NOVEL APPROACHES TO IRON CHELATION THERAPY: NOVEL COMBINATIONS AND NOVEL COMPOUNDS Dr Evangelia Vlachodimitropoulou Koumoutsea BSc, MBBS King’s College London A thesis submitted for the degree of PhD University College London (UCL) From work performed at the Department of Haematology, University College London, 72 Huntley Street, London, WC1E 6BT ‘I, Evangelia Vlachodimitropoulou Koumoutsea confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis.’ 1 ITHACA by C.P Cavafy As you set out for Ithaka hope the voyage is a long one, full of adventure, full of discovery. Laistrygonians and Cyclops, angry Poseidon—don’t be afraid of them: you’ll never find things like that on your way as long as you keep your thoughts raised high, as long as a rare excitement stirs your spirit and your body. Laistrygonians and Cyclops, wild Poseidon—you won’t encounter them unless you bring them along inside your soul, unless your soul sets them up in front of you. Hope the voyage is a long one. May there be many a summer morning when, with what pleasure, what joy, you come into harbors seen for the first time; may you stop at Phoenician trading stations to buy fine things, mother of pearl and coral, amber and ebony, sensual perfume of every kind— as many sensual perfumes as you can; and may you visit many Egyptian cities to gather stores of knowledge from their scholars. Keep Ithaka always in your mind. Arriving there is what you are destined for. But do not hurry the journey at all. Better if it lasts for years, so you are old by the time you reach the island, wealthy with all you have gained on the way, not expecting Ithaka to make you rich. Ithaka gave you the marvelous journey. Without her you would not have set out. She has nothing left to give you now. And if you find her poor, Ithaka won’t have fooled you. Wise as you will have become, so full of experience, you will have understood by then what these Ithakas mean . 2 ACKNOWLEDGEMENTS After a 5 year journey, the day to put the finishing touch to my thesis has come. It has been a period of intense learning for me, not only scientifically but also personally. I am extremely happy to be in a position to write this note of thanks, as I consider this one of my biggest achievements to date. This journey has allowed me to consolidate a number of relationships. I would like to first and foremost express my sincere gratitude to my supervisor Prof. John Porter for welcoming me into his team. Despite my slightly unconventional research path he has been unwavering in his support, patience, motivation, enthusiasm, and encouragement. I could not have imagined having a better mentor. I would like to express my appreciation to Dr. Anna David for her support and advice in both my clinical and academic work. A special thank you goes to Dr. Panicos Shangaris for his encouragement and invaluable support with the mouse work. Special thanks also goes to Prof. Robert Hider for his constant support throughout this time with his enthusiasm and immense knowledge on medicinal chemistry as well as Dr. Yu-Lin for his contribution of invaluable compound combination speciation plots. Many thanks to Dr. Daniel Stuckley and Laurence Jackson from the UCL Centre for Advanced Biomedical Imaging (CABI) for a brilliant collaboration. I would also like to acknowledge Prof. Richard Naftalin, who introduced me to the world of research and who has encouraged and supported me every step of the way, up until today. I would like to thank all my fellow labmates, particularly Dr. Maciej Garbowski and Dr. Edith Weiner for all the guidance, stimulating discussions, and sleepless nights spent working together, plus all the fun we have had collaborating over the last 5 years. Huge thanks to Dr. Srichairatankool and our team from Thailand, Pimpi, and Adchara who spent many months tirelessly working with us. I would like to express my profound gratitude to my mother, dog Hector, aunt Stamatia and cousin Dimitri and to all of my family for their unconditional love and support. I would also like to thank my friends Lizzie, Neil, Igor, Maria and my Kolyan for putting up with me when times were rough, supporting me during my write up and seeing me through this journey. Finally, I gratefully acknowledge the Onassis Foundation, Greece, and Novartis UK, for their financial support which has made this thesis possible. 3 SUMMARY Iron overload is an inevitable consequence of repeated blood transfusions required to sustain life in a wide array of haematological conditions such as thalassaemia, aplastic anaemia, and myelodysplastic syndromes (MDS). Without iron chelation therapy, death from cardiotoxic effects of iron overload usually ensues in the second decade. Iron chelation therapy with subcutaneous Desferrioxamine (DFO) infusions at least 5 nights per week has been shown unequivocally to prolong life expectancy in thalassaemia major. However, although this molecule is remarkably free of toxic side effects at treatment doses, patient compliance is often poor, and iron overload still leads to death today. One of the scopes of this Ph.D. was to develop a cellular model that would allow the testing of novel chelating agents used alone or in combination with established chelators. Our in vitro model of iron overload was able to elucidate several principles regarding the interactions of chelators within cells. It allowed for the first time a detailed interrogation of synergy as opposed to additivity of action of licensed chelators when used in combination, which has now been published. This model is also relevant to the development of new chelators and was used to demonstrate the iron binding properties of Eltrombopag (ELT), a drug used to manage ITP, at clinically achievable concentrations. ELT is a powerful intracellular iron chelator that decreases storage iron and enhances iron removal when in combination with commercially available iron chelators. In clinical use, donation of chelated iron by ELT to these chelators offers established routes for elimination of chelated iron. Furthermore, we extensively investigated the iron mobilising properties of the naturally occurring flavonoid quercetin and its principle metabolites. For the first time we showed that quercetin and its metabolites can act as a shuttle when combined with licensed chelators and provided a unique structure-function analysis of flavonoids with regards to iron and ferritn mobilisation and antioxidant capacity as a function of Fe(II) binding. A further goal of this thesis was to establish an iron-overloaded humanised thalassaemia mouse model that could be used to examine whether the same principles which determine iron release from cell cultures also influence the oral efficiency of iron chelators, in vivo. We utilised iron dextran to achieve cardiac iron loading confirmed by histology and MRI investigations. Iron overloaded mice were treated with a combination of the flavonoid quercetin and the iron chelator Deferasirox (DFX), and we established the value of this in combination in terms of cardiac iron mobilisation. Our novel humanised β thalassaemia ion-overloaded mouse model demonstrating cardiac iron loading is a first-in-kind development, and the novel application of MRI will provide a useful tool for studying iron chelators, the pathophysiology and disease progression, blood transfusion regimens and cellular/gene therapy in iron overload in the future. Our findings in vivo support the contention that our cellular model is a useful screening tool for new compounds, both for toxicity and efficacy. Keywords: Iron, Chelator, Hepatocyte, Cardiomyocyte, Thalassaemia 4 Table of Contents NOVEL APPROACHES TO IRON CHELATION THERAPY: NOVEL COMBINATIONS AND NOVEL COMPOUNDS..................................................... 1 ACKNOWLEDGEMENTS ......................................................................................... 3 LIST OF ABBREVIATIONS .................................................................................... 16 Chapter 1 INTRODUCTION ................................................................................. 17 1.1 Objective of this work ................................................................................ 17 1.2 The need for safe and effective iron chelation ........................................... 17 1.3 Principles of iron chelation therapy ........................................................... 22 1.4 Design of chelators: properties for maximal oral efficacy ......................... 27 1.5 Properties to minimise toxicity: design of chelators .................................. 31 1.6 Chelator Evaluation ................................................................................... 34 1.7 Commercially available iron chelators ...................................................... 44 1.8 Other chelators that have reached clinical trials ........................................ 51 1.9 Conclusions ................................................................................................ 53 Chapter 2 MATERIALS AND METHODS ........................................................... 54 2.1 Iron chelators and substances tested for iron chelation activity ................ 54 2.2 Cell Lines ................................................................................................... 54 2.3 Cellular Methods ........................................................................................ 54 5 2.4 Flowchart of