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Copper-Topotecan Complexation: Development of a Novel Liposomal Fromulation of Topotecan

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Copper-Topotecan Complexation: Development of a Novel Liposomal Fromulation of Topotecan COPPER-TOPOTECAN COMPLEXATION: DEVELOPMENT OF A NOVEL LIPOSOMAL FROMULATION OF TOPOTECAN by AMANDEEP S. TAGGAR B.Sc. (Chemistry, Honours), University of British Columbia, 2003 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (PATHOLOGY AND LABORATORY MEDICINE) The University of British Columbia April, 2006 © Amandeep S. Taggar, 2006 ABSTRACT Previously it has been reported that transition metal complexation reactions can be used as a method to encapsulate the anticancer drug doxorubicin into liposomes. It has also been reported that a therapeutically promising formulation of topotecan could be prepared using methods that relied on encapsulated manganese(II) ions (Mn ) and a divalent cation/proton ionophore A23187. In this formulation, however, it was not clear whether topotecan encapsulation was achieved as a result of an established pH gradient or as a consequence of transition metal cation complexation with topotecan. The studies described here assess the role of transition metal ions in the encapsulation of topotecan into liposome prepared from l,2-distearoyl-^n-glycero-3-phosphocholine (DSPC) and cholesterol (CH) (55:45, mole ratio). Liposomes with Mn2+, copper(II) (Cu2+), zinc(II) (Zn2+) or cobalt(II) (Co2+) ion gradients (metal inside) were prepared. Subsequently, topotecan was added to the outside of these liposomes (final drug to lipid ratio (mol:mol) of 0.2) and drug encapsulation was measured as a function of time and temperature. Consistent with previous results, topotecan could be encapsulated into Mn2+-containing liposomes only in the presence of A23187. This result suggested that a transmembrane pH gradient was necessary for topotecan loading. No drug loading was achieved with liposomes containing Co2+ or Zn2+. However, Cu2+-containing liposomes, in the presence or absence of an imposed pH gradient, efficiently encapsulated topotecan. It has been 2"F reported that Cu can form a complex with camptothecin and for this reason the complexation reaction between topotecan and Cu was characterized in solution as a function of pH. These studies demonstrated that topotecan inhibited formation of an insoluble copper(II) hydroxide precipitate. Furthermore, analysis of the topotecan loaded liposomes indicated that only the active lactone form of the drug was encapsulated and that the inactive, carboxylate form, could not be encapsulated. Therefore, transition metal ion complexation reactions define a viable methodology to prepare liposomal topotecan formulation. TABLE OF CONTENTS ABSTRACT ii TABLE OF CONTENTS iv LIST OF TABLES vi LIST OF FIGURES vii ABBREVIATIONS viii ACKNOWLEDGEMENTS x DEDICATIONS xi CHAPTER 1 - INTRODUCTION 1 1.1 Project Overview 1 1.2 Transition metals in medicine 3 1.2.1 Transition metal pharmaceuticals 4 1.2.2 Influence of transition metals on therapeutic 6 outcome 1.3 Liposomes 7 1.3.1 Liposomes as drug carriers 7 1.3.2 Components of Liposomes 11 1.3.2.1 Phospholipids 11 1.3.2.2 Cholesterol 15 1.3.2.3 Non-conventional excipients used in bilayers 16 1.3.3 Liposome Classification 17 1.3.4 Formation of liposomes: thermodynamic considerations 19 1.3.5 Gel-to-liquid crystalline phase transition 21 1.3.6 Drug Encapsulation and retention 23 1.3.6.1 Encapsulation methods 24 1.3.6.1.1 Passive encapsulation 26 1.3.6.1.2 Active Encapsulation (remote loading) 26 1.3.6.1.2.1 Ionophores 27 1.4 Complexation reactions with active anticancer agents: their use in 28 liposome formulations 1.5 Camptothecins 31 1.5.1 Camp to thecin Chemistry 32 1.5.2 Camptothecins in liposomes 34 iv 1.5.3 Camptothecin-metal interactions 1.6 Hypotheses and research objectives CHAPTER 2 - MATERIALS AND METHODS 2.1 Materials 2.2 Liposome Preparation 2.3 Ion gradient preparation and topotecan loading 2.4 Topotecan assays 2.5 pH gradient and trapped volume determination 2.6 pH titrations of topotecan and/or copper containing solution 2.7 Cryogenic transmission electron microscopy (Cryo-TEM) CHAPTER 3 - RESULTS CHAPTER 4 - DISCUSSION CHAPTER 5 - FUTURE DIRECTIONS REFERENCES LIST OF TABLES Table 1.1 List of FDA approved liposomal formulations of some therapeutic 8 agents Table 1.2 Melting points of some saturated alkanes and their 1-unsaturated 14 derivatives Table 3.1 Conversion of topotecan from ring closed to ring open form at 60°C 58 in pH 7.4 buffer vi LIST OF FIGURES Figure 1.1 Chemical structures of drugs that can complex with metal ions 5 Figure 1.2 Liposome target site accumulation - EPR effect. 10 Figure 1.3 Structure of a phospholipid molecule and some examples of head 13 groups, acyl chains, and other excipients found in lipid bilayers Figure 1.4 Basic structures of phospholipids 20 Figure 1.5 Gel-to-liquid crystalline phase transition 22 Figure 1.6 Drug encapsulation methods 25 Figure 1.7 Structures of ionophore 29 Figure 1.8 Topotecan structure and E-ring hydrolysis 33 Figure 3.1 Topotecan encapsulation into DSPC/CH (55:45) liposomes with pre- 45 entrapped MnS04 at 40°C or 60°C, (A) in the presence of ionophore A23187, (B) in the absence of ionophore Figure 3.2 Topotecan encapsulation into DSPC/CH (55:45) liposomes with pre- 47 entrapped MnS04, C0SO4, CuS04 and ZnS04 at 60°C Figure 3.3 Determination of transmembrane pH gradient before and after 49 addition of drug/ionophore to DSPC/CH (55:45) liposomes formulated with unbuffered MnS04 Figure 3.4 Topotecan encapsulation into DSPC/CH (55:45) liposomes with pre- 51 entrapped MnS04, CoS04, CuS04 and ZnS04 at 60°C in the presence of ionophore Nigericin Figure 3.5 Topotecan encapsulation into DSPC/CH (55:45) liposomes with pre- 53 entrapped CuS04 at 40°C or 60°C or 20°C, (A) in the presence of ionophore A23187, (B) in the absence of ionophore, (C) encapsulation in liposomes formulated with buffered pH 7.5 CuS04 solution. Inset: representative Cryo-TEM images of liposomes loaded with topotecan under various conditions Figure 3.6 NaOH induced precipitation of copper in the presence or absence of 56 topotecan vii Figure 3.7 Influence of lactone ring hydrolysis on topotecan loading into 59 DSPC/Ch liposomes prepared in unbuffered CuS04 solution Figure 4.1 Possible coordination sites in topotecan 67 vm ABBREVIATIONS ao area of phospholipid head group CH Cholesterol Co2+ cobalt(II) ions CPP critical packing parameter CPT camptothecin Cryo-TEM cryogenic-transmission electron microscopy Cu2+ copper(II) ions DNA deoxyribonucleic acid dsDNA double strand DNA DOTAP 1,2-dioleoyl-3 -trimethylammonium-propane DPSC l,2-distearoyl-src-glycero-3-phosphocholine EDTA ethylene diamine tetraacetic acid EPR enhanced permeability and retention ESR electron spin resonance FATMLV freeze and thawed multilamellar vesicles FDA Food and Drug Administration HBS HEPES buffered saline HEPES N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) HPLC high performance liquid chromatography IR infrared resonance iv intravenous lc critical chain length LUV large unilamellar vesicles MLV multilamellar vesicles Mn2+ manganese(II) ions MPS mononuclear phagocytic system NMR nuclear magnetic resonance NSAID non-steroidal anti-inflammatory drugs PC phosphocholine PE phosphoethanolamine PEG polyethanol glycol PI phosphoinositol PS phosphoserine SEC size exclusion column SHE sucrose HEPES EDTA viii SUV small unilamellar vesicles TEA triethylamine transition temperature Tm Topo-I topoisomerase-I enzyme UV ultra-violet V volume of a phospholipid molecule V volume trapped within liposomes (ul/umol) Vis visible light Zn2+ zinc(II) ions 3H-CHE 3H-cholesterylhexadecyl ether ACKNOWLEDGEMENTS This thesis in no part is an individual effort. Rather it is love and support of many people through out my life that made it possible for me to achieve this. First of all I would like to thank Marcel Bally for taking me on as a graduate student and believing in me, although there were instances even I thought I wouldn't finish this project. Thank you Marcel, for guidance, helpful discussions, resources and not letting me work on 5-FU stuff. I would also like to thank other members of Bally lab especially Euan Ramsay for answering my countless pestering questions, Anitha Thomas for all the helpful suggestions, Malathi Anantha for teaching me the art of AA, HPLC and number of other machines and gadgets around the lab, Catherine Tucker for that wonderful smile and positive attitude to lift me up whenever I encountered a problem, Janet and Brian for just being there. I would like to send special thanks to Spenser Kong for fixing "all the problems" and whose presence was direly missed in the lab during my second year. To all the members of the Advanced therapeutics group - thanks for the wonderful discussions, lunches, parties (and for not trashing my place during those parties) and lots of other fun times. Special thanks to Fuloi restaurant for shanghai noodles, Wrap-zone and GGW.. .kept me going for wee hours into the night I am also very grateful to Dr. Chris Orvig for many discussions on metals, metal chemistry and explaining me some key elements crucial to this work. I would like to extend my warmest thanks to my family. Mom, dad thank you very much for everything you have done for me. Your love and support has made it possible for me to achieve everything in my life. Deep & Jatinder thank you for always supporting me and believing in me and don't worry, I am getting somewhere and won't always be a student. Lastly, I am very grateful to Paveen for listening to all my concerns, cheering me up with her brilliant smile, editing my work, helping me practice my talks and for everything else in my life. Thank you. x DEDICATIONS This thesis is dedicated to my mother and father Kewal and Kuldev Taggar Thank you for your patience and support And also to the loving memory of my uncle Darshan Singh Taggar Who passed away due to cancer in 2005 Chapter 1 INTRODUCTION 1.1 Project overview Cancer is a disease characterized by heterogeneous populations of cells that have the ability to generate their own growth signals, thereby reducing dependence on growth inhibitory signals; signals released both internally and externally [1].
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