv List of Figures .............................................................................................. xvi List of Tables ............................................................................................... xxi Abbreviations .............................................................................................. xxii CHAPTER 1 - INTRODUCTION ..................................................................... 1 1.1 General Introduction to Osteoarthritis ...................................................... 1 1.1.1 Introduction ....................................................................................... 1 1.1.2 Risk Factors ....................................................................................... 1 1.1.3 Prevalence .......................................................................................... 2 1.1.4 Impact ................................................................................................ 3 1.2 Synovial Joint Physiology ........................................................................ 3 1.2.1 General Structure ............................................................................... 4 1.2.2 Cartilage............................................................................................. 5 1.2.2.1 Extracellular matrix .................................................................... 5 1.2.2.2 Chondrocytes .............................................................................. 8 1.2.3 Synovial Membrane and Joint Capsule ............................................. 9 1.2.3.1 Synovial membrane .................................................................... 9 1.2.3.2 Joint capsule ............................................................................. 10 1.2.4 Synovial Fluid.................................................................................. 10 1.2.4.1 Hyaluronic acid ......................................................................... 11 1.2.4.2 Other constituents ..................................................................... 13 1.2.4.3 Functions .................................................................................. 14 iv 1.3 Disease Progression in Osteoarthritis ..................................................... 14 1.3.1 Early Osteoarthritis .......................................................................... 15 1.3.2 Progression to Clinical Osteoarthritis .............................................. 15 1.3.3 Cytokines ......................................................................................... 16 1.3.4 Enzymes........................................................................................... 17 1.3.5 Synovial Inflammation .................................................................... 19 1.3.6 Chondrocytes ................................................................................... 21 1.3.7 Reactive Oxygen Species ................................................................ 22 1.3.8 Synovial Fluid.................................................................................. 24 1.3.9 Osteophytes, Subchondral Bone Sclerosis and Capsular Fibrosis .. 26 1.3.9.1 Osteophytes .............................................................................. 26 1.3.9.2 Subchondral bone sclerosis ...................................................... 27 1.3.9.3 Capsular fibrosis ....................................................................... 28 1.3.10 Vicious Cycle ................................................................................ 28 1.4 Drug Treatments for Osteoarthritis ......................................................... 29 1.4.1 Current Treatments .......................................................................... 29 1.4.2 Intra-articular Delivery for Osteoarthritis ........................................ 30 1.4.2.1 Current treatments .................................................................... 30 1.4.2.2 Challenges for intra-articular delivery ...................................... 33 1.4.2.3 Drug delivery systems for intra-articular delivery.................... 34 1.4.3 Disease Modifying Drugs for Osteoarthritis ................................... 42 1.4.3.1 DMOAD potential of current treatments .................................. 42 1.4.3.2 Novel DMOADs ....................................................................... 43 1.4.3.3 DMOAD potential of other drugs ............................................. 45 1.5 Plan for Delivery System ........................................................................ 46 v 1.6 Introduction to Hydrogels and Injectable Hydrogels .............................. 47 1.6.1 Hydrogels......................................................................................... 47 1.6.2 Injectable Hydrogels ........................................................................ 48 1.6.3 Polyelectrolyte Complexes .............................................................. 49 1.6.3.1 Introduction to polyelectrolyte polymers ................................. 49 1.6.3.2 Polyelectrolyte complexation ................................................... 50 1.7 Introduction to Nanoparticle Drug Delivery Systems ............................ 52 1.7.1 General Introduction 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