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Copper-based therapeutics : creating a formulation platform to facilitate development of an emerging… Wehbe, Mohamed 2017

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COPPER-BASED THERAPEUTICS: CREATING A FORMULATION PLATFORM TO FACILITATE DEVELOPMENT OF AN EMERGING DRUG CLASS by MOHAMED WEHBE BSc, University of Ottawa, 2012  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Pharmaceutical Sciences)  THE UNIVERSITY OF BRITISH COLUMBIA (VANCOUVER) October 2017 © Mohamed Wehbe, 2017 ii  Abstract: Copper-based therapeutics (CBTs) are a promising class of drug candidates that have been shown to have anti-cancer activity. One of the major challenges associated with developing this emerging class of medicines, however, is their poor aqueous solubility (< 1 mg/mL). This has impeded the transition of these agents from preclinical candidate drugs to lead candidates suitable for use in humans. This thesis describes a novel method for preparing copper-based therapeutics suitable for parenteral administration. The method involves synthesis of the copper complexes in the core of liposomes (nanoscaled spherical structures with an aqueous core surrounded by a lipid bilayer). The method provides a simple, transformative solution enabling the development of CBTs as viable candidate anti-cancer drugs—a brand new class of therapeutics for cancer patients.  The potential of this technology was exemplified with several compounds. Diethyldithiocarbamate (DDC) is the primary metabolite of disulfiram, an approved drug for alcoholism that is being repurposed for cancer. The anti-cancer activity of DDC is dependent on complexation with copper (Cu(DDC)2),  which is a highly insoluble complex. An injectable Cu(DDC)2 formulation was prepared through synthesis of Cu(DDC)2 inside the aqueous core of liposomes, and the resultant formulation exhibited significant therapeutic activity in two different rodent models of cancer. Clioquinol (CQ) is an approved anti-microbial agent that has potential anti-cancer activity. The activity of CQ is enhanced in some cancer cell lines when complexed to copper. The copper-CQ complex (Cu(CQ)2) is sparingly soluble in aqueous solution but it is demonstrated that Cu(CQ)2 can be synthesized inside liposomes. The therapeutic activity of the resultant formulations was, however, not significant. In an effort to identify cancer indications that may benefit most from treatment with CBTs, the therapeutic potential of DDC, pyrithione (Pyr), plumbagin (Plum), 8-hydroxyquinoline (8-HQ) and CQ copper complexes was determined in a panel of cancer cell lines that differed in their sensitivity to cisplatin. Cu(DDC)2, Cu(Pyr)2, Cu(Plum)2 and Cu(8-HQ)2 showed IC50 values less than that of cisplatin in all tested cell lines. This work suggests that future studies with CBTs for cancer should focus on platinum refractory cancers.iii  Lay Summary: Copper complexes have been shown to be anti-cancer, anti-microbial, anti-viral and anti-inflammatory agents. While this highlights the benefits of these types of compounds, their insolubility has thwarted their transition to clinic. This thesis describes a method for solving this challenge through the synthesis of copper complexes inside liposomes (tiny bubbles composed of fats). Through this technology it was possible to evaluate the copper complexes of diethyldithiocarbamate (a metabolite of the anti-alcoholism drug disulfiram) and clioquinol (an FDA approved anti-fungal drug) as anti-cancer agents. These are the first studies to use these copper complexes in animal models, contributing valuable insight into possible approaches to treating cancer with copper-containing medicines. A platform technology has been developed which provides a transformative solution to solve solubility challenges of metal complexes, thus enabling the development of this novel class of drugs for the first time.              iv  Preface:  The studies presented in this dissertation were conceived and designed by M. Wehbe and MB. Bally.  Chapter 1 was adapted from the following review and perspective: Wehbe M, Leung A, Abrams M, Orvig C and Bally MB (2017) A perspective – Can copper complexes be developed as a novel class of therapeutics? Dalton Transactions (In Press). Wehbe M, Chernov L, Chen K, and Bally MB (2016). PRCosomes: pretty reactive complexes formed in liposomes. Journal of Drug Targeting. 24(9):787-96. Both manuscripts were organized, written and edited by Wehbe M. The perspective was edited by Leung, A, Abrams M, Orivg C and Bally MB. Figures 1.1, 1.2 and 1.6 were illustrated by Leung A and conceived by Wehbe M, Leung A and Bally MB. The review was edited Chernov L, Chen K and Bally MB. Figures (1.4 and 1.5) were all prepared and illustrated by Wehbe M.  Studies in Chapter 3 are published in the following article: Wehbe M, Anantha M, Backstrom I, Leung A, Chen K, Malhotra A, Edwards K, Bally MB (2016) Nanoscale Reaction Vessels Designed for Synthesis of Copper-Drug Complexes Suitable for Preclinical Development. Plos One 11(4): 1-16. All Cu(DDC)2 experiments were conducted by Wehbe M with assistance from Anantha M (analytical support) and Malhotra A . The cell culture experiments were produced by Wehbe M, the HBEpC cell line was performed by Leung A and A549 was performed by Backstrom I. The CX-5461 and quercetin experiments were conducted by Leung A and Chen K, respectively. The cryo-transmission electron microscopy images were taken at Uppsala University by Wehbe M and Edwards K. This publication was written by Wehbe M with editing by Bally MB.  Studies in Chapter 4 are published in the following article: Wehbe M, Anantha M, Shi M, Leung AWY, Dragowska WH, Sanche L and Bally MB (2017). Development and optimization of an injectable formulation of copper diethyldithiocarbamate, an active anti-cancer agent. International Journal of Nanomedicine. Wehbe M performed all experiments in Figures 4.1, 4.3, 4.5, 4.6, 4.7 and 4.8 with assistance from Anantha M (analytical support). Figure 4.2 was produced by Wehbe M, Leung A and Dragowska W. Leung A conducted the reactive v  oxygen species assay and performed the Western blot with Wehbe M. The flow cytometry experiment was conducted by Wehbe M with Dragowska W and Leung A. The design and formulations were prepared by Wehbe M while Shi M performed the convection enhanced delivery study at the Université du Sherbrooke as shown in Figure 4.4. Sanché L contributed to the design of the convection enhanced delivery study and editing of the manuscript. This publication was written by Wehbe M with editing by Bally MB. The research studies in Chapter 5 have been submitted as a manuscript: Wehbe M, Malhotra A, Anantha M, Lo C, Backstrom I, Dos Santos N and Bally MB (2017). Repurposing the topical antifungal clioquinol for the treatment of cancer. All Cu(CQ)2 experiments were performed by Wehbe M with assistance from Anantha M (analytical support) and Malhotra A . The cell culture experiments were conducted by Wehbe M, the U251 and MV-4-11cell lines were performed by Lo C and Backstrom I. The model development for the A2780-CP model was conceived by Wehbe M with Dos Santos N. This publication and figures were written and prepared by Wehbe M with editing by Bally MB.  Studies in Chapter 6 are published in: Wehbe M, Lo C, Leung A, Dragowska WH, Ryan G, Bally MB (2017). Copper complexes of bidentate ligands exhibit potent anti-cancer activity regardless of platinum sensitivity status. Investigational New Drugs (In press). Wehbe M selected copper complexes and designed experiments. Wehbe M and Leung A selected the cancer cell lines for these studies. The screen was conducted by Lo C with assistance from Wehbe M. Data analysis was performed by Wehbe M and Lo C with assistance from Dragowska and Ryan G. The liposomes were prepared by Wehbe M for the in vivo study. This publication and figures were written and prepared by Wehbe M with editing by Dragowska W and Bally MB.  This thesis was written by Wehbe M with editing provided by Burt H, Ensom M, Webb M, Yip S and Bally MB. The Animal Care Committee at the University of British Columbia examined and approved the use of animals for the studies in this thesis under the following Animal Care Certificates: A10-0171 and A14-0290. vi  Table of Contents: Abstract: ................................................................................................................................................................... ii Lay Summary: ........................................................................................................................................................ iii Preface:..................................................................................................................................................................... iv Table of Contents: ................................................................................................................................................ vi List of Tables: ......................................................................................................................................................... xi List of Figures: ...................................................................................................................................................... xii List of Abbreviations: ....................................................................................................................................... xiv Acknowledgements: .......................................................................................................................................... xvi 1. Introduction ....................................................................................................................................................... 1 1.1 Thesis Overview ........................................................................................................................................ 1 1.2 Copper-Based Therapeutics as an Alternative to Platinum ..................................................... 3 1.3 Copper in Biology ...................................................................................................................................... 6 1.3.1 The Importance of Copper Homeostasis ................................................................................. 6 1.3.2 Cellular Components for Copper Regulation ......................................................................... 7 1.4 Copper Chemistry .................................................................................................................................. 10 1.5 Therapeutically Active Copper Complexes as Anti-cancer, Anti-Inflammatory, and Antimicrobial Agents ................................................................................................................................... 11 1.5.1 Copper Chelation Therapy ......................................................................................................... 14 1.5.2 Copper Radiopharmaceuticals ................................................................................................. 15 1.6 Classification of Copper Complexes ................................................................................................ 16 1.6.1 Copper-Ligand Complexes (CLCs)........................................................................................... 16 1.6.2 Copper-Drug Complexes (CDCs) .............................................................................................. 19 1.7 Approaches to Solve Copper Complex Solubility Challenges: .............................................. 21 1.7.1 High-Throughput Screening ...................................................................................................... 21 vii  1.7.2 Computer-Aided Drug Design ................................................................................................... 22 1.8 Nanoscale Synthesis Reactions Inside Liposomes .................................................................... 23 1.9 Liposomes ................................................................................................................................................. 23 1.10 Remote Loading Using pH Gradients ........................................................................................... 25 1.10.3 Use of A23187 (Divalent Metal Ionophores) to Facilitate Drug Loading in Liposomal Formulations Prepared with Metal Ion Gradients ................................................ 27 1.11 Summary ................................................................................................................................................. 27 2. Materials and Methods ................................................................................................................................ 29 2.1 Materials .................................................................................................................................................... 29 2.2 Cell Lines ................................................................................................................................................... 29 2.3 In Vitro Cell-Based Assays .................................................................................................................. 30 2.3.1 Cytotoxicity Assay of Pt Drugs, Disulfiram, Ligands and Copper Complexes ......... 30 2.3.2 Western Blot Analysis for Ubiquitinylated Protein .......................................................... 30 2.3.3 Flow Cytommetric Analysis of Cu(DDC)2 Treated Cells ................................................. 31 2.3.4 Reactive Oxygen Species Assay ................................................................................................ 31 2.3.5 Phen Green™ FL Copper Ionophore Testing ....................................................................... 32 2.4 Liposome Preparation and Characterization .............................................................................. 32 2.4.1 Liposome Preparation ................................................................................................................. 32 2.4.2 Copper Complexation Synthesis: ............................................................................................. 33 2.4.3 Cryo-Electron Microscopy: ........................................................................................................ 34 2.4.4 Cu(DDC)2 and Cu(CQ)2 Release From Liposomes ............................................................. 34 2.5 Analytical Assays: .................................................................................................................................. 35 2.5.1 Cu(DDC)2 Quantification ............................................................................................................. 35 2.5.2 Cu(CQ)2 Quantification ................................................................................................................ 35 2.5.3 CuQu and CuCX-5461 Quantification ..................................................................................... 36 viii  2.5.4 Copper Quantification .................................................................................................................. 36 2.5.5 Liposomal Lipid Quantification ................................................................................................ 36 2.6 In Vivo Characterization of Liposomal Formulations: ............................................................. 37 2.6.1 Dose Range Finding Studies ...................................................................................................... 37 2.6.2 Pharmacokinetic Studies ............................................................................................................ 38 2.7 In Vivo Efficacy Studies ........................................................................................................................ 38 2.7.1 Convection Enhanced Delivery in F98 Glioma Model ..................................................... 38 2.7.2 Cu(DDC)2 Efficacy Studies in the MV-4-11 and A2780-CP Subcutaneous Tumour Models ........................................................................................................................................................... 39 2.7.5 Cu(CQ)2 Efficacy Studies in the U251 and A2780-CP Subcutaneous Tumour Models ........................................................................................................................................................... 40 2.7.6 Cu(CQ)2 and DSF Combination Efficacy Studies in A2780-CP Subcutaneous Tumour Models ......................................................................................................................................... 40 2.8 Statistical Analysis ................................................................................................................................. 41 3. Nanoscale Reaction Vessels Designed for Synthesis of Copper-Drug Complexes Suitable for Preclinical Development .......................................................................................................................... 42 3.1 Introduction ............................................................................................................................................. 42 3.2 Results ........................................................................................................................................................ 43 3.2.1 DSF, DDC and Cu(DDC)2 Cytotoxicity ..................................................................................... 43 3.2.2 Formation of Cu(DDC)2 by Addition of DDC to Pre-formed Copper Containing Liposomes ................................................................................................................................................... 46 3.2.3 Use of Copper Containing Liposomes to Prepare CBTs Suitable for Intravenous Injection ....................................................................................................................................................... 50 3.3 Discussion ................................................................................................................................................. 53 3.4 Conclusion ................................................................................................................................................ 55 4. Development and Optimization of an Injectable Formulation of Copper Diethyldithiocarbamate, an Active Anti-cancer Agent ........................................................................ 57 ix  4.1 Introduction ............................................................................................................................................. 57 4.2 Results ........................................................................................................................................................ 58 4.2.1 Cu(DDC)2 Characterization ........................................................................................................ 58 4.2.2 Studies to Assess the Mechanism of Cu(DDC)2 Activity .................................................. 60 4.2.3 Plasma Elimination of Cu(DDC)2 Prepared in DSPC/Chol (55:45) Liposomes ..... 62 4.2.4 Cu(DDC)2 Efficacy in Models of Cancer ................................................................................. 63 4.2.5 Enhancing the Circulation Longevity of Cu(DDC)2 ........................................................... 66 4.2.6 Characterizing Cu(DDC)2 Prepared in DSPC/DSPE-PEG2000 (95:5) Liposomes .... 69 4.2.7 Therapeutic Activity of Cu(DDC)2 Prepared in DSPC/DSPE-PEG2000 (95:5) Liposomes ................................................................................................................................................... 71 4.3 Discussion ................................................................................................................................................. 73 4.4 Conclusion ................................................................................................................................................ 76 5. Development of a Copper-Clioquinol Formulation Suitable for Intravenous Use ............... 77 5.1 Introduction ............................................................................................................................................. 77 5.2 Results ........................................................................................................................................................ 78 5.2.1 Cytotoxicity of CQ and its Copper Complex ......................................................................... 78 5.2.2 Copper CQ Liposome Characterization ................................................................................. 79 5.2.3 Tolerability and Pharmacokinetics Following Intravenous Administration of the Cu(CQ)2 Formulation............................................................................................................................... 82 5.2.4 Anti-tumor Efficacy Following Intravenous Administration of Cu(CQ)2 ................. 85 5.2.5 Efficacy of Cu(CQ)2 in Combination with Disulfiram ....................................................... 86 5.3 Discussion ................................................................................................................................................. 88 5.4 Conclusion ................................................................................................................................................ 90 6. Copper Complexes of Bidentate Ligands Exhibit Potent In Vitro Anti-Cancer Activity Regardless of Platinum Sensitivity Status ................................................................................................ 92 6.1 Introduction ............................................................................................................................................. 92 x  6.2 Results ........................................................................................................................................................ 95 6.2.1 Pt-Resistance Does not Impact Sensitivity of Cancer Cells to Cu(DDC)2 Complex 95 6.2.2 Activity of Copper Complexes in Cancer Cells with Different Pt Sensitivity........... 96 6.2.3 Efficacy of Liposomal Cu(DDC)2 in a Pt-resistant A2780-CP Tumour Xenograft Model ........................................................................................................................................................... 100 6.3 Discussion ............................................................................................................................................... 101 6.4 Conclusion .............................................................................................................................................. 103 7. Summarizing Discussion .......................................................................................................................... 104 7.3 Novelty of Metal-based Complexation Reactions to Prepare Liposomal Drug Formulations ................................................................................................................................................. 107 7.4 Future Work ........................................................................................................................................... 112 7.4.1 Other Metals .................................................................................................................................. 112 7.4.2 Other Diseases and Complexes .............................................................................................. 114 7.5 Conclusion .............................................................................................................................................. 115 References .......................................................................................................................................................... 117      xi  List of Tables: Table 1. 1: Examples of copper ligand complexes with antimicrobial and anti-cancer activity ................................................................................................................................................................................... 17 Table 1. 2: Examples of drug classes which form copper drug complexes.................................. 18 Table 3. 1: Donor systems that can be used in Copper(II)-complex loading.. ............................ 52 Table 6. 1: Correlation between CDDP and copper complex sensitivity based on the IC50 values ...................................................................................................................................................................... 99                 xii  List of Figures: Figure 1. 1 Overcoming the pharmaceutical challenge of developing copper-based therapeutics. ........................................................................................................................................................... 5 Figure 1. 2: Cellular regulation of copper levels. ...................................................................................... 8 Figure 1. 3: Venn diagram showing the possible copper chelating functional groups composed of nitrogen (N-donor), oxygen (O-donor) and sulfur (S-donor) atoms. ................. 11 Figure 1. 4: Doxorubicin possesses structural motifs that facilitate remote loading in liposomes through use of transmembrane pH gradients and metal complexation. ................ 24 Figure 1. 5:  Remote loading approaches to encapsulate doxorubicin into liposomes........... 26 Figure 3.1: The anti-cancer activity of diethyldithiocarbmamate (DDC) is dependent on copper ..................................................................................................................................................................... 45 Figure 3.2: Diethyldithiocarbamate (DDC) loading into DSPC/Chol (55:45) liposomes prepared with encapsulated 300 mM CuSO4. ......................................................................................... 47 Figure 3.3: Characterization of copper-complex loading method. ................................................. 49 Figure 3. 4: Preliminary tolerability and plasma elimination profiles for liposomal formulations of Cu(DDC)2, Cu(CQ)2 , CuQu and CuCX-5461 after intravenous injection into CD-1 mice. ............................................................................................................................................................. 53 Figure 4. 1: Characterization of Cu(DDC)2 prepared in a liposomal formulation (DSPC/Chol (55:45)) wherein the Cu(DDC)2 was synthesized inside the copper containing liposomes. 59 Figure 4. 2: Cu(DDC)2 acts primarily as a proteosome inhibitor and induces cell death in MV-4-11 cells. ...................................................................................................................................................... 62 Figure 4. 3: Cu(DDC)2 (prepared in DSPC/Chol (55/45) liposomes) elimination after intravenous injection in CD-1 mice.. ........................................................................................................... 63 Figure 4. 4: Cu(DDC)2 efficacy studies. ...................................................................................................... 65 Figure 4. 5: Examining the role of factors within the DSPC/Chol liposomes that may affect Cu(DDC)2 levels in the plasma compartment 30 min after administration. ............................... 68 Figure 4. 6: Examining the role of lipid composition on Cu(DDC)2 levels in the plasma compartment 30 min after administration. ............................................................................................. 69 Figure 4. 7: Characterizing Cu(DDC)2 prepared inside DSPC/DSPE-PEG2000 (95:5) liposomes. ............................................................................................................................................................. 71 xiii  Figure 4. 8: Cu(DDC)2 anti-tumour activity determined in mice bearing established sc MV-4-11 tumours. ...................................................................................................................................................... 72 Figure 5. 1: The cytotoxicity of CQ and Cu(CQ)2 against 5 different cancer cell lines. ............ 79 Figure 5. 2: Synthesis of Cu(CQ)2 in liposomes prepared with encapsulated 300 mM CuSO4 ................................................................................................................................................................................... 81 Figure 5. 3: Cu(CQ)2 and copper liposome plasma elimination following intravenous injection in CD-1 mice. ..................................................................................................................................... 84 Figure 5.4: Efficacy of Cu(CQ)2 in animals bearing subcutaneous A2780-CP and U251 tumour xenographs. .......................................................................................................................................... 85 Figure 5. 5: Cu(CQ)2 mediated increase in copper delivery to cells and its in vitro activity when combined with disulfiram (DSF). .................................................................................................... 87 Figure 5.6: Efficacy of DSF in combination with Cu(CQ)2 and copper liposomes (liposomes prepared in 300mM CuSO4) determined in NRG mice with sc A2780-CP tumors.. ................. 88 Figure 6.1: Ligands and their respective copper complex structures. .......................................... 94 Figure 6. 2: Structures of commonly used platinum drugs. (A) CDDP and (B) CBDCA. ......... 94 Figure 6. 3: Cytotoxicity profiles of Pt sensitive A2780-S () and resistant A2780-CP (■) ovarian cancer cells (or) following 72 hr treatment with (A) CDDP, (B) CBDCA and (C) Cu(DDC)2. .............................................................................................................................................................. 95 Figure 6. 4: Ligand/drug and copper complex cytotoxicity. ............................................................. 98 Figure 6. 5: Screen of copper-based therapeutics in cancer cells with different Pt sensitivities. .......................................................................................................................................................... 99 Figure 6. 6: In vivo activity of liposomal Cu(DDC)2 in animals bearing Pt resistant A2780-CP tumours. ............................................................................................................................................................... 100 Figure 7. 1: Schematic representation of MetaplexTM. The lipid composition of interest is dissolved into chloroform and this is then dried using a rotary evaporator. ........................... 111 Figure 7. 2: The cytotoxicity of DDC (●), Cu(DDC)2  (■) and Zn(DDC)2() in A2780-S (A) and A2780-CP (B) cells. .......................................................................................................................................... 113 Figure 7. 3: The cytotoxicity of CQ (●), Cu(CQ)2  (■) and Zn(CQ)2() in A2780-S (A) and A2780-CP (B) cells. .......................................................................................................................................... 114 Figure 7. 4: Structures of the copper based proteosome inhibitor NCI-109268 ..................... 115 xiv  List of Abbreviations: 3H-CHE    3H-cholesteryl hexadecyl ether AAS  Atomic absorption spectroscopy AD  Alzheimers disease ALDH1   Acetaldehyde dehydrogenase 1 ATSM  Diacetyl-2,3-bis(N4-methyl-3-thiosemicarbazone) AUC  Area under the curve BD  Biodistribution Bpy  2,2 -bipyridine  BTIC  Brain tumour initiating cell BTZ  Bortezomib CBDCA  Cyclobutane dicarboxylic acid (Carboplatin) CBT  Copper-based therapeutic CC  Copper chaperone CDC  Copper drug complex CDDP  cis-diamminedichloroplatinum (II) (Cisplatin) CED  Convection enhanced delivery CEM  Cryo-electron microscopy Chol  Cholesterol CLC  Copper ligand complex COPD  Chronic obstructive pulmonary disease CQ  Clioquinol CTR1  Copper transporter 1 CTR2  Copper transporter 2 Cu  Copper CuSO4  Copper sulfate DC-Chol  3ß-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol DDC  Diethyldithiocarbamate DMSO  Dimethyl sulfoxide DMT  Divalent metal transporter DOTA  1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid DOX  Doxorubicin DSF  Disulfiram DSPC 1,2-distearoyl-sn-glycero-3-phosphocholine DSPE-PEG2000   1,2-distearoyl-sn-glycero-3-phosphoethanolamine-n-[carboxy(polyethylene glycol)-2000] DSPG 1,2-distearoyl-sn-glycero-3-phospho-(1'-rac-glycerol) EDTA    Ethylenediaminetetraacetic acid xv  GTSM    Glyoxal-bis(N4-methylthiosemicarbazone) H4ML    2-acetylpyridine-4-methylthiosemicarbazone HBEpC   Human bronchial epithelial cells  HD    Huntington’s disease HIF-1    Hypoxia inducible factor - 1  HIV    Human immunodeficiency virus HPLC    High performance liquid chromatography HQ    Hydroxyquinoline HSA    Human serum albumin I.V.    Intravenous Mg    Magnesium Mn     Manganese MST    Median survival time MTD    Maximum tolerated dose NSAID    Non-steroidal anti-inflammatory drug PBS    Phosphate buffered saline Phen    1,10-Phenanthroline PK    Pharmacokinetic Plum    Plumbagin Pt    Platinum Py    Pyrrolidine dithiocarbamate Pyr    Pyrithione QELS    Quasi-electric light scattering ROS    Reactive oxygen species Sala    N-(2-hydroxybenzyl)-D,L-alanine SEM    Standard error of the mean Ser    L-serine SH    Sucrose Hepes SHE    Sucrose Hepes EDTA SM    Sphingomyelin SOD    Superoxide dismutase SD    Standard deviation Tdp    2-[(2-(2-hydroxyethylamino)- ethylimino)methyl]phenol TEA    Triethanolamine TETA    1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid) TMZ    Temozolomide VEGF    Vascular endothelial growth factor Zn    Zinc   xvi  Acknowledgements: I would like to thank my supervisor Dr. Marcel Bally for his patience and support throughout this and other projects. While we are both “negative” thinkers, thank you for being slightly more positive and allowing me the independence to chase after the science that most interested me (despite my constant state of over-caffeination).  I would like to express my gratitude to my co-supervisor Dr. Helen Burt who took me on as a student and allowed me to enter the Faculty of Pharmaceutical Science. I would also like to thank Dr. Burt and the members of my committee, Dr. Murray Webb, Dr. Mary Ensom and Dr. Stephen Yipp, for their intellectual support and mentorship throughout my PhD.  I would like to give a special thank you to Dr. Wayne Riggs for his mentorship in pharmacokinetics and his continued support (even while retired). I have appreciated your kind words in times of stress.  I have been very fortunate to be a member of the Bally Lab and to collaborate with the lab members during my time at BC Cancer Research Center. I would like to thank my lab manager, Dr. Brent Sutherland, for his friendship and dedication to ensuring the safety and maintenance of the lab. My first day in the lab I met Ms. Malathi Anantha; you are an outstanding teacher and analytical scientist. Whether I have needed advice or a sounding board for ideas, you always take the time to be available. Dr. Gemma Ryan, although you joined the lab near the end of my time here, I appreciate our long discussions at your desk. Dr. Ada Leung, you have been the biological “voice” that literally sat behind me for the last four years. Our collaborations have led to useful technology and lots of laughs, thank you. I would like to thank my students (Ian Backstrom, Cody Lo and Armaan Malhotra) for their technical and intellectual support, as well as the valuable experience as a mentor.  I would like to acknowledge the BC Cancer Research Agency and the Faculty of Pharmaceutical Science for their support. The people always make the place better, and so I would like to thank my student cohort: Jonathon Campbell, Jacob Gordon, Ankur Midha and Sahan Ranamukhaarachchi. xvii  I would like to thank my parents (Nawal & Abdullah Wehbe) for calling me to check that I’m eating—it never fails to make me laugh. I cannot imagine completing this process without the love and support of my siblings, Nariman, Lamis, Hanadie, Khaled and Heba. Finally, to my oldest and dearest friend Mariajosé Aguilera; you are the reason they make unlimited phone plans. Thank you for always being a phone call away.                      xviii         To my family,  I only did this  to be the favourite.            1  1. Introduction 1.1 Thesis Overview Liposomes, small vesicular structures comprising a lipid bilayer surrounding an aqueous core, have emerged as a successful drug delivery platform, whereby associated drugs or drug candidates are improved in terms of their stability, biodistribution and ultimately therapeutic potential[1, 2]. Liposomes have been used to formulate a variety of agents including those used to treat cancer[3-5], microbial infections[6, 7], inflammatory conditions[8, 9], etc [10, 11]. The methods used to prepare and characterized liposomal drug formulations have proven to be robust, and many formulations have successfully transitioned from the research lab to the clinic[2]. To date, much of the focus on development of liposomal drugs has been directed to reformulation of approved anti-cancer agents. Further, part of the success of these formulations was due to the development of robust ways to prepare liposomes at a scale and in a manner suitable for use in humans. In this context, a method, whereby dried lipids are hydrated in a solution of choice and then extruded under moderate pressure through filters which have defined pore sizes has been developed [12]. The liposomes produced in this way exhibit a mean diameter that is close to the size of the pores, i.e. if the pore sizes are 0.1 µM, then the resultant liposomes will be about 0.1 µM. Further, a method, referred to as remote loading, facilitated the encapsulation of selected drugs into preformed liposomes[13-15]. As exemplified by drugs such as doxorubicin, vincristine, and irinotecan, they can be added to the outside of liposomes exhibiting a transmembrane pH gradient (inside acid)[14, 16, 17]. These drugs have protonizable amine groups which at pHs close to the pKa are maintained in a neutral form. The uncharged form of the drug can cross the liposomal membrane but once in the acidic interior the drug becomes protonated, charged and membrane impermeable. Greater than 98% of the drug added to the liposomes can become trapped inside using this remote loading methods[13]. A number of different methods have been used to generate transmembrane pH gradients including encapsulation of a low pH buffer (e.g. citrate buffers at pH 4.0)[14, 18]; ammonium sulfate solutions (the ammonium ion can leave the liposomes leaving behind a proton and thus generating a pH gradient)[19, 20]; 2  and the use encapsulated divalent metals (Mn2+, Mg2+, Cu2+, etc) in combination with a divalent metal ionophore (e.g. A23187 aka- Calcimycin)[21, 22] which exchanges a metal ion for 2 protons and thus generates a pH gradient. Previous studies, however, have shown that certain drugs with functional groups capable of coordinating with divalent metals can be used to achieve drug encapsulation through the formation of a metal complex[23, 24]. These studies have been conducted using drug agents with both protonizible amine and metal binding functional groups. Thus, there exists a potential for using metal complexation reactions to associate compounds of interest inside liposomes which offers an opportunity to formulate drug candidates that were previously considered unsuitable for liposome formulations through remote loading.  The research summarized in this thesis explored the potential for using metal complexation reactions to prepare formulations of novel agents with anti-cancer activities. More specifically, the thesis research focuses on the development of copper-based therapeutics (CBTs), which define any compounds with functional groups that can bind Cu(II) and exhibit some therapeutic activity in particular for the treatment of cancer. There already exists a plethora of agents developed through the use of inorganic chemistry approaches to create compounds with anti-cancer activity. Many of these are consider class IV compounds in the biopharmaceutical classification system; exhibiting low permeability and low solubility. Class IV compounds are perhaps the most challenging to develop for therapeutic applications and would typically not be consider for liposomal formulations. The research described in this thesis suggests that CBT specifically and perhaps metal-based therapeutics in general can be formulated in liposomes. It was hypothesized that CBTs can be synthesized inside liposomes and that the resulting formulations would be suitable for further pharmaceutical development as a novel class of anti-cancer agents. For the first time, it is shown that copper complexes can be synthesized inside liposomes, where a reaction occurs between encapsulated copper and a compound with known metal binding groups that have been added to the outside of the liposome. The use of CBTs targeting cancer required the development of strategies to formulate these sparingly soluble compounds in a manner suitable for parenteral administration. This 3  approach has solved the solubility challenges faced when considering the development of CBTs. The thesis research was built around four experimental aims: Aim 1: To determine if CBTs can be synthesized inside liposomes which contain copper and to determine if the method defined a platform approach for developing CBT formulations suitable for intravenous administration.  Aim 2: To evaluate the therapeutic potential of a novel copper diethyldithiocarbamate formulation discovered as a result of efforts designed to repurpose disulfiram, for use in the treatment of cancer.     Aim 3: To evaluate the anti-cancer potential of clioquinol, a topical antifungal, when prepared as a copper drug complex. Aim 4: To determine whether CBTs are effective in platinum resistant cancers. 1.2 Copper-Based Therapeutics as an Alternative to Platinum In the 1970s, platinum-based complexes emerged as the focus of metal-based synthesis efforts owing to the widely successful drug cisplatin (CDDP). This effort spawned the development of carboplatin (CBDCA), oxaliplatin and other platinum agents that exhibit potent anti-cancer activity when used as a single agent and in combination [25]. Efforts focused on developing copper-based therapeutics (CBTs) have been directed, in part, towards replacing platinum therapeutics, which are incredibly successful in cancer therapy but do suffer from major drawbacks [26, 27]. The use of platinum, which is not native to the human body, results in many serious adverse effects such as nephrotoxicity [28], neurotoxicity[29] as well as ototoxicity and myelosuppression [30]. Furthermore, the development of platinum resistance has created a major clinical problem [31]. Platinum-based therapeutics such as CDDP[32], CBDCA[33, 34] and oxaliplatin[35] are best known for their DNA damaging abilities[30, 36]. It is important to note that oxaliplatin is the only platinum agent that is able to overcome platinum resistance through the formation of DNA adducts that are distinct from those formed with cisplatin and carboplatin [37, 38]. In contrast, copper-complexes developed as anti-cancer agents have been noted to induce cancer cell death through a variety of mechanisms including proteosome inhibition,[39-42] 4  generation of reactive oxygen species (ROS) [43-45] and DNA damage [46, 47], amongst others.  Therefore, there has been a great deal of interest in using copper (Cu) in place of platinum as it would be better tolerated due to the existence of natural biological pathways to regulate Cu levels and to detoxify the metal where necessary. Recently, Cu has re-emerged as the focus of inorganic synthesis programs wherein Cu-complexes have been prepared for different indications including cancer[48-50], viral infections [51], inflammatory diseases[52, 53], microbial infections[54-57] and cosmetic applications[58, 59]. Although there has been some success with Cu(II)-indomethacin complexes (currently used in veterinary medicine as an anti-inflammatory drug) [53, 60, 61], no CBT has been approved for medical use in humans. Despite the increase in the number of articles describing Cu-complexes as therapeutics, there has been limited transition from in vitro to pre-clinical in vivo studies[62].  Although not a single copper complex has gained regulatory approval for human use it should be noted that the casiopeínas are a class of copper complex compounds (specifically, casiopeina III-ia (aqua, 4,4-dimethyl-2,2′-bipyridine, acetylacetonato copper(II) nitrate)) that have entered Phase I clinical trials in Mexico[63]. In general, this class of compounds exhibits very low aqueous solubility. While it is relatively easy to perform in vitro experiments using solvent systems such as dimethyl sulfoxide (DMSO; a class 3 solvent not suitable for parenteral injection in concentrations >0.05%/day)[64] the poor aqueous solubility creates a pharmaceutical barrier for further pre-clinical and clinical evaluations. As noted in section 1.4, Cu(II) interacts with N, O and/or S binding moieties and this interaction results in the removal of two hydrogen bond-acceptor groups from the system, leading to a net-increase in hydrophobicity. Thus, many CBT candidates are insoluble in aqueous solutions at therapeutically relevant concentrations. To address the solubility issue associated with CBTs, classical medicinal chemistry approaches have been used; approaches that involve chemical synthesis of more water soluble analogues of the CBTs of interest [65]. The other approach involves a discovery that is the topic of this thesis; whereby the synthesis of CBT of interest can be done within liposomes (Figure 1.1) [66]. Essentially the liposomes serve as nanoscale reaction vessels 5  supporting the synthesis of the Cu-complexes in a form that keeps the complex in a solution that is suitable for administration (referred to as MetaplexTM complex synthesis).       Figure 1. 1 Overcoming the pharmaceutical challenge of developing copper-based therapeutics. This figure illustrates the major solubility issue associated with most copper/metal complexes. The problem of metal complexes being insoluble in aqueous solutions under physiological conditions can be addressed via two main methods: modification of chemical structure or synthesis of the metal complex within lipid-based nanoparticles. High-throughput medicinal chemistry can be performed in two ways: 1) chemical synthesis of analogue libraries of the complex of interest followed by high-throughput screening for biological activity, 2) or computer-aided drug design of analogues of the complex of interest using techniques such as molecular 6  docking, followed by high-throughput screening for biological activity. This result of these processes is a list of candidate drugs that may or may not be soluble in aqueous solutions. Insoluble drug candidates would require further chemical modifications until a more soluble and at least equally active candidate can be identified. In contrast, the use of the MetaplexTM technology requires no chemical modification of the metal complex. It involves the preparation of copper/metal-containing liposomes by adding the appropriate drug/ligand to liposomes which contain entrapped copper/metals. Depending on liposomal lipid composition, temperature and copper to drug/ligand ratio, this process will result in the generation of the copper drug complex/copper ligand complex inside the liposomes; forming a solution that is suitable for parenteral administration. MetaplexTM can also be used to prepare formulations of poorly water soluble drug candidates identified from medicinal chemistry-based screen thus substantially reducing the amount of time required to generate a lead candidate for in vivo testing. 1.3 Copper in Biology 1.3.1 The Importance of Copper Homeostasis  The dietary requirement of copper is only 0.7-0.9 mg/day and yet it is vital for the growth and development of all aerobic organisms [67, 68]. Cu is a key player in a diverse number of biological pathways owing in part to its presence as either a reduced (+1) or oxidized (+2) state[67]. Deregulation of Cu homeostasis has led to a better understanding of the significance of Cu in human physiology[69]. For instance, X-linked mutations in ATP7A, which codes for a Cu-transporting ATPase, lead to the production of a truncated, dysfunctional enzyme, resulting in poor distribution of copper and is linked to a fatal condition known as Menkes Syndrome. Since ATP7A is responsible for the delivery of copper as co-factors and regulators of certain enzymes as well as the efflux of Cu from cells, the loss of ATP7A function leads to accumulation of Cu in certain tissues such as the small intestine and systemic Cu deficiency, leading to neurodegeneration and growth abnormalities [70-72]. If diagnosed early, neurodegeneration from Menkes Syndrome can be prevented and the disease can be managed through parenteral administration of Cu-histidine. Nonetheless, the treatment outcome is largely dependent on the residual activity of the mutant ATP7A enzyme and the time of initial treatment [73, 74]. In contrast, Wilson’s disease is a disorder caused by mutations in ATP7B, which is primarily expressed in the liver and brain. Dysfunctional ATP7B results in impaired excretion of excess Cu from the body and increased accumulation leading to tissue damage particularly in the liver, brain, kidneys, and cornea[75]. Wilson’s disease is usually treated with chelation therapy with agents such as penicillamine and trientine[70]. In recent years, treatment with zinc has been introduced as zinc can induce metallothionein, a protein that binds to copper in the 7  gut and facilitates copper excretion[69, 76, 77]. The life-threatening impact of Cu deficiency and overload found with Menkes and Wilson’s disease, respectively, exemplifies the importance of Cu homeostasis to human health.  1.3.2 Cellular Components for Copper Regulation   It is known that Cu is a co-factor for metalloenzymes including superoxide dismutase, cytochrome C oxidase, tyrosinase, peptidylglycine alpha-amidating mono-oxygenase, and lysyl oxidase[78, 79]. The diverse role of Cu in biology is in part a result of its existence as either reduced Cu(I)  or oxidized Cu(II)  with each oxidation state having a preferred affinity for different coordinating groups[67]. Cu(I)  is known to bind thiol and thioester groups whereas Cu(II)  prefers coordinating with oxygen and nitrogen groups[80]. Therefore, the different oxidation states can interact with a variety of proteins that drive a multitude of biochemical reactions. Given its many biological roles, Cu levels are tightly regulated at all levels including cellular uptake, distribution, detoxification, and elimination [79, 81]. These mechanisms are vital to maintaining Cu homeostasis and avoiding Cu associated toxicities. Cu transporters are present on cell surfaces to mediate the influx and efflux of Cu [79, 82, 83]. Further, copper chaperones (CC) are present to deliver the imported Cu to specific target proteins within the cell [84]. Figure 1.2 summarizes the trafficking of Cu and the cellular components involved in regulating Cu levels within a cell. 8   Figure 1. 2: Cellular regulation of copper levels. CTR1 is the copper transporter that is primarily responsible for delivering copper into the cell. Once copper enters the cell, it is bound to chaperones for further distribution. For instance, ATOX 1 is responsible for delivering copper to the copper efflux pumps ATP7A/B, which are localized to the trans-golgi apparatus where copper is transferred to metalloenzymes that require copper as a co-factor. If copper levels are high, ATP7A/B will redistribute to the cell surface via the secretory pathway where copper is pumped out of the cell. Intracellular copper can also bind to COX17, a chaperone which is important for transporting copper to the mitochondria. This is vital to organisms that require aerobic respiration to survive as the copper here is crucial to the proper functioning of the cytochrome c oxidase and the electron transport chain. Finally, CTR2, another copper transporter, resides in the late endosomes and lysosomes. This transporter aids in the regulation of copper levels inside the cytosol.      9  Copper transporter 1 (CTR1) contains 3 transmembrane domains and is believed to be the primary protein responsible for the import of dietary Cu [85, 86]. When Cu levels are elevated, CTR1 proteins at the plasma membrane are endocytosed and degraded to prevent Cu overload. A second isoform of the copper transporter known as CTR2 has been identified. The function of CTR2 remains to be fully elucidated. Nonetheless, it has been shown that while CTR1 is primarily expressed on cell surfaces, CTR2 is localized to endosomes and lysosomes, and partially to the plasma membrane[83, 87, 88]. It is postulated CTR2 is important for regulating Cu levels in the cytosol and hence plays a role in intracellular Cu homeostasis [89]. Additionally, some have suggested that the divalent metal transporter 1 (DMT1), which is typically involved the iron transport, may also be involved in Cu influx [90, 91]. While Cu uptake through CTR1 and 2 is an energy independent process, the efflux of cellular Cu requires the hydrolysis of ATP [83, 92, 93]. Two well-characterized Cu efflux proteins are known as ATP7A and ATP7B [93, 94]. ATP7B is highly expressed in the liver and expressed at lower levels in other organs; whereas, ATP7A is expressed in most tissues but its function is most important in the small intestine [94]. The ATP7A gene is encoded on the X-chromosome whereas ATP7B is found on chromosome 13. As indicated above, mutations in these genes are associated with Menkes and Wilson’s disease, respectively [95-97]. Normally, ATP7A and ATP7B reside at the trans Golgi network (TGN) where Cu is incorporated into Cu-dependent enzymes [98]. ATP7A, particularly in the small intestine, is important for trafficking Cu out of the Golgi and into portal circulation, allowing systemic distribution of Cu. In the liver, ATP7B is responsible for incorporating Cu into ceruloplasmin, an enzyme that is important for regulating iron levels [99, 100]. When the intracellular Cu level reaches a threshold, the efflux pumps are redistributed to plasma membrane where their primary role is copper excretion from the cell.    CCs are responsible for trafficking Cu intracellularly and also for ensuring that the cell is not exposed to highly reactive Cu ions in transit which could result in the generation of ROS [101]. ATOX1 was the first chaperone discovered as a cytosolic protein that binds and delivers Cu(I) ions to ATP7A/B [93, 102, 103]. Another CC known as CCS (copper chaperone for superoxide dismutase) forms a heterodimer with Cu/Zn superoxide dismutase (SOD) and is essential for activating mammalian SOD [93]. Similarly, COX17 is 10  the CC that delivers Cu to the mitochondria; this chaperone is required for the activation of cytochrome c oxidase (COX), which is essential for aerobic respiration [104]. Other chaperones such as COX1, 2, 11 and 17, as well as SCO1 and SCO2, are also involved in the assembly of COX [104, 105]. CCs such as ATOX1 and COX17 are essential as knockout mice with loss of Atox1 or Cox17 were embryonically and perinatally lethal [93, 98, 106]. Cu is capable of coordinating with N, O and S groups in general and these interactions are involved in many biochemical reactions [67]. Additionally, Cu can displace other metals such as Zinc (Zn) in metalloproteins which could alter the protein-metal structure rendering the metalloenzyme inactive [107].Thus, it is reasonable to expect that in the presence of excess Cu, this affinity could result in the formation of undesirable coordinated systems. The transition between Cu(I) and Cu(II)  can result in the generation of hydroxyl radicals which can damage proteins, nucleic acids and lipids [67, 108, 109].  1.4 Copper Chemistry  Research in Cu chemistry and drug synthesis is dominated by compounds using the most common oxidation states [62]. Cu(I) ions have a d10 configuration and are able to form complexes with ligands that have “soft” donor characteristics such as those containing thioether S, P, and aromatic nitrogen  groups [48]. These complexes tend to exhibit a linear, trigonal or tetrahedral geometry [110, 111]. Cu(II) ions have a d9 configuration and complexes with coordination geometry, favour 4-6 coordinate geometries (square-planar, trigonal bipyramidal, and octahedral) and allow for a large variety of ligands with different denticity and sizes [48, 111]. The ligands for Cu(II) are favoured towards N,O,S donors [112]. The stability of divalent metal complexes can be predicted to an extent using the Irving-Williams series[113]; Thus, Cu(II) is a good choice of metal as the complexes would be more stable in comparison to other potential metal species.  There are several excellent reviews, including those written by Santini et al.[62], Marzano et al.[48] and Iakovidis et al.[112] that discuss different ligands and functional groups that form Cu- complexes along with their various in vitro activities. These reviews are extensive and truly highlight the vast therapeutic opportunity for Cu-complexes in respect to both ligand heterogeneity and therapeutic application diversity. A familiarity 11  with copper chelating groups is required to fully appreciate the range of binding partners that are possible (Figure 1.3). These can be adapted and altered for different disease indications or targets.   Figure 1. 3: Venn diagram showing the possible copper chelating functional groups composed of nitrogen (N-donor), oxygen (O-donor) and sulfur (S-donor) atoms. Copper binding groups can contain a single donor type or a mixture of N, O and S. The illustration was constructed based on a review article by Santini et al.[62] 1.5 Therapeutically Active Copper Complexes as Anti-cancer, Anti-Inflammatory, and Antimicrobial Agents The therapeutic window for anti-cancer agents has always been dependent on the selectivity of these agents for the cancer cells over normal, healthy cells. While Cu chelation may reverse the metal imbalance and remove the tumour’s growth advantages provided by elevated Cu levels, another approach is to administer agents that upon Cu binding, would elicit potent cytotoxic effects. As an example, disulfiram (DSF), an FDA-approved agent for 12  the treatment of alcoholism, has garnered much attention in recent years as a Cu-activated anti-cancer agent [114]. While DSF alone has little effect against cancer cells, its use in the presence of Cu in vitro has been shown to have IC50 values in the nano-molar range against a wide range of cancer cell lines [66, 115, 116]. In vivo, the administration of DSF into animals with subcutaneous breast cancer xenograft models known to have abnormally high levels of Cu resulted in decreased tumour growth [117, 118]. Some have suggested that the formation of a Cu-DSF complex as the means by which cytotoxicity is achieved. However, a thorough examination of DSF metabolism has led to the conclusion that the Cu-DSF complex does not exist [115, 119]. Lewis et al. have deduced that even in  aqueous solution, DSF does not form a copper complex but rather it degrades into diethyldithiocarbamate (DDC), which reacts with Cu to form copper diethyldithiocarbamate (Cu(DDC)2) [120]. Dithiocarbamates are a known class of Cu chelators and they have been shown to be very active in vitro against a variety of cancer cell lines [65]. Interestingly, the mechanism of action of Cu(DDC)2 was found to be proteasome inhibition, but this action was through a mechanism that was distinct from bortezomib (BTZ), an FDA-approved proteasome inhibitor for cancer treatment [42, 121, 122]. It should be noted that clinical trials have attempted to use DSF for the treatment of liver tumours with adjuvant Cu given in the form of copper gluconate [123]. Aside from generating novel chemotherapeutics by metal coordination of existing drugs, researchers have also begun to explore the utility of metal coordination in the development of new drugs. For example, with the emergence of drug-resistant Plasmodium falciparum, there is an urgent need for new anti-malarials. Sekhon and Bimal have described various metal complexes that have demonstrated anti-malarial activity [124]. One example is the Cu(II)-complex with pyridine-2-carboxamidrazone. This copper complex has exhibited enhanced anti-malarial activity in vitro compared to the parent compound [125]. Inflammatory diseases, such as rheumatoid arthritis, are another area that could benefit from treatment with Cu-complexes. While multiple non-steroidal anti-inflammatory drugs (NSAIDs) are available in the market, they are associated with renal, gastrointestinal, and cardiovascular toxicities [53]. Over the past two decades, there is increasing evidence that Cu-complexes of NSAIDs are more effective and less toxic 13  compared to Cu or the parental NSAID compound used alone [53]. In many cases, chronic inflammation such as asthma, chronic obstructive pulmonary disease (COPD) and rheumatoid arthritis are associated with reduced superoxide dismutase (SOD) activity, leading to chronic oxidative stress and inflammation [53, 88, 126]. Although the exact anti-inflammatory mechanisms of Cu-NSAIDs remain to be elucidated, these Cu-NSAIDs have exhibited marked SOD-like anti-inflammatory activity which differentiates them from their parent compounds [53, 127, 128]. This SOD-mimetic activity of Cu-NSAIDs may also be useful in the treatment of cardiovascular diseases and skin disorders [53, 127, 129].  With the emergence of multi-drug resistant pathogens and a lack of novel effective antibiotics, metal complexes have also become of interest as antibacterial agents. As an example, Beeton et al. have synthesized nine Cu(II)-complexes of the aromatic ligand 1,10-phenanthroline and tested their antimicrobial and antibiofilm activity [130]. These Cu-complexes have demonstrated better antimicrobial activity than the free ligand against both Gram-Positive and Gram-Negative bacterial strains, as well as enhanced antibiofilm activity compared to the standard drug vancomycin against a clinical strain of methicillin-resistant Staphylococcus aureus (MRSA). Facing a similar multi-drug resistant problem in the clinic, the need for novel tuberculosis drug has led to the synthesis of Cu-complexes of pyruvate-isoniazid conjugate analogues and isoniazid-derived hydrazones which have demonstrated greater anti-tubercular activity than isoniazid in vitro[130, 131].  Finally, while Menkes Syndrome and Wilson’s disease are known to be associated with Cu deficiency and overload, respectively, there is now strong evidence that disruption of biometal homeostasis plays an important role in certain neurodegenerative diseases including Alzheimer’s (AD) and Huntington’s Disease (HD) [132]. Formation of plaques comprising the misfolded amyloid-β (Aβ) protein in the brain is characteristic of AD. Several studies have demonstrated that AD patients have abnormally high levels of Cu in the amyloid plaques and these metal-bound Aβ proteins are more prone to self-aggregation [133], which correlates with increased oxidative stress and more severe disease progression [134]. Similarly, Cu accumulation has also been detected in the brains of HD patients and the metal has been found to promote the formulation of mutant Huntington protein aggregates, potentiating disease progression [135]. As such, CQ and later, PBT2, a second generation 8-HQ analogue, have been used to treat AD and HD [136-138]. PBT2 14  became the drug of choice as it is easier to synthesize, more soluble, and exhibits greater permeability across the blood-brain barrier [138]. It is important to note that while many consider this copper chelation therapy, the role that these agents play is to correct the metal imbalance in the brain. Unlike Wilson’s Disease, the Cu accumulation occurs extracellularly rather than intracellularly in AD and HD. Trafficking of Cu with PBT2 reduces extracellular Cu levels and increases intracellular bioavailability, which leads to delayed protein aggregation and allows for activation of neuroprotective pathways [138]. Unfortunately, PBT2 has failed to meet the efficacy goals for both AD and HD in phase 2 clinical trials [139]. It has been suggested that successful treatment of AD and HD would require targeting multiple hallmarks of these diseases [140]. 1.5.1 Copper Chelation Therapy Copper chelation is a standard approach for treating Wilson’s disease, wherein drugs/chelators are administered to bind Cu, resulting in the formation of complexes that can be eliminated through the kidneys [141-143]. In the last two decades, there is increasing evidence that many cancer patients have elevated levels of Cu within sites of tumour growth and plasma (when compared to plasma levels of Cu in healthy subjects) [144, 145]. Gupte and Mumper have written a comprehensive review demonstrating the link between Cu imbalance and cancer [144]. It is also known that one of the hallmarks of cancer is induction of angiogenesis [146-148]. Cu is required for the activation of hypoxia-inducible factor 1 (HIF-1), a major transcription factor regulating the expression of vascular endothelial growth factor (VEGF), which is primarily responsible for angiogenesis [147, 149]. Additionally, angiogenin is a potent inducer of blood vessel formation which binds to the endothelial cell receptors. It has been shown that the presence of Cu can lead to an increase in angiogenin binding to endothelial cells by more than 4-fold [150]. Thus, copper chelators can be used as part of a “de-coppering” approach to treat cancer similar to what has been done for Wilsons’ disease [151]. Copper chelation has been shown to selectively kill colon cancer cells via ROS generation and a similar strategy has been proposed to be effective for targeting cancers, particularly melanoma, that harbour oncogenic BRAF mutations [44, 152]. Multiple clinical trials are ongoing investigating the 15  effectiveness of combining copper chelators such as tetrathiomolybdate and penicillamine with standard chemotherapy or radiotherapy; however, given the heterogeneous nature of cancer, it is unlikely that Cu depletion alone will be sufficient to cure the disease [153]. 1.5.2 Copper Radiopharmaceuticals 64Cu has garnered significant attention in the context of radiopharmaceuticals applications, owing in part to its utility in molecular imaging and targeted radiation therapy [154, 155]. Other Cu radioisotopes, including 60Cu, 61Cu, 62Cu and 67Cu have been reviewed by Asabella et al. Of these, 67Cu is the only other isotope that can be used for radiation therapy [155]. Production of 64Cu is through a 64Ni(p,n)64Cu reaction and is obtained as 64CuCl2 [156]. The radioisotope possesses a half-life of 12.7 h and decays by positron emission (17.8%) and beta decay (38.4%).[157] These decay properties render it a useful isotope for PET imaging and/or radiotherapy.[154] Due to the lack of kinetic stability of Cu(I) complexes and the rarity of Cu(III), the radiopharmaceutical field is dominated by Cu(II) complexes [154]. Classes of Cu chelators such as sarcophogine[158], cross-bridged tetraamine[159-161] and thiosemicarbazone[162, 163] ligands have been developed. As an example, the ligand ATSM (diacetyl-2,3-bis(N4-methyl-3-thiosemicarbazone)) forms a Cu(II) complex useful as a marker of chronic hypoxia. Lewis et al. performed a side by side comparison of 64Cu/60Cu-ATSM and they showed equivalent patterns and magnitude of tumor uptake. These authors, however, concluded that 64Cu-ATSM was better due to lower noise in images [164]. One major limitation of Cu-ATSM is that the kinetics of uptake appears to be cell dependant and thus this complex may not be relevant for all cancers [165].   Conjugation of 64Cu to biologic agents has been of great interest. In this context, the most widely used chelators are based on tetraazamacrocylic[154, 166] ligands (typically DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) and TETA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid)). This chelator class can be successfully conjugated to antibodies, protein and peptides but transchelation was noted to be a problem in vivo [167]. The conjugation of 64Cu through DOTA has been performed and evaluated for cetuximab (EGFR antibody) [168, 169], trastuzumab (HER2 antibody) [170]  16  and Abegrin (human integrin αvβ3 antibody) [171]. The small cyclic peptide RGD which targets human integrin αvβ3 was conjugated to 64Cu through DOTA and a novel bifunctional chelate 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetic acid [172]. Octreotide, a peptide targeting the somatostatin receptor, was conjugated to 64Cu through TETA and was used to detect neuroendocrine tumours [173]. Additionally, a dextranated and DTPA-modified nanoparticle was labeled with 64Cu and used for imaging by Nahrendorf et al.[174]. To date, nothing has transitioned into humans due to challenges linked to poor in vivo stability and poor biodistribution attributes [166].  1.6 Classification of Copper Complexes The information summarized above and in other more comprehensive review articles highlight the therapeutic potential of CBTs. When considering these, it is useful to define the complexes into two categories: copper-ligand complexes (CLCs) and copper-drug complexes (CDCs). In general, CDCs typically may not require Cu for activity whereas CLCs are significantly more active as the Cu-complexes. Table 1.1 and Table 1.2 provide examples for each of these two classes of Cu-complexes which are further described below.  1.6.1 Copper-Ligand Complexes (CLCs) CLCs are CBTs comprising ligands that have little or no therapeutic activity at relevant concentrations (e.g. IC50 > 10 µM). Examples of CLCs include Cu-(N-salicylidene-selenomethionine) [175], Cu-(NCI-109268) [39], and Cu(1-pyrenethiosemicarbazone) [176]. Cu-(N-salicylidene-selenomethionine has been shown to act as an antimicrobial agent while the latter two CLCs have demonstrated anti-cancer activity. Similar to the platinum-based chemotherapies, the ligands attached can alter the activity of the drug but the ligand itself does not have any therapeutic activity in the absence of the metal. A number of CLCs, including ligands like ethyl 2-[bis(2-pyridylmethyl)amino] propionate (ETDPA) [177], 2-[(2-(2-hydroxyethylamino)- ethylimino)methyl]phenol (tdp) [178], 1,10-phenanthroline-L-serine (Phen-Ser) [98], and 2-acetylpyridine-4-methylthiosemicarbazone (H4ML) [179], have been identified as DNA binding and cleavage agents, making them suitable anti-cancer drug candidates. Although these CBTs have been 17  characterized extensively and evaluated in vitro, no in vivo study with these compounds has been published.  Table 1. 1: Examples of copper ligand complexes with antimicrobial and anti-cancer activity Drug/Ligand(s): Copper Complex Structure: Indication(s): Ref: (A) N-salicylidene-selenomethionine  Antimicrobial [175] (B) NCI-109268  Anti-cancer [39] (C) PyTSC  Anti-cancer [176] (D) 2-acetylpyridine-4-methylthiosemicarbazone  Anti-cancer [179] (E) GTSM  Anti-cancer / Antimicrobial [180-182] (F) SAla and phen  Antimicrobial [183] (G) SAla and bpy  Antimicrobial [183] (H) Diethyldithiocarbamate  Anti-cancer [184] 18  APPC:N1-(2-acetylpyridine)pyridine-2-carboxamidrazone Bpy: 2,2 -bipyridine GTSM: glyoxal-bis(N4-methylthiosemicarbazone) Phen: 1,10-Phenanthroline PyTSC: 1-pyrenethiosemicarbazone SAla; N-(2-hydroxybenzyl)-D,L-alanine  Table 1. 2: Examples of drug classes which form copper drug complexes (I) APPC  Anti-cancer [185] Chemical Class: Indication(s): Copper Binding Moiety: Example(s): Ref: Quinolones Antimicrobial  ciprofloxacin, norfloxacin,  cinoxacin  [186-189] 8-Hydroxyquinoline and derivatives Antimicrobial/ Anti-cancer  8-hydroxyquinoline, clioquinol  [190-193] Salicylate derivates Anti-inflammatory  salicylic acid,  diflunisal  [186, 194] Propionic acids  Anti-inflammatory  ibuprofen, fenoprofen, naproxen, ketoprofen  [186, 195] Heteroarylacetic acids Anti-inflammatory  indomethacin  [61, 186] Anthranilic Acid derivatives Anti-inflammatory   mefenamic acid, diclofenac,  [186, 195-197] Oxicam derivatives Anti-inflammatory  meloxicam, piroxicam,  [186, 198, 199] 19  *Copper binding moiety labelled in red  To identify novel antibiotics, Haeili et al. performed a high-throughput screen using Cu-complexes against MRSA [180]. The bactericidal activity of the copper bis(thiosemicarbazonato) complexes identified were  copper-dependent. In another study, complexes of N-(2-hydroxybenzyl)-D,L-alanine (SAla) and 1,10-phenanthroline (phen) or 2,2 -bipyridine (bpy) were found to have antibacterial/antifungal activity[183] when screened against nine bacterial and nine fungal pathogens using ampicillin and amphotericin B, respectively, as positive controls. The ligands alone did not show any growth inhibition whereas the CLCs show activity against a variety of bacterial and fungal pathogens. Despite in vitro activity of these compounds, among others [54, 57, 99, 202, 203], there does not appear to be any further study of these compounds in relevant disease models. The pharmacological properties, such as tolerability or pharmacokinetics, of these CLCs have yet to be explored.  1.6.2 Copper-Drug Complexes (CDCs) Cu(II) is able to coordinate with a large variety of functional groups and these groups are present in many drugs already approved by the FDA. Some examples include Cu-complexes of ciprofloxacin [189], isoniazid [204, 205], doxorubicin [205], indomethacin [61, 206] and clioquinol[190, 207]. For these agents, Cu is not required for the drug to exert its activity, however the Cu-drug interactions can be used to alter the solubility of the drug (i.e. making an insoluble drug more soluble by Cu coordination) [208]. The resulting complex defines a new formulation; one that will have differing pharmacokinetic properties and associated changes in therapeutic activity [53]. The Cu-indomethacin complex, for example, has been shown to have an enhanced activity as a topical anti-inflammatory in comparison to indomethacin [209]. This can be attributed, in part, to a Anthracycline Anti-cancer  daunorubicin, doxorubicin, idarubicin  [200, 201] 20  differentiation in the physico-chemical properties of the copper complex [208]. Interestingly, Okuyama et al. hypothesized that Cu(indomethacin)4 is able to activate Cu-dependent opioid-receptor which may help to explain the enhanced anti-inflammatory activity [210]. Cu-complexes have been made with other NSAIDs, including diclofenac [196, 211], fenoprofenate [212], mefenamic acid[197] as well as others [53, 186, 210]. Although many of these Cu-NSAID complexes have demonstrated some success in relevant animal models, none have been approved for human use.  Zinc pyrithione, which is used in dandruff shampoos, is an example of an agent with antimicrobial activity [213]. The ligand pyrithione on its own has no activity. Interestingly, zinc pyrithione and copper pyrithione complexes have been shown to have anti-cancer activity as well [214, 215]. Additionally, copper complexation can alter the mechanism of action of the associated drug. Other examples would include Cu-complexes of the 8-HQ class of drugs which are typically used as anti-fungals or antimicrobials [55]. When these drugs are complexed to Cu, they exert novel anti-cancer activities [216]. Clioquinol (CQ), an analogue of 8-HQ which is traditionally used as an antibiotic, has demonstrated enhanced activity when complexed with Cu as an anti-cancer agent [217]. As suggested above for Cu(DDC)2, the principal mechanism of activity for Cu-CQ and Cu-8-HQ involves proteasome inhibition [216]. These compounds have also been shown to behave as metal ionophores [218]. Previous reports have shown that copper-complexed 8-HQ is at least 10 times more cytotoxic than the drug given alone when tested against HeLa and PC3 cell lines over 48 hr time course [219]. The Cu-8HQ complex has also been tested on bacteria and has shown some success as a more potent antibiotic compared to its parent compound [55, 220].  Schimmer et al. demonstrated that CQ has Cu dependent and independent mechanisms of action when added to cancer cells [191]. Thus, an important consideration when attempting to repurpose drugs such as CDCs is that the inherent mechanism of action of the metal-free drug may not play an important role if the metal complex stays intact in vivo but this activity may become apparent should the complex fall apart.  21  1.7 Approaches to Solve Copper Complex Solubility Challenges: 1.7.1 High-Throughput Screening High-throughput screening (HTS) is ubiquitously used in the pharmaceutical industry as a means to identify candidate compounds for development. This approach has been used to identify candidate compounds selected on the basis of phenotypic screens; where measurable changes in cell appearance (e.g. autophagy)[221] linked to a particular behaviour (e.g. cell survival)[222] or biochemical screens [223]; where inhibition of a purified target protein is the goal. HTS have been used to indentify active anti-cancer and anti-microbial copper complexes. A HTS was performed by Feldman et al. wherein ~320,000 compounds were screen to identify caspase inhibitors [224]. This screen led to the discovery of a new class of allosteric copper-based caspase inhibitors (NSC321205, NSC277584, NSC321206, and NSC310547) with submicromolar IC50 values. A screen was also devised to identify copper-activated ligands  that were efficacious in methicillin-resistant Staphylococcus aureus (MRSA) [180]. The compounds were tested against the bacteria in media containing trace levels of copper or media supplemented with 50 µM copper using optical density as a marker for bacterial cell growth. This screen indentified the bis(thiosemicarbazones) ligands (GTSM, PTSM, ATSM) as active in MRSA in the presence of copper. It should be noted that upon further study of this class of copper complexes as antibacterial agents solubility issues were identified making it difficult to test the activity of these copper complexes in the absence of DMSO [225]. Thus, while the HTS approach allows for the identification of candidate compounds further chemical modifications may be required to create chemical entities with improved solubility while retaining activity. Such traditional medicinal chemistry programs are highlighted by the studies described by Wang et al. who were examining the development of analogues of two popular Cu-complexes that have garnered much attention over recent years.[65] The first is DDC. The Cu(DDC)2 complex suffers from solubility issues [42, 184, 226], so this group chose to make analogues of DDC, maintaining the dithiocarbamate backbone and altering the end groups from ethyl groups to functional groups that would make the compound more water soluble. Interestingly, they were able to increase the water solubility of the 22  complex but this came at the expense of therapeutic activity; the more water soluble compounds were less active. The chemical modification altered the ability of the complex to fit into the 19S cap of the proteasome; an interaction that is crucial for its activity [184]. This chemical modification approach was also used with 8-HQ [193], where 64 analogues of 8-HQ were synthesized and screened for proteasome inhibition. The authors attempted to increase the solubility of the complex by substituting the hydroxyl group with N-phenylacetamide, which resulted in decreased potency. It was found, in this case, that the polarity of the substituent played a role in 8-HQ-induced cytotoxic activity. 1.7.2 Computer-Aided Drug Design Computer-aided drug design is a computational approach which allows for the development of molecular entities which are specific to a structure of interest. This approach is limited in that it does require the three-dimensional structure of the target of interest. This can be achieved through bioinformatic methods, which predict the structure based on an amino acid sequence, or alternatively, a crystal structure determined by X-ray crystallography methods or single-particle electron cryo-microscopy. One example in the CBT literature using such methods is described by Zuo et al. who utilized docking analysis of the proteosome to analyze the binding affinity of four amino acid Schiff base-Cu(II) complexes [227]. This approach allowed the authors to molecularly model the copper complexes and compare the binding to that of BTZ, the clinically available proteosome inhibitor. Another example concerns human serum albumin (HSA) which has a well defined crystal structure and has been used in molecular docking analysis. Li et al. examined how four Schiff-base copper complexes would interact with the protein [228]. HSA plays an important role in drug transport and distribution and thus through docking analysis it was possible to gain an understanding of how the copper complex behaves in vivo. Owing to the success of DNA damaging agents in treatment of cancer some researchers have directed their efforts on the creation of copper complexes with DNA intercalation capabilities [229, 230]. This was accomplished through the use of docking analysis used to examine how the copper complexes interact with DNA. This computer-aided approach allows one to directly compare where in the active site of the complexes are binding and additionally one could 23  predict how alterations of the compound may affect binding affinity. Again, if the candidate compounds identified prove to be pharmaceutically challenging because of their solubility properties, more classical medicinal chemistry programs can be applied to define more water soluble compounds. 1.8 Nanoscale Synthesis Reactions Inside Liposomes As noted already, this thesis describes a new approach which allows for the formulation of CBTs inside liposomes. This method is reliant on metal complexation reactions. Provided that the reaction does not impact the integrity of the liposome, the resulting product will be suitable for administration. In this context, it is important at this stage in this introduction to provide a brief description of liposomes and how the approaches developed in my thesis can be differentiated from previous disclosure involving the use of encapsulated metal ions.  1.9 Liposomes Liposomes represent one of the most successful nanoparticle platforms with numerous products already approved by the FDA [11]. Since the original structure was described by Bangham, liposomes as a technology have clearly blossomed with the success of liposomal doxorubicin formulations (Doxil® [5],and Myocet® [231]) and several new iterations of liposomes (e.g. thermosensitive [232] and combiplex formulations [233]) which are being produced today. They are typically composed of phospholipids and cholesterol and form spontaneously when hydrated. Liposomes have become a go-to technology useful for many different therapeutics including anti-cancer, antibiotic, natural products and NSAIDs [17, 234-236]. Much of the success of liposomes as drug delivery vehicles has to do with advancements made in manufacturing methods. Today, liposomes can be created in large batches and candidate drug loading is efficient when using active “remote” loading processes reliant on pH gradients (Figure 1.4A) or metal complexation (Figure 1.4B) [23, 237-239]. Technologies such as extrusion and more recently the development of microfluidic devices (e.g. NanoAssemblr) have allowed for the reproducible production of liposomes with defined attributes [12, 240, 241].   24   Figure 1. 4: Doxorubicin possesses structural motifs that facilitate remote loading in liposomes through use of transmembrane pH gradients and metal complexation. (A) pH gradient loading: uncharged doxorubicin on the liposome exterior crosses the lipid bilayer and becomes charged in the low pH environment. The charged form of doxorubicin is less membrane permeable and remains encapsulated. (B) Metal complexation loading: doxorubicin on the exterior of the liposomes crosses the lipid bilayer in its neutral form and forms complexes with the encapsulated divalent metal (e.g. Mn2+, Cu2+).         25  1.10 Remote Loading Using pH Gradients Many factors contribute to the success of liposomes as a platform for drug delivery. In this context, the development of remote candidate drug loading processes was a significant “game changer”; allowing for a shift away from passive encapsulation approaches. This process allowed candidate drugs to be loaded into pre-formed liposomes with high trapping efficiency, minimizing waste of costly drugs and eliminating the need to work with drugs during liposomal preparations. One driving force for remote loading relies on the presence of transmembrane pH gradients (acidic interior) (Fig.1.5). Candidate drugs (e.g. doxorubicin) with weakly basic amines (R-NH3+) could be added to the outside of liposomes and, at appropriate pHs, the neutral form of the candidate drug would redistribute across the lipid bilayer and in the low interior pH the candidate drug becomes protonated. The protonated form of the drug is much less membrane permeable and becomes trapped in the liposomes. This approach used liposomes with encapsulated citrate buffer (pH 4.0, Figure 1.5A) or ammonium sulfate (pH 5.5, Figure 1.5B). Despite the limitation that remote loading using pH gradients works for only a subset of drug structures, for those candidate drugs with appropriate chemical attributes the method facilitates efficient trapping (>98%). The loading efficiency eliminates waste and allows for practical scale-up processes. Remote loading of drugs with a protonizable amine function, as one example, has already been widely discussed [13, 15, 237]. Hence, the focus here will be on the use of metal based loading methods, and differentiation between metal complexation reactions from pH gradient loading to remote load candidate drugs into preformed liposomes. As suggested above, another variation of remote loading was developed that relied on the use of liposomes with encapsulated divalent metal-containing solutions. For example, unbuffered MnSO4 solution (pH 3.5) could be trapped inside liposomes and following the addition of a divalent cation ionophore (e.g. A23187, an ionophore which exchanges metal ions for protons) a pH gradient could be generated to engender drug loading (Figure 1.5C). This was first reported in publications using doxorubicin. What was interesting about doxorubicin as a drug choice was the fact that it can form dimeric Mn-26  (Dox)2 complexes (Figure 1.5D). Studies demonstrated remote loading of doxorubicin into liposomes with entrapped Mn2+ even in the absence of transmembrane pH gradients. Metal complexation reactions can be used as an alternative to encapsulate drugs into pre-formed liposomes. In this thesis, copper-dependent loading methods are differentiated from pH-driven loading processes where the metal ions are removed from the interior of the liposomes by addition of A23187. While this method has been effective, it is limited to drugs with the protonizable amine functions.   Figure 1. 5:  Remote loading approaches to encapsulate doxorubicin into liposomes. Liposomes can be prepared in (A) citrate (pH 3.5) or (B) ammonium sulfate (pH 5.5) solutions which maintain an acidic interior within the liposome. Doxorubicin within the acidic interior becomes protonated at the tertiary amine functional group. Doxorubicin encapsulation methods that rely on the use of trapped metal ions have evolved from the use of MnSO4, which can be used (C) to create a pH gradient by addition of A23187 an ionophore, which exchanges protons for Mn2+ ions. (D) Direct metal complexation can also be used to encapsulate doxorubicin in liposomes. The drug–metal complex forms in the liposome and the metal-complexed drug is less membrane permeable and trapped.    27  1.10.3 Use of A23187 (Divalent Metal Ionophores) to Facilitate Drug Loading in Liposomal Formulations Prepared with Metal Ion Gradients The use and success of divalent metal ionophores such as A23187 (calcimycin) to create and maintain pH gradients in liposomes with encapsulated divalent metals such as manganese and copper is well established and characterized [242-244]. The ionophore facilitates the movement of two protons from the external buffer inside the liposome in exchange for one divalent cation. In addition to Mn2+ and Cu2+ other metals such as Mg2+ and Zn2+ can be used [17, 245]. When using this approach, the liposomes are prepared in the metal containing solutions which are typically acidic (pH <4.5). Thus the initial driving force for encapsulation of a compound added to the outside of liposomes prepared with a metal ion gradient is the pH gradient even in the absence of the ionophore. The addition of the ionophore facilitates maintenance of the pH gradient. Thus this method has been consistently applied to candidate drugs that have protonizable amine functions, albeit some of those compounds also carry metal binding motifs such as the camptothecins and anthracyclines. It should be noted that published reports indicate that A23187 addition to liposomes with encapsulated copper can result in a candidate drug loaded liposome that exhibits improved drug retention when compared to other metal ions [246]. Although, this may be a result of copper-candidate drug interactions, it is unlikely that this contributes significantly to improved retention since the encapsulated copper concentration is reduced substantially when adding the ionophore to the liposomes. The remaining encapsulated copper, however, may interact with the phospholipids in the inner leaflet of the liposomal membrane and this copper-lipid interaction may improve drug retention [247, 248]. Notably, this is not observed for all metals as the improved retention is not observed for Mg2+, Zn2+ or Mn2+ solutions. 1.11 Summary The development of copper-based therapeutics has been hindered due to their very poor aqueous solubility. As an example, diethyldithiocarbamate (DDC) is the primary metabolite of disulfiram, an approved drug for alcoholism that is being repurposed for cancer. The 28  anti-cancer activity of DDC is dependent on complexation with copper to form copper bis-diethyldithiocarbamate (Cu(DDC)2), a highly insoluble complex that  has not been possible to develop for indications requiring parenteral administration. I have resolved this issue by synthesizing Cu(DDC)2 inside liposomes (see Chapters 3 and 4). DDC crosses the liposomal lipid bilayer, reacting with the entrapped copper; a reaction that can be observed through a colour change as the solution goes from a light blue to dark brown. This method was successfully applied to other CDCs including the anti-parasitic drug clioquinol, the natural product quercetin and the novel targeted agent CX-5461 (see Chapters 3 and 5).  The studies described in Chapter 6 supports the potential use of copper-based therapeutics to treat cancers that are insensitive to platinum drugs. The method, whereby CBTs are synthesized inside liposomes, provides a simple, transformative solution enabling, for the first time, the development of CDCs as viable candidate anti-cancer drugs; drugs that would represent a brand new class of therapeutics for cancer patients.               29  2. Materials and Methods 2.1 Materials 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) , cholesterol (Chol), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-n-[carboxy(polyethylene glycol)-2000] (DSPE-PEG2000), Sphingomyelin (SM), 1,2-distearoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DSPG) and 3ß-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol) was purchased from Avanti Polar Lipids (Alabaster, AL). Sephadex G-50 beads were purchased from GE healthcare (Boston, MA). 3H-cholesteryl hexadecyl ether (3H-CHE) and Pico-Fluor 40 scintillation cocktail were purchased from PerkinElmer Life Sciences (Woodbridge, ON, Canada). Plumbagin (Plum) was obtained from Plumbago Indica, pyrithione (Pyr;2-mercaptopyridine N-oxide sodium salt), 8-Hydroxyquinoline, Sodium diethyldithiocarbamate trihydrate, clioquinol (5-Chloro-7-iodo-8-quinolinol), quercetin (Qu), DSF,  Nigericin sodium salt, copper sulfate (CuSO4), HEPES, and all other chemicals (Reagent grade) were purchased from Sigma Aldrich (Oakville, ON, Canada). CBDCA and CDDP were obtained from Hospira (Lake Forest, IL). CX-5461 was purchased from Selleck Chemicals (Houston, TX). 2.2 Cell Lines The A549, FaDu, Cal-27, SCC-25, U87, U251MG, F98 and MV-4-11 cell lines were purchased from ATCC. A2780-S and A2780-CP cell lines were obtained from Dr. Mark W. Nachtigal at the University of Manitoba (Winnipeg, Canada) and the H1933 cells were provided by Dr. William Lockwood at the BC Cancer Agency’s Research Centre (British Columbia, Canada). HBEpC (Human Bronchial Epithelial Cells) was obtained from Cell Applications (San Deigo, California) and MDA-231-BR was kindly donated by Patricia Steeg, NIH/NCI. All cell lines were used for 15-18 passages. A549 and H1933 cells were maintained in RPMI (Gibco). A2780-S, A2780-CP and SCC-25 were maintained in DMEM/F12 (Gibco). MV-4-11 and FaDu cells were maintained in IMDM (Gibco), and MEM (Gibco), respectively. U-87, U251MG, Cal-27 and MDA-231-BR were maintained in DMEM (Gibco). Media for all cell lines was supplemented with 2mM L-glutamine (Gibco) and 10% fetal bovine serum 30  (Gibco) and maintained at 37°C and 5% CO2 in a humidified incubator. Media for the SCC-25 cell line was also supplemented with 400 ng/mL hydrocortisone. All cell lines tested negative for mycoplasma when obtained by the laboratory.  2.3 In Vitro Cell-Based Assays 2.3.1 Cytotoxicity Assay of Pt Drugs, Disulfiram, Ligands and Copper Complexes For in vitro testing, copper complexes were synthesized prior to cell treatment by mixing CuSO4 and the ligand at a fixed 2:1 ratio in DMSO (50%). Disulfiram and/or CuSO4/Cu(CQ)2 were mixed at a 1:1 ratio in DMSO (50%). Disulfiram or the ligands DDC, CQ, 8-HQ, Plum were solubilized in DMSO and Pyr was dissolved in sterile water. CBDCA and CDDP were dissolved in sterile saline (0.9%).  Prior to drug treatment, the cells were seeded into 384 well plates and incubated 24 hrs in media prior to treatment. The adherent cell lines (A549, A2780-S, A2780-CP, FaDu, Cal-27, SCC-25, H1933, U-87, U251, F98, MDA-231-BR and HBEpC) were exposed to the indicated compounds in triplicate wells for 72 hrs (unless indicated). Following treatment, cells were stained in situ with Hoechst 33342 and ethidium homodimer-I to differentiate between viable (Hoechst-positive/ethidium homodimer-negative) and dead cells that had lost membrane integrity (Hoechst-positive/ethidium-positive). Cells were imaged with the IN Cell Analyzer 2200 (GE Healthcare Life Sciences) and 4 images/well were collected. Images were analyzed with the ToolBox Developer 1.9 software (GE Healthcare Life Sciences) to obtain viable and dead cell counts based on differential staining of cell nuclei. The suspension cell line (MV-4-11) was treated for 72 hrs and viability was assessed using the Presto Blue™ assay (Life Technologies) following the manufacturer’s instructions. The viability data were normalized to vehicle control (0.5% DMSO in media) and expressed as fraction affected where value of 1 corresponded to 100% loss of cell viability relative to vehicle controls and 0 corresponds to a viability comparable to control cells in culture.  2.3.2 Western Blot Analysis for Ubiquitinylated Protein  Cells were seeded in 6-well plates (400,000 cells per well) and treated with the IC30 of the indicated test compound for 24 hrs. Cell lysates were prepared using lysis buffer 31  comprising 50 mM Tris-HCl (pH 7.4), 150mM NaCl, 0.25% sodium deoxycholate, 1% NP-40, 0.1% SDS, 1mM EDTA, and Mini Protease Inhibitor Cocktail tablets (Roche Diagnostics) for 1 hr on a shaker at 4°C. Cell lysates were centrifuged at 14000 x g for 10 min to collect total protein. A BCA Protein Assay Kit (Pierce) was used to determine protein concentrations and 10 µg of lysate protein were run on a 4-12% Bis-Tris gel (Life Technologies) at 170V for 1 hr before being transferred to a 0.2 µm nitrocellulose membrane using the Trans-Blot Turbo Transfer System (Bio-Rad Laboratories). The membranes were blocked with 5% skim milk in TBST (20mM Tris-base, 140mM NaCl, 0.1% Tween 20) and then  probed for Ubiquitin (Cell Signaling Technology, 1:1000) and β-Actin (Sigma-Aldrich, 1:50,000) overnight at 4°C. Blots were then washed with TBST (3 x 5 min) and incubated with horseradish peroxidase-conjugated secondary antibody (Promega) for 1 hr at room temperature (1:10,000 for β-actin, 1:5000 for Ubiquitin). After washing with TBST (3 x 5 min), the blots were developed using Clarity Western ECL Substrate (Bio-Rad) for 5 min before imaging with the ChemiDoc MP Imaging System and ImageLab software (Bio-Rad).  2.3.3 Flow Cytommetric Analysis of Cu(DDC)2 Treated Cells MV-4-11 cells were seeded in 6-well plates for 24 hrs and then treated with vehicle, DDC, CuSO4 or Cu(DDC)2 (5µM final concentration). At 24 hrs post-treatment cells were washed 3 times with cold HBSS and fixed in 70% ethanol. The final concentration of cells was adjusted to 106 cells/mL. The samples were left for 1 hr on ice followed by an overnight incubation at -20°C. Cells were centrifuged and the pellet was stained using phosphate buffered saline (PBS, pH 7.4) buffer containing 50 μg/mL propium iodide (Invitrogen), 1 mg/mL RNase A (Sigma), and 0.1% Triton X-100 (Bio-Rad) for 15 mins at 37°C followed by an incubation for 1 hr on ice. Data were acquired and analyzed using a FACS Calibur flow cytometer and WINMDI 2.9 software, respectively.  2.3.4 Reactive Oxygen Species Assay The reactive oxygen species (ROS) assay was performed using the ROS-GloTM H2O2 Assay Kit (Promega) as per manufacturer instructions. Briefly, MV-4-11 cells were seeded at 14,000 cells/well in 96-well, white, clear-bottom plates. Treatment was added subsequently along with 20 µL substrate per well as per the supplier’s protocol. Cells were 32  treated with either menadione (positive control, 50μM) or Cu(DDC)2  (200μM) for 4 hrs. All treatments were completed with and without cells to account for ROS generation as a result of interaction with vehicle and medium components. Finally, the H2O2 detection solution was added at 100 µL/well and incubated at room temperature for 20 mins. ROS generation was measured based on luminescence signal using a FluorStar Optima plate reader.  2.3.5 Phen Green™ FL Copper Ionophore Testing Phen Green™ FL was used to assess the amount of copper in the presence and absence of CQ in A2870-CP cells. Cu reduces the fluorescence intensity of Phen Green™ and thus allows for the identification of Cu entering the cell. Cells were grown to 80-90% confluency and treated with vehicle (0.01% DMSO), CQ (25µM), CuSO4 (100µM) and CQ/CuSO4 (25/100µM) for 1 hr. The cells were washed three times with HBS prior to media replacement with fresh medium containing 5µM Phen Green™ FL for 0.5 hr. Cells were then washed three times with Hanks Buffered Saline Solution and imaged using InCELL Analyzer 2200 (excitation 420 nm and emission 538 nm).  2.4 Liposome Preparation and Characterization  2.4.1 Liposome Preparation  The extrusion method for liposome preparation has been well documented by others [240]. Briefly, for the cholesterol containing formulations (DSPC/Chol (55:45), SM/Chol (55:45), DSPC/DSPG/Chol/ (70:20:10),DSPC/DC-Chol/DSPE-PEG2000 (30:50:20) or , DSPC/Chol/DSPE-PEG2000 (45 to 50: 45: 0 to 5 mole ratio) the lipids were removed from the freezer (-80oC) and placed in a desiccator for 2 hrs before being weighed and dissolved in chloroform at the appropriate ratios. A non-exchangeable and non-metabolizable lipid marker, 3H-CHE, was incorporated into the chloroform mixture to achieve a specific activity of approximately 0.025 µCi/mmol total lipid. The solution was dried from chloroform using nitrogen gas and the thin film generated was further dried under high vacuum for 3 hrs. The lipid film was then rehydrated at 65oC with unbuffered 300mM CuSO4 (pH 3.5) or 300mM Cu-Gluconate (pH 3.5) for at least 2 hrs. To prepare Cu(DDC)2-liposomes with final 33  Cu(DDC)2 to lipid ratios of 0.1 or 0.05, copper salt solutions of 150mM and 75mM were used. The resulting multilamellar vesicles underwent 5 freeze (in liquid nitrogen) and thaw (65oC water bath) cycles [249]. These were then placed in an extruder (Evonik Transffera Nanosciences, Vancouver) and extruded through stacked 0.1 µm polycarbonate filters at least 10 times at 65°C. All formulations were characterized for size and polydispersity (ZetaPals, Brookhaven). Samples were diluted to 1-5 mM in filtered 0.9% NaCl or SH buffer for size and polydispersity analysis. For cholesterol-free formulations (DSPC/DSPE-PEG2000 or SM/DSPE-PEG2000, 95:5), the indicated lipids were removed from the freezer and placed in a desiccator for at least 2 hrs before being weighed and dissolved in ethanol. 3H-CHE was incorporated in the dissolved lipids in ethanol. The final ethanol solution with dissolved lipids was added dropwise to a solution of 300mM unbuffered CuSO4 (pH 3.5) in a 65oC water bath with constant mixing. The final ethanol concentration of the aqueous ethanol mixture was approximately 15% (v/v). This solution was then extruded using the same process as written above.  For all liposomal preparations the unencapsulated copper was removed by first exchanging the sample into a sucrose (300mM), HEPES (20mM) and EDTA (15mM) buffer (SHE buffer, pH 7.4) by passing the sample through a Sephadex G-50 column equilibrated with the buffer. The resulting solution was then dialyzed against a sucrose (300mM) and HEPES (20mM) buffer (SH buffer, pH 7.4) and concentrated to the desired concentration for experimental studies using tangential flow. Liposomal lipid concentration was determined by measuring 3H-CHE using liquid scintillation counting (Packard 1900TR Liquid Scintillation Analyzer).  2.4.2 Copper Complexation Synthesis: Copper loaded-liposomes were mixed with DDC (4 or 25oC), CQ (40oC), Qu (50oC) or CX-5461 (60oC) at the indicated compound to liposomal lipid ratio in the SH buffer (pH 7.5) and incubated over a 60-min time course. Liposome and associated compound were separated from unassociated (free) compound using a Sephadex G-50 column equilibrated with SH buffer. The eluted liposome fractions (collected with the excluded volume of the 34  column) were analyzed for copper, compound (as the copper complex or after dissociation of the bound copper) and liposomal lipid concentrations. Lipid concentrations were measured by assaying for [3H]-CHE by liquid scintillation counting (Packard 1900TR Liquid Scintillation Analyzer) where 20 μL of eluted liposome sample was mixed into 5 mLs Pico-Fluor Plus (Perkin Elmer). For the spectrophotometric assay samples were diluted into 1 mL methanol for Cu(DDC)2 and Cu(CQ)2 and absorbance was measured at 435nm (1-10 μg/mL) or 275nm (0.25-2.5 μg/mL), respectively. CuQu and CuCX-5461 were dissolved in 1 mL of 3% acetic acid in methanol and Qu and CX-5461 were measured by assessing absorbance at 372nm (1-10 μg/mL) or 288nm (1-10 μg/mL), respectively.  Copper was measured using atomic absorption spectrophotomer (AAnalyst600, Perkin Elmer).  The Cu-containing liposomes were diluted in 10 mLs of 0.1% HNO3. A copper (Cu2+) standard curve was generated using Cu2+ (0- 100 ng/mL) in 2% nitric acid (Sigma Aldrich).  2.4.3 Cryo-Electron Microscopy: Further analysis of the Cu(DDC)2 formulations was done by cryo-electron microscopy (CEM). CEM analysis was performed using a Zeiss Libra 120 transmission electron microscope at the University of Uppsala, Sweden. Briefly, liposomes were prepared as described above containing either CuSO4 or Cu(DDC)2 with SH buffer at pH 7.4. In a controlled chamber for humidity and temperature (25oC) samples of 1-2 µL of the sample were deposited on copper grids coated with a holey cellulose acetate butyrate polymer. Excess liquid was blotted away carefully with filter paper and then samples were quickly vitrified by plunging into liquid ethane. This was then transferred to liquid nitrogen to maintain the temperature below 108 K, which minimizes formation of ice crystals. Images were taken in a zero-loss bright-field mode and an accelerating voltage = 80 kV.  2.4.4 Cu(DDC)2 and Cu(CQ)2 Release From Liposomes Cu(DDC)2 or Cu(CQ)2 containing liposomes (final liposomal lipid concentration 5 mM) were suspended in SH buffer with 50% (v/v) FBS and incubated with constant mixing at 37oC in a water bath. At the indicated time points, an aliquot (100 μL) of the liposome solution was passed through a 1 mL Sephadex G-50 spin column equilibrated with SH buffer. The columns were centrifuged at 680 x g for 3 min at 25°C. The eluate was assayed for 35  Cu(DDC)2 or CQ using HPLC and lipid concentration was determined by measuring 3H-CHE using scintillation counting. An aliquot (50 µL) of eluate was mixed with 950 µL methanol and the sample was then centrifuged at 10000 x g for 10 mins at 4oC to pellet precipitated proteins. The supernatant was assayed for Cu(DDC)2 or CQ using HPLC. 2.5 Analytical Assays: 2.5.1 Cu(DDC)2 Quantification For the copper complexation experiments an aliquot of liposomal Cu(DDC)2 was diluted into 1 mL methanol and absorbance was measured 435 nm (1-10 μg/mL) on an Agilent 8453  UV-Vis spectrophotometer (Waldbronn, Germany). For in vivo and release studies, Cu(DDC)2 was measured on a Waters Alliance HPLC Module 2695 with a photodiode array detector (model 996, Milford, MA)  and the resulting chromatograms were analyzed by Empower 2 software. A Pronto SIL 120-3-C18ace-EPS (3.0 μm, 4.6 x 150 mm) column was used with a mobile phase composed of 90% methanol and 10% water. A 100 μL sample volume was injected, the flow rate was 1 mL/min and column temperature was set to 40oC. Only samples with >90 ng/mL could be detected. Thus, HPLC assay developed for Cu(DDC)2 was not sensitive enough for the in vivo studies completed; therefore Cu was used as a surrogate marker for Cu(DDC)2. Samples were diluted in 0.1% HNO3 and subsequently the Cu concentration was measured using atomic absorption spectrophotometer (AAS; AAnalyst600, Perkin Elmer) see below. 2.5.2 Cu(CQ)2 Quantification For the copper complexation experiments an aliquot of liposomal Cu(CQ)2 was diluted into 1 mL methanol and absorbance was measured at  275 nm (0.25-2.5 μg/mL) on an Agilent 8453  UV-Vis spectrophotometer (Waldbronn, Germany). For in vivo and release studies, Cu(CQ)2 was measured on a Waters Alliance HPLC Module 2695 and Empower 2 Software. A 30 μL sample volume was injected, and an isocratic mobile phase of water (pH 3 phosphoric acid) and acetonitrile (60:40) at a flow rate of 1 mL/min was used over a Luna C18 column (5 μm, 4.6 x 150 mm) heated to 55°C. CQ was 36  detected post column with a Waters 996 photodiode array detector (Milford, MA) at 254 nm. Pyrrolidine diethyldithiocarbamate was added to samples and standards at an excess of 3 mol equivalents prior to injection to ensure dissociation of CQ from Cu. 2.5.3 CuQu and CuCX-5461 Quantification For copper complexation synthesis experiments, liposomal CuQu and CuCX-5461 were dissolved in 1 mL of 3% acetic acid in methanol and Qu and CX-5461 were measured by assessing absorbance at 372 nm (1-10 μg/mL) or 288 nm (1-10 μg/mL) on an Agilent 8453  UV-Vis spectrophotometer (Waldbronn, Germany), respectively. The quantification of drugs in plasma was performed using HPLC. Qu was measured at 368 nm following separation on a symmetry C18 column (3.5 μm, 3.0 x 150 mm) using a mobile phase of 0.1% TFA in water and acetonitrile (2.3:1). A 25 μL sample volume was injected, the flow rate was set at 1 mL/min and the column temperature was 30oC. Samples and standards were prepared in acidified methanol so as to dissociate the CuQu complex prior to HPLC analysis. Similarly, the quantification of CX-5461 was performed in acidified methanol to dissociate the complex and CX-5461 was measured at 300 nm following separation on a Luna C18 column (5 μm, 4.6 x 150 mm). The mobile phase contained a 1:1.2 mixture of 0.1% TFA in water and 0.1% TFA in methanol. A 5 μL sample volume was injected, the flow rate was set at 1 mL/min and the column temperature was 35oC. 2.5.4 Copper Quantification  Liposomal and plasma copper quantification was performed using AAS. The Cu-containing liposomes were diluted in 0.1% HNO3. A Cu2+ standard curve was generated using Cu2+ (0- 100 ng/mL) in 2% nitric acid (Sigma Aldrich).  2.5.5 Liposomal Lipid Quantification Lipid concentrations were measured by assaying for [3H]-CHE by liquid scintillation counting (Packard 1900TR Liquid Scintillation Analyzer) where 20 μL of eluted liposome sample was mixed into 5 mLs Pico-Fluor Plus (Perkin Elmer). 37  2.6 In Vivo Characterization of Liposomal Formulations: 2.6.1 Dose Range Finding Studies 2.6.1.1 Single Dose Female CD-1 mice were given a single bolus tail vein i.v.(intravenous) injections of Cu(DDC)2 (15 mg/kg, drug-to-lipid ratio 0.2 mol:mol), Cu(CQ)2 (30 mg/kg, drug-to-lipid ratio 0.2 mol:mol), CuQu (70 mg/kg, drug-to-lipid ratio 0.2 mol:mol), or CuCX-5461 (50 mg/kg, drug-to-lipid ratio 0.2 mol:mol). All formulations were prepared using DSPC:Chol (55:45 mole ratio) liposomes with encapsulated 300 mM copper sulfate as described above. To define tolerability of the CBT formulations, mice (n=3) were given an intravenous injection (lateral tail vein) of the indicated formulation at the specified dose. The health of the animals was measured over a 14 day period after administration and a full necropsy was performed at that time to assess changes in tissue/organ appearance.  2.6.1.2 Multi-Dose  To define the maximum tolerated dose of Cu(DDC)2 formulations, mice (n=3) were given an i.v. injection (lateral tail vein) of Cu(DDC)2 using a Monday, Wednesday and Friday x 2 dosing schedule. To define a dose of the Cu(CQ)2 formulation that was well tolerated, mice (n=3) were given an i.v. injection (lateral tail vein) of Cu(CQ)2 using a Monday, Wednesday and Friday x 2 dosing schedule. These studies also assessed the tolerability of Cu(CQ)2 when combined with DSF; where DSF was dosed orally at 100 mg/kg, once daily Monday through Friday for 2 weeks). In these studies Cu(CQ)2 was dosed at 30 mg/kg, Monday, Wednesday and Friday x 2 weeks.  For all dose range finding studies the health status of the animals was monitored following an established standard operating procedure. In particular, signs of ill health were based on body weight loss, change in appetite, and behavioral changes such as altered gait, lethargy and gross manifestations of stress. When signs of severe toxicity were present, the animals were terminated (isoflurane overdose followed by CO2 asphyxiation) for humane reasons. Necropsy was performed to assess other signs of toxicity. The surviving animals 38  were monitored for two weeks (14 days) after administration of the last dose and full necropsies were completed on all treated mice at that time to assess whether there were gross changes in tissue/organ appearance.  2.6.2 Pharmacokinetic Studies DSPC/Chol (55:45) liposomes containing Cu(DDC)2 (15 mg/kg, drug-to-lipid ratio 0.2 mol:mol), Cu(CQ)2 (30 mg/kg, drug-to-lipid ratio 0.2 mol:mol), CuQu (70 mg/kg, drug-to-lipid ratio 0.2 mol:mol), CuCX-5461 (50 mg/kg, drug-to-lipid ratio 0.2 mol:mol) or DSPC/DSPE-PEG2000 (95:5) Cu(DDC)2 (15 mg/kg, drug-to-lipid ratio 0.25 mol:mol) were injected into CD-1 mice. At selected time points mice (n=4 per time point) were terminated by isoflurane followed by CO2 asphyxiation and blood was collected by cardiac puncture directly into EDTA coated tubes kept on ice. Blood samples were centrifuged (Beckman Coulter Allegra X-15R) at 1500 x g for 15 min at 4°C. Plasma was collected and placed into a separate tube prior to assaying for copper, drug and liposomal associated lipid. The copper was measured using AAS by diluting plasma into 0.1% HNO3 and drugs were measured using the HPLC method described above. Plasma (30 µL) was added to Pico-Fluor 40 scintillation cocktail and the amount of [3H]-CHE was determined as described above.  2.7 In Vivo Efficacy Studies 2.7.1 Convection Enhanced Delivery in F98 Glioma Model Male Fischer rats were purchased from Charles River Laboratories International Inc. (Wilmington, MA). The implantation method was described previously [250]. F98 cells (10,000 cells in 5 μL) were prepared and implanted into the right caudate nucleus (1 mm anterior, 3 mm right of the bregma, and 6 mm deep) of the brain in 5 min. The CED procedure [251] was performed 10 days after implantation of F98 cells, at the same injection site using a 33 gauge Hamilton syringe. Before infusion, the burr was filled with bone wax and the needle was inserted 6.5 mm deep, retained their for 5 min and then, withdrawn to 6 mm. Test articles and a control vehicle to be injected were vehicle (SH Buffer), CuSO4 (300mM)- DSPC/Chol  liposomes  (copper 0.1 mg/mL, lipid 3.5 mg/mL) lipid  or Cu(DDC)2  formulated in DSPC/Chol liposomes (0.5 mg/mL, lipid 3.5 mg/mL). A 39  total volume of 10 μL was infused at an infusion rate of 0.5 μL/min for 20 minutes (5µg Cu(DDC)2 per rat). After the infusion, the needle was left in, to reduce backflow and increase convection volume, for 5 min prior to being withdrawn.  2.7.2 Cu(DDC)2 Efficacy Studies in the MV-4-11 and A2780-CP Subcutaneous Tumour Models Animals were monitored at least three times weekly for body weight and tumour growth which was measured with calipers and tumour volumes were calculated based on a formula of (tumour length x tumour width2)/2. Animals were terminated by isoflorane followed by CO2 asphyxiation when animals reached a humane endpoint for these studies was defined when tumours exceeded 800 mm3 or when tumours ulcerated. 2.7.2.1 MV-4-11 Subcutaneous Tumour Model As indicated above MV-4-11 cells used for sc implantation were between passages 3-10 and were always collected when maintained at a confluence of 80-90% at the time of harvesting for implantation. RAG-2M mice (up to 9 per group) were inoculated with 1 x 106 cells in an injection volume of 50 μL per animal using a 28-gauge needle. Treatment was initiated on day 12 and treatment included the vehicle control (SH buffer), copper liposomes or Cu(DDC)2 prepared in liposomes composed of either DSPC/Chol (55:45) or DSPC/DSPE-PEG2000 (95:5). . 2.7.2.2 A2780-CP Subcutaneous Tumour Model A2780-CP cells that were used for subcutaneous (sc) implantation were between passages 3-10 and maintained in DMEM/F12 (Gibco) at a confluence of 80-90%. NRG mice (7 per group) were inoculated sc with 1 x 106 cells in a volume of 50 μL using a 28-gauge needle. Treatment was initiated on day four and treatment groups included the vehicle control (SH buffer), CuSO4-liposomes (1.7 mg/kg copper, 50 mg/kg lipid), and Cu(DDC)2 liposomes (8 mg/kg Cu(DDC)2 and 50 mg/kg lipid) on a Monday, Wednesday and Friday x2 weeks dosing schedule. The copper dose of the CuSO4 liposome control was equivalent to that of Cu(DDC)2.   40  2.7.5 Cu(CQ)2 Efficacy Studies in the U251 and A2780-CP Subcutaneous Tumour Models Animals were monitored at least three times weekly for body weight and tumour growth which was measured with calipers and tumour volumes were calculated based on a formula of (tumour length x tumour width2)/2. Animals were terminated by isoflorane followed by CO2 asphyxiation when animals reached a humane endpoint for these studies was defined when tumours exceeded 800 mm3 or when tumours ulcerated. 2.7.5.1 U-251 Subcutaneous Tumour Model U-251 cells were grown in culture for 4-8 passages prior to inoculation. Rag2M mice (n=6 per group) were inoculated subcutaneously using a 28-gauge needle into the right flank of the mouse with 5 x 106 cells in a total volume of 50 µL. When the tumours reached 50-100 mm3,as measured using digital calipers, they were treated with vehicle (SH buffer), CuSO4-liposomes (Cu = 3.2 mg/kg) or Cu(CQ)2-liposomes (CQ = 30 mg/kg) via iv injection on a Monday, Wednesday and Friday x 2 weeks treatment schedule. The copper dose of 3.2 mg/kg was selected as it is the equivalent amount of copper found in the Cu(CQ)2 formulation.  2.7.5.2 A2780-CP Subcutaneous Tumour Model A2780-CP cells that were used for subcutaneous (sc) implantation were between passages 4-8 and maintained in DMEM/F12 (Gibco) at a confluence of 80-90%. NRG mice (7 per group) were inoculated sc with 1 x 106 cells in a volume of 50 μL using a 28-gauge needle. Treatment was initiated four days after cell inoculation with either vehicle (SH buffer), CuSO4-liposomes (Cu = 3.2 mg/kg) or Cu(CQ)2-liposomes (CQ = 30 mg/kg) via iv injection on a Monday, Wednesday and Friday x 2 weeks treatment schedule. The copper dose of 3.2 mg/kg was selected as it is the equivalent amount of copper found in the Cu(CQ)2 formulation. 2.7.6 Cu(CQ)2 and DSF Combination Efficacy Studies in A2780-CP Subcutaneous Tumour Models For combination studies, A2780-CP cells were grown and inoculated in NRG mice (n=13 per group) as outlined above. On day 4 mice were treated iv with vehicle (SH buffer), 41  CuSO4-liposomes (Cu = 3.2 mg/kg) or Cu(CQ)2-liposomes (CQ = 30 mg/kg) on a Monday, Wednesday and Friday x 2 weeks schedule. Additionally, DSF (100 mg/kg) was dosed by oral gavage Monday through Friday for 2 weeks alone and in combination with CuSO4-liposomes or Cu(CQ)2-liposomes given intravenously. Tumor size and body weight were measured three times weekly throughout the study. The health status of the animals was monitored daily following an established standard operating procedure as described above. Animals were terminated by CO2 asphyxiation following isoflurane anaesthesia when tumors reached a maximum size of 800 mm3 or underwent ulceration. 2.8 Statistical Analysis All data were plotted using the Prism 6.0 software (GraphPad) as mean ± SEM or mean ± SD as described in the figure legends. The IC50 values and 95% confidence intervals (CI) were extrapolated from nonlinear regression (curve fit) of the cytotoxicity curves using Prism 6.0 (GraphPad software). To determine whether the cytotoxic effects of copper complexes are associated with platinum sensitivity, the IC50 values of each copper complex was plotted against the CDDP IC50 for each cell line. Each data point represents one cell line. The Pearson Correlation coefficient and corresponding two-tail p-values were then determined using Prism 6.0 (GraphPad software). A P-value < 0.05 was considered statistically significant. Survival study statistical analysis was performed using the log rank test; a P-value < 0.05 was considered statistically significant. Statistical analyses comparing Cu(DDC)2  plasma levels, PhenGreen Cu ionophore comparison or tumour growth studies were performed using one-way ANOVA followed by Tukey adjustments to correct for multiple comparisons.      42  3. Nanoscale Reaction Vessels Designed for Synthesis of Copper-Drug Complexes Suitable for Preclinical Development* 3.1 Introduction Over the past decade, the number of copper complexes with anti-cancer activity has been increasing and a number of reviews have detailed their synthesis and development [25, 48, 62]. However, CBTs have not been approved for use in patients. A significant challenge associated with the development of CBTs is their extremely low aqueous solubility, thus making it difficult to establish the utility of these copper-complexes in preclinical models or patients. Further, development of intravenous dosage formulations requires the use of solubilising agents to create products suitable for use. For example, the therapeutic promise of CBTs has largely been based on data obtained with compounds solubilized in DMSO [252-255]. Here we describe, for the first time, a novel method to prepare formulations of CBTs, formulations that are suitable for intravenous administration.  Methods that rely on copper complexation to encapsulate water-soluble drugs including anthracyclines and camptothecins have already been described; however, optimal drug loading and drug retention in these formulations relied on use of both the encapsulated metal and a transmembrane pH gradient [14, 242, 256, 257]. In fact, it is established that drugs with protonizable amine functions, can readily be encapsulated in liposomes using a transmembrane pH gradients [238]. Here, liposomes prepared with encapsulated copper were mixed with compounds that have copper-binding moieties. The compounds selected exhibit common attributes of extremely low (< 1 mg/mL) water solubility prior to or after complexation with copper.  The method was characterized using DDC (see Figure 1) as a model compound, which is known to be the active metabolite generated following administration of DSF                                                         *Adapted from : M. Wehbe, M. Anantha, I. Backstrom, A. Leung, K. Chen, A. Malhotra, K. Edwards, M.B. Bally, Nanoscale Reaction Vessels Designed for Synthesis of Copper-Drug Complexes Suitable for Preclinical Development, Plos One, 11 (2016) e0153416 43  [118]. It has become a drug of interest for use in the treatment of human immunodeficiency virus (HIV) and cancer [116, 258-261]. DSF is metabolized to DDC, a well-known copper chelator [262-264]. DDC forms a copper complex at a 2:1 mole ratio (DDC:Cu2+), a reaction that is detected by eye as a brown precipitate forms (Figure 3.1E). Unlike DDC, Cu(DDC)2 is highly insoluble in water. Thus, pursuing opportunities related to repurposing DSF for oncology indications should focus on Cu(DDC)2. The formulation relies on the fact that DDC would be membrane permeable and upon addition to preformed liposomes with entrapped copper salts, complexation would occur within the interior of the liposome. The Cu(DDC)2 complex would not be membrane permeable and hence trapped. The resulting formulation was characterized and defined as suitable for intravenous administration. Importantly, the method for preparing Cu(DDC)2 in liposomes proved suitable for other copper complexing compounds. Three types of copper-binding moieties were evaluated including S-Donor, O-Donor and N,O-Donor systems. Examples of drugs that are described here, in addition to DDC (an S-Donor), include Qu (an O-Donor), CQ (an N, O donor) as well as a compound, CX-5461, previously not identified as a copper complexing agent. CQ is an analogue of 8-hydroxyquinole and an approved antibacterial agent. It forms a Cu(II) complex which inhibits proteosome function and is known as a copper ionophore [50, 191]. Qu is a natural product belonging to the class of compounds known as flavanoids. In recent work it was shown to exhibit anti-cancer effects through generation of reactive oxygen species [265]. CX-5461 is a RNA polymerase inhibitor being evaluated in clinical trials [266, 267] and its use exemplifies the versatility of this method as CX-5461 is a high molecular weight compound with many functional groups capable of binding copper. With the existence of other donor systems not described here, it is likely that this method can be applied to a broad range of drugs and drug candidates with a variety of structures, sizes and functional copper-binding moieties [48, 62].  3.2 Results 3.2.1 DSF, DDC and Cu(DDC)2 Cytotoxicity DSF is metabolized to DDC (Figure 3.1A) and DDC is a well-established copper chelator [42, 268]. The cytotoxic activity of DSF when added to cancer cells is copper 44  dependent. As shown in Figure 3.1B, the IC50 of DSF against U87 glioblastoma cells is >10 μM in the absence of copper. In the presence of copper there is a substantial shift (2-orders of magnitude) in cytotoxicity when copper is added with DSF at a 1:1 molar ratio. DSF is unable to interact with copper; thus, activity of DSF depends on its degradation to DDC. As shown in Figure 3.1C, the activity of DDC in the absence of copper is also >10 μM and in the presence of copper (2:1 molar ratio of DDC to copper) is approximately 220 nM. Similar results were obtained in 3 other cell lines where the IC50 of copper + DDC was 345, 329 and 880 nM when used against U251 (glioblastoma line), MDA-231BR (a triple negative breast cancer line selected for its propensity to metastasize to the brain) and A549 (lung cancer line) cells, respectively. DDC as well as Cu(DDC)2 exhibited little activity when added to normal human bronchial epithelial cells (HBEpC), suggesting specificity of Cu(DDC)2 against cancer cells. It is argued that attempts to repurpose DSF as an anti-cancer drug should focus on Cu(DDC)2; however, Cu(DDC)2 is almost completely insoluble in aqueous solution (Figure 3.1E).   45   Figure 3.1: The anti-cancer activity of diethyldithiocarbmamate (DDC) is dependent on copper. (A) Disulfiram is metabolized to diethyldithiocarbamate (DDC) and DDC complexes with Copper (Cu) (II). (B) Cytotoxicity curves for DSF (●)  and DSF + CuSO4 (■) were obtained with the IN CELL Analyzer using U87 glioblastoma cells where cell viability was assessed based on loss of plasma membrane integrity 72 hours following treatment; i.e. total cell count and dead cell count were determined using Hoechst 33342 and ethidium homodimer staining, respectively. (C) Cytotoxicity curves for DDC (●) and DDC + CuSO4 (■); where cytotoxicity was measured as described above. (D) DDC and Cu(DDC)2 IC50 for U251, MDA-231-BR, and A549 cancer cell lines as well as HBEcP a normal cell line; averages (±SEM) are reported from three separate experiments each done in triplicate. (E) Photograph of  DDC, CuSO4 and Cu(DDC)2 solutions in water.   46  3.2.2 Formation of Cu(DDC)2 by Addition of DDC to Pre-formed Copper Containing Liposomes As illustrated in Figure 3.2, within minutes of DDC addition to copper containing lipid vesicles a color change indicative of Cu(DDC)2 formation is visible (Fig. 3.2A). The rate of Cu(DDC)2 formation inside the liposome can be quantified by separating liposome-associated Cu(DDC)2 from unassociated DDC and then assaying for Cu(DDC)2 spectrophotometrically. As shown in Fig. 3.2B, the amount of liposome-associated Cu(DDC)2, measured as the Cu(DDC)2 to liposomal lipid ratio, is temperature-dependent. Cu(DDC)2 association is rapid when DDC is added to copper-containing liposomes at 25oC (RT) and 40oC, where the maximum Cu(DDC)2 to lipid ratio of 0.2 (mol ratio) is achieved within 3 minutes. If the temperature is decreased to 4oC, the Cu(DDC)2 to lipid ratio of 0.2 (mol ratio) is achieved at 60 minutes. The movement of DDC from the external media into the copper-containing liposomal core is not affected by external pH. As shown in Fig. 3.2C, when the external pH is adjusted to 3.5 the loading rate is comparable to that observed at pH 7.4. To determine the maximum Cu(DDC)2 to liposomal lipid ratio that can be achieved when using liposomes prepared in 300 mM copper sulfate, the amount of external DDC was titrated from  0.04 to 0.40 (moles DDC to moles liposomal lipid) and the results suggest that the maximum Cu(DDC)2 to lipid ratio achievable under these condition was 0.2 (mol:mol). This was achieved when the initial DDC to liposomal lipid ratio was 0.4 (mol:mol). As indicated, Cu(DDC)2 forms an insoluble precipitate and it was possible that the synthesis of Cu(DDC)2 inside the liposomes may have engendered formation of a precipitate within the liposomes. To evaluate this, the liposomes were visualized by CEM (Fig. 3.2E). The results illustrate two points: (1) the Cu(DDC)2 liposomes exhibited a mean particle size that was comparable to that observed with the copper containing liposomes before addition of DDC, and (2) the formation of Cu(DDC)2 inside the liposomes did not result in the formation of an electron dense core. It should be noted that while a precipitate was not seen in the cryo-TEM images it does not indicate that Cu(DDC)2 has not precipitated in the liposome core. It is possible that a Cu(DDC)2 precipitate is present but does not exist in a physical state which could effectively absorb electrons such that it could be seen using cryo-TEM.   These points do suggest that there is a homogenous distribution 47  of CDCs across all liposomes in solution. It should be noted that the liposome size estimated by CEM analysis was comparable to that determined by QELS (Fig. 3.2F).    Figure 3.2: Diethyldithiocarbamate (DDC) loading into DSPC/Chol (55:45) liposomes prepared with encapsulated 300 mM CuSO4. (A) Photograph of solutions consisting of DDC (5mg/mL) and added to CuSO4-containing DSPC/Chol (55:45) liposomes (20 mM liposomal lipid) over a 1 hour at 25oC. (B) Formation of Cu(DDC)2 inside DSPC/Chol liposomes (20 mM) as a function of time over 1 hour at 4(●), 25(■) and 40(▲)oC  following addition of DDC at a final DDC concentration of (5 mM); Cu(DDC)2 was measured using a UV-Vis spectrophotometer and lipid was measured using scintillation counting. (C)  Cu(DDC)2 formation inside DSPC/Chol (55:45) liposomes over time where the external pH was 7.4 (▲) and 3.5 (▼). (D) Measured Cu(DDC)2 as a function of increasing DDC added, represented as the theoretical Cu(DDC)2  to total liposomal 48  lipid ratio; where the lipid concentration was fixed at 20 mM and final DDC concentration was varied. (E) Cryo-electron microscopy photomicrograph of CuSO4- containing DSPC/Chol (55:45) liposomes and the same liposomes after formation of encapsulated Cu(DDC)2. (F) Size of the CuSO4- containing liposomes and liposomes with encapsulated Cu(DDC)2 as determined by quasi-electric light scattering and cryo-electron microscopy; data points are given as mean ± SD.    The only change in liposomal lipid composition considered in these studies was incorporation of polyethylene glycol (PEG2000) modified DSPE. This was considered for two reasons: (1) DSPE- PEG2000 is a negatively charged lipid and its use should increase the amount of encapsulated copper when preparing the liposomes; and (2) DSPE- PEG2000 is known to prevent surface-surface associations that can influence liposome-liposome aggregation and liposome-cell interactions which, in turn, can effect elimination rates in vivo [269]. When DSPE- PEG2000 was added to our base lipid formulation of DSPC:CHOL (55:45) ranging from 0.5 to 5% (based on reductions of DSPC content) the maximum amount of liposome-associated Cu(DDC)2 as measured by the Cu(DDC)2 to liposomal lipid ratio increased from 0.2  to 0.4 (Figure 3.3A, gray bars). When analyzing the amount of copper associated with these liposomes (black bars) it was clear that the Cu(DDC)2 to liposomal lipid ratio was related to the amount of copper encapsulated in the liposomes. The addition of DSPE- PEG2000 increased copper encapsulation, likely due to the introduction of an anionic change that is known to enhance liposome trapped volume [240]. The DSPC/CHOL/DSPE-PEG2000 (50/45/5 mol ratio) was selected to establish the relationship between amount of encapsulated copper and final Cu(DDC)2 to liposomal lipid ratio. These liposomes were prepared using copper sulfate solutions with copper concentrations ranging from 0 to 300 mM. The osmolarity (~300 mOs/kg) of these solutions was balanced with MgSO4. These liposomes were analyzed for copper content prior to DDC addition and after addition of DDC in excess (>2-fold molar excess to the measured liposome associated copper for liposomes prepared in the 300 mM copper sulfate solution). The results (Fig. 3.3B and 3.3C) are consistent with the data in Fig. 3A; the Cu(DDC)2 to liposomal lipid ratio achieved was directly proportional to the amount of copper retained in the liposomes. A plot of encapsulated copper vs encapsulated Cu(DDC)2 demonstrated a linear regression fit of R2=0.9754. This is consistent with a 1:2 mol ratio between copper and added DDC.  49   Figure 3.3: Characterization of copper-complex loading method. (A) Measured (AAS) copper to liposomal lipid ratio (black bars) compared to measured Cu(DDC)2 (UV-Vis spectrophotometer) to liposomal lipid ratio (grey bars) after DDC was added to CuSO4-containing DSPC/Chol liposomes prepared with different amounts of DSPE-PEG2000 (ranging from 0 to 5 mole%). (B) Formation of Cu(DDC)2 inside CuSO4-containing DSPC/ Chol/DSPE-PEG2000 (50:45:5) liposomes as a function of the CuSO4 concentration used to prepare the liposomes (ranging from 0 to 300 mM); where the measured copper (AAS) to liposomal lipid ratio (black bar) is compared to the measured Cu(DDC)2 (UV-Vis spectrophotometer) to liposomal lipid ratio (grey bar). (C) Linear regression analysis comparing measured (AAS) copper concentration (assuming encapsulated copper was free in solution) to measured Cu(DDC)2 (UV-Vis spectrophotometer) concentration (assuming encapsulated Cu(DDC)2 was free in solution); R2= 0.9754; each data point represents a mean ± SEM determined from at least three separate experiments done in duplicate.  50  3.2.3 Use of Copper Containing Liposomes to Prepare CBTs Suitable for Intravenous Injection The approach is compatible to other copper-binding drugs and candidates. As a platform approach, this method would address one of the most important problems limiting the development of CBTs. To assess the breadth of this approach, other compounds that contain functional groups known to bind copper were evaluated. These are summarized in Table 3.1 and include, but are not limited to, S-Donor, O-Donor and N,O-Donor systems. Examples tested, in addition to DDC (an S-Donor), include Qu (an O-Donor), CQ (an N,O donor) as well as a compound, CX-5461, previously not identified as a copper complexing agent. The indicated drugs are all sparingly soluble in aqueous solutions at pH 7.4 and all can be encapsulated when added to lipid vesicles (DSPC/CHOL (55:45 mole ratio)) prepared with encapsulated copper. It should be noted that due to their very low aqueous solubility Qu and CQ were added in a solid/powdered form. CX-5461 is also poorly soluble in aqueous solutions but could be prepared as a metastable solution in low pH (4.5) phosphate buffer as described elsewhere [270]. As noted in Table 3.1 (far right column) all formulations could be designed to achieve a final Cu-complexed drug to liposomal lipid ratio of 0.2 (mol:mol). In every example formation of the CDC was rapid, but the temperature needed for CDC was empirically determined. Cu(DDC)2 formation was optimal at 25oC, Cu(CQ)2 formation was optimal at 40oC, the Cu(Qu) and Cu(CX-5461) formations were optimal at 50oC and 60oC, respectively. The goal of this method was to facilitate the ability to test copper complex compounds as therapeutics following intravenous administration. Preliminary data to support this is provided in Figure 3.4. The formulations described in Table 3.1, were prepared for single dose safety studies in mice and once a safe dose was defined, the elimination of the copper complex compound was determined as described in the Methods. Figure 3.4A summarizes the change in body weight of mice injected with the indicated formulation at the determined maximum tolerated dose. The formulations caused <15% body weight loss and other health status indicators suggested mild and reversible changes in animal health status. The elimination behaviors of the intravenously injected compounds were dependent on the compound tested. Cu(DDC)2 was eliminated the most rapidly with 51  <10% of the injected dose remaining after 1 hr. Cu(CX-5461) exhibited the longest circulation longevity with > 30% of the injected dose remaining in circulation after 8 hr.                                                    52  Table 3. 1: Donor systems that can be used in Copper(II)-complex loading. Copper is able to form complexes with compounds containing S-Donor, O-Donor and N,O-Donor systems as well as other mixed donor systems. Examples of drugs that are described here, in addition to DDC (an S-Donor), include quercetin (Qu) (an O-Donor), clioquinol (CQ) (an N,O donor) as well as CX-5461, previously not identified as a copper complexing agent. Each was loaded into DSPC/Chol (55:45 mol ratio) liposomes prepared with 300 mM CuSO4. The loading temperature used in these examples was 25, 50, 40 and 60oC, respectively.        53   Figure 3. 4: Preliminary tolerability and plasma elimination profiles for liposomal formulations of Cu(DDC)2, Cu(CQ)2 , CuQu and CuCX-5461 after intravenous injection into CD-1 mice. Mice were injected with a single dose of 15 mg/kg Cu(DDC)2 (-●-), 30mg/kg Cu(CQ)2 (-■-), 70mg/kg CuQu (-▲-) and 50 mg/kg Cu-CX-5461 (-▼-). (A) Changes in body weight following administration of the indicated liposomal formulation where body weights were measured over 14 days after injection (n=3). (B) Preliminary plasma elimination profiles of the indicated liposomal formulations where the copper-complexed compound was measured at 1, 4, 8 and 24 hrs after administration (n=4); concentrations were measured using HPLC or AAS as described in the Methods. 3.3 Discussion The therapeutic (anti-cancer) activity of DDC (a metabolite generated after administration of DSF) is inactive in the absence of copper; further, DDC is not stable in acidic solutions leading to rapid degradation [226]. The complex formed when mixing CuSO4 and DDC is charge neutral and forms as a dark brown precipitate [271]. It was hypothesized that lipid vesicles (liposomes) could behave as “nano-scaled reaction vessels” for Cu(DDC)2-complex formation. Provided that the liposome structure remains intact, synthesized Cu(DDC)2 would remain in suspension and would be suitable for intravenous administration. This proved to be the case as illustrated by the results in Figure 3.2 and 3.4. While the cryo-TEM images do not confirm the formation of an electron dense precipitate inside the liposomes it does not rule out the possibility that such a precipitate exists. To confirm that Cu(DDC)2 was being formed in the liposome and not as a nano-precipitate, fractionated column chromatography (data not shown) was performed and shows that Cu(DDC)2 does elute with the liposome fraction. In aggregate, these data confirm the formation of Cu(DDC)2 inside liposomes.  54  The process described here is distinct from other liposomal formulation methods which rely on compounds considered somewhat soluble in aqueous solution (>1mg/mL). Comparisons may be drawn to pH gradient loading methods [13, 14] or comparable methods relying on use of encapsulated ammonium sulfate [19, 244] or encapsulated ions and an added ionophore designed to generate a pH directly [242] or indirectly through formation of a transmembrane potential [272]. In these previous examples, the agents formulated into the liposomes have protonizable amine functions and encapsulation and/or drug retention is influenced by the presence of the transmembrane pH gradient (acidic interior) and/or the presence of residual liposome associated metal. The method described here works for compounds that do not have protonizable amine functions and are sparingly soluble in water. In fact, changing the pH from 7.4 to 3.5 showed no effect on the ability for DDC to complex copper trapped in the lipid vesicles. For the Cu(DDC)2 formulation, evidence shows that the amount of Cu(DDC)2 formed is directly related to the amount of encapsulated copper (see Fig. 3.3). For DDC, one copper molecule binds two DDC molecules to form Cu(DDC)2. This appears to be the case of CQ as well. Whereas, Qu or CX-5461 complexation appears to be defined at a 1 to 1 ratio. Future studies will further characterize what factors influence the nature of the copper complex formed within the liposome.  The rate of copper-complex formation is likely dependent on the rate at which the externally added compound crosses the liposomal lipid bilayer, which in the examples here were prepared from DSPC/Chol. These formulations are generally considered stable and exhibit reduced permeability at physiological temperatures. Formation of Cu(DDC)2 is rapid, occurring in minutes, while the reaction between CX-5461 and lipid vesicle entrapped copper is more gradual. The rate of loading may, however, be dependent on several factors, including temperature, divalent metal ion, the counter ion, the ionic strength, pH etc. Using Qu as an example, this compound is added as a powder to the pre-formed copper containing liposomes. The amount of Qu in free solution, albeit low, will increase with increasing temperature. Solubilized Qu will be free to move across the liposomal lipid bilayer (from the outside to the inside), and the permeability of Qu across the membrane will be lipid composition and temperature dependent. Once inside the 55  liposome, Qu will complex with copper, creating a complex that has reduced solubility and reduce membrane permeability.  The preliminary data provided in Figure 3.4 demonstrate that the rate of release of the CDCs is dependent on the agent used. Each CDC formulation is unique and will be examined more specifically in ensuing publications focusing on in vitro and in vivo behaviour, including stability of the CDC as well as the retention of the active agents. Cu(DDC)2 is released rapidly from the liposomes following i.v. administration of the formulations as evidenced by the rapid elimination of Cu(DDC)2. The rate of elimination of Cu-complexed CX-5461 is considerably slower. Although not shown, it is important to note that both copper and complexed agent are released when administering the Cu(DDC)2 and  Cu(CQ)2 formulations. In contrast, it appears that only Qu and CX-5461 are released from the CuQu and CuCX-5461 formulations in a form that is not complexed to copper. This has only been established in lipid vesicles prepared from DSPC/Chol (55:45, mole ratio) and further study into the effects of lipid composition and copper salt will be performed.  By formulating complexes through an inorganic synthesis reaction occurring within the liposomal core, one can obtain high drug-to-lipid ratios that are dependent on the number of copper ions inside and the nature of the complex formed. The formulations prepared this way appear stable; all formulations described here were stable with respect to particle size, polydispersity and complex to liposomal lipid ratio for at least 21 days at 4oC (results not shown). The method is scalable and suitable for manufacturing a pharmaceutical product. The inability to test poorly soluble complexes is a problem that has limited the ability of promising copper-based therapeutics to transition from the bench to the clinic. There are studies that show efficacy in tumour models using solubilising agents that have been formulated with other strategies like very low pH or Cremphor/DMSO/Ethanol mixtures [273-275]; solutions that are not particularly suitable for human use.  3.4 Conclusion This method provides a simple, transformative solution enabling, for the first time, the development of CDCs as viable candidate anti-cancer drugs; drugs that would represent a brand new class of therapeutics for cancer patients. The focus of this method 56  development was on the synthesis of Cu(DDC)2 in lipid vesicles and the resulting product is the first injectable Cu(DDC)2 formulation described in the literature. The process has been extended to other compounds including CQ, Qu and CX-5461. In each case the candidate CDC formulation appears stable and addresses problems related to the solubility for each agent. This allowed for the preclinical assessment of these CDCs and it will be expanded to other agents.           57  4. Development and Optimization of an Injectable Formulation of Copper Diethyldithiocarbamate, an Active Anti-cancer Agent* 4.1 Introduction  DSF is an FDA approved drug for the treatment of alcoholism. It functions to inhibit acetaldehyde dehydrogenase 1 (ALDH1) which in turn produces an acute sensitivity to alcohol via the uninhibited production of acetaldehyde [118]. DSF has recently become of interest in the treatment of HIV and cancer through independent mechanisms [116, 258-261]. This has sparked an interest in repurposing DSF for indications other than alcoholism and this chapter focuses on the potential repurposing of this drug or its metabolites for treating cancer.  A high-throughput chemical screen demonstrated that DSF was active against glioma stem cells; an activity that was substantially increased in the presence of Cu(II) [277]. These results have been reported by others with a series of in vitro experiments highlighting this combinatorial effect [116, 118, 277, 278]. Although some publications have speculated that DSF and Cu form a complex [116, 279, 280], studies in vitro and in vivo show DSF is reduced to form a Cu binding ligand [120, 281]. Since DSF has been used clinically for over 60 years, the pharmacokinetic properties of the drug have been explored in depth [268]. There have been reports of the formation of a copper complex in the stomach as a result of DSF conversion to DDC, a well-known copper chelator [262-264, 282, 283]. Interestingly, Cu(DDC)2  shows the same cytotoxic activity against gliomablastoma cells as when disulfiram and copper are administered concurrently. This suggests clearly that the copper complex of DDC is the therapeutically active agent [66, 115]. Cvek et al. have shown that Cu(DDC)2  acts as a proteosome inhibitor [42], specifically through                                                         * Adapted from: M. Wehbe, M. Anantha, M. Shi, A. Leung, W.H. Dragowska, L. Sanche, M.B. Bally, Development and optimization of an injectable formulation of copper diethyldithiocarbamate, an active anticancer agent, Int J Nanomed, (2017) In Press[276] M. Wehbe, M. Anantha, M. Shi, A. Leung, W.H. Dragowska, L. Sanche, M.B. Bally, Development and Optimization of an Injectable Formulation of Copper Diethyldithiocarbamate, an Active Anticancer Agent, International Journal of Nanomedicine, (2017) In Press. 58  binding to the 19S lid of the proteosome rather than the 20S subunit, which is targeted by bortezomib [184].  Prior to developing any therapeutic application for Cu(DDC)2, there are formulation issues that need to be addressed if its use in vivo is to be evaluated. Specifically, the complex is a slightly soluble precipitate (<0.1 mg/mL)[66] and, to date, there has not been any pharmaceutically appropriate formulations of Cu(DDC)2 suitable for in vivo use. In Chapter 3, a method wherein the copper-complexation reaction occurs inside copper-containing liposomes was described which solves the solubility challenges [66]. This Chapter examines, for the first time, the anti-cancer activity of the resultant Cu(DDC)2 formulation. This method allows for the development of a parenterally suitable Cu(DDC)2 formulation and can take advantage of the well-established potential for liposomes to modulate the pharmacokinetic characteristics of an associated drug candidate. These Cu(DDC)2 formulations are the first of their kind to be used to assess the therapeutic potential of this interesting Cu complex.       4.2 Results 4.2.1 Cu(DDC)2 Characterization A general method for preparing metal complexed drug candidates has been described in Chapter 3 [66]. One of the examples included the use of DSPC/Chol (55:45) liposomes, prepared in 300mM copper sulfate, as nano-scale reaction vessels for the synthesis of Cu(DDC)2. This process solved solubility issues because the highly insoluble complex was formed when the ligand was added to the outside of copper-containing liposomes. This synthesis reaction (Figure 4.1A) was completed at 25oC within 5 mins and was easily detected by eye as the absorbance of the solution changed to exhibit a broad peak around 435nm. The resultant product appeared stable, as the reaction could be carried out for 1 hr with no significant change in the Cu(DDC)2 to lipid ratio (Figure 4.1B). The Cu(DDC)2 formulation showed negligible dissociation from the liposomes in vitro when the samples were incubated at 37oC over 72 hrs in a solution containing 50% FBS in SH buffer (pH 7.4) (Figure 4.1C). The in vitro activity of this novel formulation of Cu(DDC)2 was compared to the complex prepared in the absence of liposomes and dissolved in DMSO by 59  generating dose response curves against M-V-411 leukemia cells (Figure 4.1D). The 72 hour IC50 determined for both formulations were identical (~0.11 µM), however in contrast to the DMSO solubilized Cu(DDC)2 there was no evidence of precipitates in the incubation wells when Cu(DDC)2 was added as the liposomal formulation.   Figure 4. 1: Characterization of Cu(DDC)2 prepared in a liposomal formulation (DSPC/Chol (55:45)) wherein the Cu(DDC)2 was synthesized inside the copper containing liposomes. (A) Schematic representation of Cu(DDC)2 complex formation inside copper liposomes  upon addition of DDC. Cu-liposomes and Cu(DDC)2-liposomes were scanned on a UV-VIS, the formation of Cu(DDC)2 results in a peak at 435 nm indicative of complex formation. (B) Formation of Cu(DDC)2 inside DSPC/Chol liposomes (final liposomal lipid concentration was 20 mM) as a function of time over 1 hr at 25oC following addition of DDC to the liposomes at a final DDC to lipid ratio of 0.4. (C) in vitro Cu(DDC)2 release from DSPC/Chol (55:45) liposomes over a 72 hr time course in the presence of 50% FBS at 37oC. Cu(DDC)2 was measured using a UV-Vis spectrophotometric assay (see Methods) and lipid was measured through use of a radioactive lipid marker (3H-CHE). (D) M-V-411 cytotoxicity curves for Cu(DDC)2 dissolved in DMSO (■) and the Cu(DDC)2  liposomal preparation (●) where cell viability was measured (using PrestoBlue®) following a 72 hr exposure to the added Cu(DDC)2. Data are presented as mean ± SEM of 3 experiments. If error bars are not seen then they are within the size of the symbol used.    60  4.2.2 Studies to Assess the Mechanism of Cu(DDC)2 Activity Previous publications [42, 284], indicated that the mechanism of DDC cytotoxicity was mediated, at least in part, through proteosome inhibition. Proteosome inhibitors, such as the FDA approved Bortezomib, have been used for the treatment of acute myeloid leukemia [285]. For this reason, an acute myeloid leukemia M-V-411 cell line was used to characterize the cytotoxicity of Cu(DDC)2 prepared within DSPC/Chol liposomes. The IC50 determined using PrestoBlue (see Chapter 2) of Cu(DDC)2 at 24 and 72 hrs was 0.42 and 0.11 µM, respectively (Figure 4.2A - B). To determine whether Cu(DDC)2 inhibits the proteosome, MV-4-11 cells were treated with vehicle, CuSO4 or Cu(DDC)2 and the accumulation of ubiquitinylated proteins, a marker of proteosome inhibition, was subsequently measured via western blotting. Only the Cu(DDC)2 treated groups showed marked accumulation of ubiquinylated protein relative to the controls (Figure 4.2C). To further investigate mechanisms involved in Cu(DDC)2 cytotoxicity, cell cycle analysis was performed using flow cytometry (see Chapter 2). The results showed no significant changes in the cell cycle after 24 hours of Cu(DDC)2 exposure. However, Cu(DDC)2 caused an increase in the sub G0/G1 phase indicative of cell death (Figure 4.2D). Since some published reports have suggested that Cu(DDC)2 treatment leads to production of  reactive oxygen species (ROS) [184], Cu(DDC)2 was evaluated to examine if cell death was a result of this mechanism. Cells were treated for 4 hrs with Cu(DDC)2 or menadione (positive control). The results showed that Cu(DDC)2 treatment did not result in ROS generation (Figure 4.2E), suggesting that it is unlikely to be an important mechanism of cytotoxicity  for the formulation described here.   61   62  Figure 4. 2: Cu(DDC)2 acts primarily as a proteosome inhibitor and induces cell death in MV-4-11 cells. (A) Cytotoxicity curves generated when MV-4-11 cells are exposed to Cu(DDC)2 (prepared inside DSPC/Chol Liposomes) for either 24 (■) or 72 (●) hrs where viability was measured using PrestoBlue®. (B) The IC50 values of MV-4-11 cells treated with Cu(DDC)2 for 24 and 72 hrs. The proteasome inhibition activity was determined as described in the Methods using MV-4-11 cells treated with the indicated doses of CuSO4 or Cu(DDC)2 (prepared inside DSPC/Chol Liposomes) for 24 h. (C) Proteasome inhibition resulted in accumulation of ubiquitinated proteins presented as long dark bands on the Western blot following Cu(DDC)2 treatment (150 and 300nM) but not for vehicle or CuSO4 (300-600nM). (D) Cell cycle analyses was completed using MV-4-11 cells treated with  CuSO4, DDC, or Cu(DDC)2  for 24 hrs and the results indicated no significant change in the cell cycle upon Cu(DDC)2  exposure. There was an increase in the subG0/G1 fraction (marked with horizontal bar) when cells were treated with Cu(DDC)2, indicative of cell death as evident by DNA fragmentation. (E) ROS formation was tested in MV-4-11 cells treated with Cu(DDC)2 (prepared inside DSPC/Chol Liposomes; where ROS formation was measured 4 hrs following initiation of treatment. Cu(DDC)2 treatment did not induce ROS formation. Menadione was used as a positive control and ROS formation in the cells was evident by a statistically significant difference in luminescence relative to the corresponding cell-free condition. Data are presented as mean ± SEM of 3 experiments.  4.2.3 Plasma Elimination of Cu(DDC)2 Prepared in DSPC/Chol (55:45) Liposomes The Cu(DDC)2 formulation prepared as described above was suitable for i.v. administration. To assess Cu(DDC)2 elimination from plasma, mice were given a single i.v. dose (15 mg/kg) of Cu(DDC)2 and plasma samples were collected as described in Chapter 2. Attempts to measure Cu(DDC)2 in the plasma compartment were, however, unsuccessful even when using a 15min. time point. The Cu(DDC)2 levels were below the detection limits of the HPLC assay (0.09 µg/mL). Plasma copper levels were measurable by AAS and in Cu(DDC)2 treated mice these levels were above the level of copper determined in plasma collected from untreated mice. For this reason, plasma copper levels (after subtraction of control plasma copper levels) were used as a surrogate marker of Cu(DDC)2. The results, summarized in Figure 4.3,  indicate that the percent of the injected copper dose remaining in plasma was 5.2% at 15 min., indicative of 95% eliminations of the injected Cu(DDC)2 (Fig. 3A; filled circles). This value decreased to 0.25% of the injected dose 1 hr after administration. In contrast, 15 minutes after administration of the formulation, 93% of the injected liposomal lipid dose was still in the plasma compartment; a value that decreased to 80% 1hr following administration (Fig. 4.3A, open circles). The liposomal lipid elimination rate was comparable to that described previously for DSPC/Chol liposomes [286].  These results suggest that Cu(DDC)2 prepared inside DSPC/Chol liposomes does not remain inside the liposomes following i.v. dosing. This result was surprising given the 63  stability of the formulation as determined using in vitro methods (see Figure 4.1). Loss of associated Cu(DDC)2 from the liposomes after administration is highlighted by the copper to lipid ratio data (Fig. 4.2B). Prior to injection, this ratio was 0.25 (time 0), but it drops to 0.02 after 15 minutes. This is indicative of 92% loss of Cu(DDC)2 from the liposome within 15 minutes after injection. In contrast, 1 hr after administration of uncomplexed CuSO4 containing DSPC/Chol liposomes, the copper to lipid ratio suggested >90% retention of the encapsulated copper. This is indicative of Cu(DDC)2 being released from the liposome as the copper complex whereas uncomplexed Cu2+ ions are, as expected, retained in the liposome owing to their charge. It can be suggested that the DSPC/Chol liposomal formulation of Cu(DDC)2 addresses the solubility challenges of this compound, but because the complex is released rapidly from the liposomes following administration it should be noted that the liposomes are not behaving as drug delivery vehicles.   Figure 4. 3: Cu(DDC)2 (prepared in DSPC/Chol (55/45) liposomes) elimination after intravenous injection in CD-1 mice. (A) The percent injected dose of copper (-●-) was measured using AAS as a surrogate for Cu(DDC)2. The percent injected dose of liposomal lipid (-○-) was measured by assessing 3H-CHE as a non-exchangeable, non-metabolizable liposomal lipid marker (see Methods). (B) The decrease in copper-to-lipid ratio suggests that the Cu(DDC)2   prepared inside the liposome dissociated from the liposomes within 15 mins. All data are plotted as mean ± SEM (n=4 per time point), if the error bars are not visible then the error bars are within the size of the symbol used. 4.2.4 Cu(DDC)2 Efficacy in Models of Cancer The results thus far demonstrate that. Cu(DDC)2 synthesized in liposomes remain in solution in a form that could be safely administered i.v. The potential for using the resultant product as an anti-cancer drug was measured in two different rodent models of cancer and 64  the results are summarized in Figure 4.4. The activity of Cu(DDC)2 (prepared in DSPC/Chol liposomes) was measured in mice with established sc tumours models generated following sc injection of the MV-4-11 cells (Fig. 4.4A). Prior to assessing activity, the MTD of Cu(DDC)2 was determine in tumour free CD-1 mice. Given the rapid elimination of Cu(DDC)2 following i.v. injection (Fig. 4.3) a dose intensive schedule was used where the animals were injected iv on Monday, Wednesday and Friday for 2 weeks (M,W,F x 2). Using this schedule the MTD of Cu(DDC)2 was determined to be 8 mg/kg. At doses greater than 8 mg/kg (e.g. 15 mg/kg) the animals were terminated at humane endpoints (5 days following initial treatment). On necropsy, these animals showed no obvious changes in tissue or organ appearance other than the occasional enlarged spleen and discolouration of the liver and kidney. It should be noted that extravasation injuries (at the site of injection) were observed with some mice. If the extravasation injury was considered severe, then the mouse was terminated for ethical reasons and that animal was excluded from the treatment group. For animals with established MV-4-11 sc tumours, dosing began on day 12 when mice were injected (i.v.) with vehicle (SH buffer), DSPC/Chol (55:45)-copper sulfate liposomes (1.3 mg copper/kg) or Cu(DDC)2 prepared in DSPC/Chol liposomes (8 mg Cu(DDC)2 /kg, copper dose 1.3 mg/kg), respectively. The liposomal lipid dose was ~50 mg/kg. The results (Fig. 4.4A) suggest that tumour growth was comparable in animals treated with the vehicle and the copper containing liposome control. The Cu(DDC)2  (prepared in DSPC/Chol liposomes) treated animals exhibited a delay in tumour growth rate. Animals in this group showed a 45% reduction in tumour volume when compared to the vehicle and copper treated animals on day 41. Although there was a treatment engendered delay in tumour growth when using the MV-4-11 sc tumour model, the results suggested that Cu(DDC)2  activity was unremarkable on the basis of RECIST criteria [287] where notable activity is reflected by stable disease, a partial response or a complete response. For this reason, Cu(DDC)2  (prepared in DSPC/Chol liposomes) was tested in an orthotopic F98 (rat glioblastoma) model, where treatment was administered directly into the site of tumour inoculation by CED [251]  The glioblastoma line was of interest because previous publications suggested that DSF and its metabolite DDC in the presence of copper were particularly active against glioblastoma cells [116, 288]. The in vivo efficacy of Cu(DDC)2 (prepared in DSPC/Chol liposomes) was evaluated in the F89 rat glioma model 65  where each rat was injected at the site of tumour cell inoculation with a 0.5 mg/mL solution of Cu(DDC)2, the vehicle or copper containing liposomes (0.08 mg/mL) (see Methods). This was defined as the MTD of the Cu(DDC)2 formulation when administered by CED methods. In these studies, control animals exhibited a median survival time (MST) of 20.5 and 19.5 days, respectively (Fig. 4.4B). In animals treated with Cu(DDC)2, the MST increased to  25 days (a 25% increase in MST). The difference between the MST of those animals treated with the controls and Cu(DDC)2 was statistically significant (p< 0.05) and comparable to previous studies demonstrating a 11.3% increase in MST when the F98 glioblastoma model was treated with lipoplatin (a liposomal formulation of cisplatin) [251].    Figure 4. 4: Cu(DDC)2 efficacy studies. (A) MV-4-11 cells (1 x 106) were inoculated subcutaneously (sc) into RAG-2M mice and the tumours were treated intravenously with vehicle (SH buffer, ●), copper containing liposomes (1.3 mg/kg, ■) or Cu(DDC)2 (8 mg/kg, ▲) formulated in DSPC/Chol liposomes using a M,W,F x 2 dosing schedule. Tumour volumes were measured three times a week and are plotted as mean ± SEM (up to 9 mice per group). As shown, there was a significant delay in tumour growth when comparing tumour sizes in animals that were treated with Cu(DDC)2  to those treated with the vehicle control. “*” indicates statistically significant difference (p<0.05) upon statistical analysis using a 2-way ANOVA followed by Tukey adjustments to correct for multiple comparisons. (B) The activity of Cu(DDC)2 (prepared in DSPC/Chol liposomes) was evaluated following CED of Cu(DDC)2 in a rat glioma model wherein 10,000 F98 cells were implanted intracranially into the right caudate nucleus (see Methods). A single 10 μL injection of vehicle (SH buffer, ∙∙∙∙∙), copper liposomes (0.08 mg/mL, ‒ ‒) or Cu(DDC)2 (0.5 mg/mL, ──) was injected 10 days after F98 implantation (n=9). Survival study statistical analysis was performed using the log rank test with GraphPad Prism 6. A P-value under 0.05 was considered statistically significant.   66  4.2.5 Enhancing the Circulation Longevity of Cu(DDC)2 Although the results presented thus far suggest that the Cu(DDC)2 formulation prepared in DSPC/Chol liposomes is therapeutically active, a significant advantage of the technology used here to create the first injectable Cu(DDC)2 formulation is that the synthesis reaction to form Cu(DDC)2 can be completed in liposomes of different lipid compositions and the environment within the liposome can be controlled. Since it is possible that the efficacy of Cu(DDC)2 may be enhanced through strategies that enhance its circulation lifetime, a number of strategies were pursued to gain an understanding of the factors influencing Cu(DDC)2  retention in the liposomes. Since the in vitro Cu(DDC)2  assay measuring the stability of the Cu(DDC)2  formulation proved to be a poor predictor of in vivo stability, thus, an in vivo assay was utilized where plasma levels of copper or Cu(DDC)2 were measured 30 minutes after i.v. administration into mice. This single time point assay allowed us to assess how changes in the formulation impacted the stability of the injected Cu(DDC)2 formulation. These results are summarized in Figures 4.5 and 4.6. First Cu(DDC)2  was synthesized in DSPC/Chol (55:45) liposomes which were prepared to contain copper sulfate or copper gluconate and, in addition, the Cu(DDC)2  to lipid ratios were varied in these formulations to determine if the amount of entrapped Cu(DDC)2  affected drug retention. As noted in Fig. 4.5A, the synthesis of Cu(DDC)2 inside the DSPC/Chol liposomes was similar when the liposomes were prepared with copper gluconate or copper sulfate. When evaluating the effect of copper salt on the plasma levels of Cu(DDC)2  30 minutes after injection (Fig.4.5B, first and fourth bar) it can be concluded that use of copper gluconate had no impact on Cu(DDC)2 elimination. These Cu(DDC)2  formulations were also prepared such that selected formulations had lower Cu(DDC)2to lipid ratios (Fig. 5B, 2nd, 3rd, and 5th column). These formulations exhibited plasma copper levels comparable to the formulations with the higher Cu(DDC)2  to lipid ratios. The elimination of liposomal lipid was unaffected by the formulation changes described above. When preparing the Cu(DDC)2  formulation using 300mM copper sulfate, it is assumed that the internal pH of the liposome is 3.5 (the pH of the copper solution used when making the liposomes). To determine whether the pH within the liposome effects Cu(DDC)2 elimination the monvalent K+ ion ionophore nigericin was added prior to 67  Cu(DDC)2 synthesis inside the liposomes. Nigericin facilitates the exchange of K+ ions in exchange for H+. Thus, CuSO4-liposomes were exchanged into a KCl-histidine containing buffer (see Methods) and subsequently nigericin was added. As noted in Fig. 4.5C, the formation of Cu(DDC)2 inside the DSPC/Chol liposomes was not affected by the change in external buffer or the addition of nigericin. Following i.v. administration into mice, the plasma copper levels 30 minutes after administration were comparable for the nigericin formulation and the formulation prepared without nigericin (Fig. 4.5D). This suggests that an increase in pH within the liposomes has no impact on the stability of the injected formulation.  To assess how liposomal lipid composition influences the in vivo elimination of i.v. injected Cu(DDC)2, formulations prepared in: (i) cholesterol containing liposomes DSPC/Chol (55:45) and SM/Chol (55:45)), (ii) in cholesterol-free liposomes (DSPC/DSPE-PEG2000 (95:5) and SM/DSPE-PEG2000 (95:5)) as well as (iii) charged liposomes (anionic (DSPC/DSPG/Chol (70:20:10) and cationic (DSPC/DC-Chol/DSPE-PEG2000 (30:50:20)) were evaluated. These studies (summarized in Fig. 4.6) used plasma Cu(DDC)2 levels determined 30 minutes following i.v. administration as a measure of whether changes in lipid composition could engender decreases in Cu(DDC)2  elimination. Although not shown, the ability to synthesize Cu(DDC)2 in the different liposomal formulations was not affected by liposomal lipid composition. As indicated in Fig. 6 (3rd and 6th bar) the only formulations which exhibited significantly higher levels of Cu(DDC)2 in the plasma when compared to Cu(DDC)2 formulated in DSPC/Chol liposomes were the DSPC/DSPE-PEG2000  and DSPC/DSPG/Chol formulations. These retained 20.8% and 11.3% of the injected copper dose in the plasma compartment at 30 min, respectively. The DSPC/DSPE-PEG2000 formulation showed the highest plasma copper levels ; a nearly 7-fold increase over that measured for animals given Cu(DDC)2 formulated in DSPC/Chol liposomes. For this reason, the remaining studies focus on Cu(DDC)2 formulations prepared in the DSPC/DSPE-PEG2000 liposomes.   68   Figure 4. 5: Examining the role of factors within the DSPC/Chol liposomes that may affect Cu(DDC)2 levels in the plasma compartment 30 min after administration. (A) Preparation of Cu(DDC)2 inside DSPC/Chol (55:45) liposomes containing either CuSO4 (●) or Cu-Gluconate (■) as a function of time at 25oC in SH Buffer. (B) The percent of injected Cu(DDC)2 dose administered i.v. to CD-1 mice (n=4) remaining in the plasma 30 minutes after injection. The formulations were prepared at different Cu(DDC)2 to lipid ratios (0.2, 0.1 and 0.05, mole: mole) and prepared using liposomes with encapsulated CuSO4 or Cu-Gluconate buffers (see Methods). Copper levels were measured by AAS and, after subtraction of background plasma copper levels, these levels were used as a surrogate for Cu(DDC)2. (C) Formation of Cu(DDC)2 inside DSPC/Chol (55:45) liposomes containing CuSO4 with (■) or without (●) Nigericin  as a function of time at 25oC. The external buffer for these liposomes was KCl (150mM) and histidine (20mM) (see Methods). (D) The percent injected dose of Cu(DDC)2 injected into CD-1 mice (n=4) remaining after 30 minutes following i.v. administration of Cu(DDC)2  prepared in DSPC/Chol liposomes in the presence and absence of Nigericin. Copper levels were measured by AAS and, after subtraction of background plasma copper levels, these levels were used as a surrogate for Cu(DDC)2. For panels A and C, n= 3 replicate experiments. In panels A and C if the error bars are not visible then the error is within the size of the symbol used. For Panels B and D, n= 4 mice per group. All data are plotted as mean ± SEM  69   Figure 4. 6: Examining the role of lipid composition on Cu(DDC)2 levels in the plasma compartment 30 min after administration. Liposomes composed of DSPC/Chol (55:45), SM/Chol (55:45), DSPC/DSPE-PEG2000 (95:5), SM/DSPE-PEG2000 (95:5), DSPC/DC-Chol/DSPE-PEG2000 (30:50:20) and DSPC/DSPG/Chol (70:20:10) were prepared in unbuffered 300mM CuSO4. Cu(DDC)2 was prepared in these liposomes to achieve a final Cu(DDC)2  to lipid ratios of 0.2 (mol:mol) (see Methods) and subsequently injected i.v. into CD-1 mice (n=4) at 15 mg/kg. The plasma levels of Cu(DDC)2 were measured 30 minutes after administration. Copper levels were measured by AAS and, after subtraction of background plasma copper levels, these levels were used as a surrogate for Cu(DDC)2. “*” indicates statistically significant difference (p>0.05) when Cu(DDC)2  levels in the plasma were compared to the levels obtained after injecting Cu(DDC)2  in DSPC/Chol liposomes (first column) . Significance was determined by a one-way ANOVA followed by Tukey's post-hoc test. For each lipid composition, n= 4 mice per group. All data are plotted as mean ± SEM. 4.2.6 Characterizing Cu(DDC)2 Prepared in DSPC/DSPE-PEG2000 (95:5) Liposomes To generate the data shown in Fig. 4.6, the ability to create Cu(DDC)2 in liposomes prepared of different lipid compositions needed to be determined. The formation of Cu(DDC)2 in DSPC/DSPE-PEG2000 (95:5) liposomes is illustrated by the data shown in Fig. 4.7. When incubated at 25oC, a Cu(DDC)2 to lipid ratio of 0.2 (molar ratio) is achieved within 10 minutes following addition of DDC to the copper-containing liposomes (Fig.4.7A, filled squares). DDC enters the liposome through passive diffusion across the liposomal 70  lipid bilayer. Since DDC is a relatively small molecule (171 g/mol) it can cross the lipid bilayer rapidly, even when the incubation temperature was reduced to 4oC. It should be noted that the rate of Cu(DDC)2 formation at 4oC in the DSPC/DSPE-PEG2000 liposomes was slower than that observed in DSPC/Chol liposomes. This is reflected by the data summarized in Fig. 4.7B where the Cu(DDC)2 to lipid ratio for DSPC/Chol and DSPC/DSPE-PEG2000 liposomes measure after a 1 hr incubation at 4oC (filled bars) suggests that 80% and 50% of the encapsulated Cu is becoming complexed with DDC over the 60 minute incubation time frame, respectively. Cryo-electron microscopy was performed on the DSPC/DSPE-PEG2000 liposomes before and after Cu(DDC)2 synthesis. As illustrated in Fig. 4.7C, there is no visible difference between the liposomes with encapsulated copper sulfate and those with encapsulated Cu(DDC)2.  Following i.v. administration of Cu(DDC)2 formulated in the DSPC/DSPE-PEG2000 liposomes, there was about 70% of the injected lipid dose in the plasma compartment at 4 hrs. In contrast, only 5% of the injected Cu(DDC)2 was detected (Fig. 4.7D). This is reflected in the change in  the Cu(DDC)2 to lipid ratio (molar) which indicates that there was 90% loss of the Cu(DDC)2 from the DSPC/DSPE-PEG2000 liposomes within 4 hrs after injection (see Fig. 4.7E). Although Cu(DDC)2 was still eliminated rapidly from the plasma compartment following injection of the formulation prepared in DSPC/DSPE-PEG2000 liposomes, this formulation retained Cu(DDC)2 significantly better than the DSPC/Chol liposomes. The plasma AUC(0-∞) for  Cu(DDC)2  inside DSPC/Chol (55:45) liposomes was found to be 4.6 µg•hr/mL while that of Cu(DDC)2 inside of DSPC/DSPE-PEG2000 (95:5) liposomes was found to 19.3 ug•hr/mL, representing a 4.2 fold increase in AUC(0-∞).   71   Figure 4. 7: Characterizing Cu(DDC)2 prepared inside DSPC/DSPE-PEG2000 (95:5) liposomes. (A) DSPC/DSPE-PEG2000 liposomes prepared in 300mM unbuffered CuSO4 were incubated at 4°C (●) or 25oC (■) with DDC (see Methods) and the amount of Cu(DDC)2  formed as a function of time was measured. (B) The Cu(DDC)2 to lipid ratio measured after a 1 hr incubation following addition of DDC to CuSO4 containing  DSPC/Chol  or DSPC/DSPE-PEG2000  liposomes after an incubation at 4°C (filled bar) or 25°C (empty bar). (C) Cryo-electron microscopy photomicrograph of CuSO4- containing DSPC/DSPE-PEG2000  liposomes and Cu(DDC)2 prepared in the same liposomes. (D) Cu(DDC)2 prepared in DSPC/DSPE-PEG2000 liposomes were injected into CD-1 mice (n=4) at 15 mg/kg and mice were sacrificed at 0.5, 1 and 4 hrs. Plasma concentration of Cu(DDC)2 and liposomal lipid were determined (see Methods) and the % injected dose of Cu(DDC)2 (■) and liposomal lipid (□) was calculated at the indicated time points. (E) Cu(DDC)2  to lipid ratios calculated based on the data provided in panel D. All data are plotted as mean ± SEM; n= 3 replicate experiments for panel A/B and n= 4 mice per group in panel D and E. 4.2.7 Therapeutic Activity of Cu(DDC)2 Prepared in DSPC/DSPE-PEG2000 (95:5) Liposomes To determine if the 4.2-fold increase in AUC(0-∞) achieved when using Cu(DDC)2 formulated in DSPC/DSPE-PEG2000 affected therapeutic activity, mice with established MV-4-11 were treated with this Cu(DDC)2 formulation and the activity was compared to animals treated with Cu(DDC)2 prepared in DSPC/Chol liposomes. As indicated above, the MTD of Cu(DDC)2 prepared in DSPC/Chol liposomes was determined to be 8 mg/kg when using the M,W,F x 2 schedule. Cu(DDC)2 prepared in DSPC/DSPE-PEG2000 liposomes was better tolerated and the MTD of this formulation was determined to be 12.5 mg/kg when given via the M, W, Fx2 schedule. To assess efficacy, Cu(DDC)2 prepared in DSPC/DSPE-PEG2000 liposomes was administered (M, W, F x2 schedule) at 8 mg/kg and 12.5 mg/kg. The 72  injected liposomal lipid doses were 46 mg/kg and 63 mg/kg, respectively. When Cu(DDC)2 was prepared in DSPC/Chol liposomes, the dose of Cu(DDC)2 was 8 mg/kg and the liposomal lipid dose was 50mg/kg. Mice bearing MV-4-11 tumours were treated 12 days after tumour cell inoculation and tumour growth was monitored over time. The results, summarized in Figure 4.8, indicated that all Cu(DDC)2 treated animals showed a statistically significant decrease in tumour volume when compared to vehicle treated animals (Fig.4.8A). When comparing tumour size on day 33, the day when some control mice were euthanized due to large tumour sizes (the animal care protocol specifies the humane endpoint for animals with sc tumours is when the tumour volume is greater than 800 mm3), animals treated with Cu(DDC)2  prepared in DSPC/Chol liposomes had tumours that were 50% the size of control animals. At the same dose of Cu(DDC)2  prepared in DSPC/DSPE-PEG2000 liposomes the tumour size was only 28% smaller than the tumours from vehicle treated mice; suggesting that at equivalent doses, the DSPC/DSPE-PEG2000 formulation was less effective. However, when efficacy was compared at the MTD of each formulation, the antitumour activity was equivalent (Fig. 4.8B). Both formulations were able to delay progression of tumour growth in this model, but further formulation changes will be required to optimize Cu(DDC)2 activity.  Figure 4. 8: Cu(DDC)2 anti-tumour activity determined in mice bearing established sc MV-4-11 tumours. 1 x 106 MV-4-11 cells were inoculated sc into RAG-2M mice (see Methods) and were treated intravenously with vehicle (-●-), copper containing DSPC/DSPE-PEG2000 (95:5) liposomes (-○-,1.7 mg/kg copper, 63 mg/kg lipid ), 8 mg/kg Cu(DDC)2 prepared in DSPC/Chol liposomes (-▲-, 8 mg/kg Cu(DDC)2  and 50 mg/kg lipid), 8 mg/kg Cu(DDC)2  prepared in DSPC/DSPE-PEG2000 liposomes (-□-, 8 mg/kg Cu(DDC)2 , 40 mg/kg lipid) or  12.5 mg/kg Cu(DDC)2 prepared in DSPC/DSPE-PEG2000 liposomes (-■-,12.5 mg/kg Cu(DDC)2 , 73  63 mg/kg lipid)  using a M,W,F x 2 schedule (see Methods). (A) Tumour volumes were measured three times a week (see Methods) and were plotted as mean ± SEM. (B) Mean tumour volume ± SEM determined on day 33 (the humane endpoint for vehicle treated animals). “*” indicates statistically significant difference p>0.05; one-way ANOVA followed by Tukey's post hoc test.  4.3 Discussion DSF and Cu have been the focus of many oncology focused research programs and clinical trials because of the surprising anti-cancer activity observed when the two components are given together in vitro [116, 118, 289]. This discovery exemplified an opportunity to repurpose DSF, an agent with over 60 years of clinical use in the context of alcohol addiction [268, 290, 291]. It has been previously shown by others and in Chapter 3 that the cytotoxicity of DSF and Cu is attributed to the formation of a Cu(DDC)2 complex [66, 120]. Attempts have been made to prepare the complex in vivo by administering DSF and Cu (usually given as Cu-Gluconate) separately. However, the pharmacokinetic and distribution behavior of copper and DSF are remarkably different and this has contributed to the lack of success using this approach. Thus, while the DSF/Cu combination shows remarkable activity in vitro this has not translated in vivo. To date, the therapeutic activity of the Cu(DDC)2 complex has not been directly evaluated in vivo owing to the insolubility of the complex in aqueous solutions. This challenge was solved by synthesizing Cu(DDC)2  inside liposomes [66]. This methods relies on liposomes serving as nano-scale reaction vessels to support the synthesis of Cu(DDC)2  when DDC is added to the outside of copper sulfate-containing liposomes. This is the first characterization of the anti-cancer activity of Cu(DDC)2 following i.v. administration.  Cu(DDC)2 was cytotoxic to cancer cells but did not have any effect in healthy bronchial epithelial cells in vitro.[66] While no precipitation was visible when cells were examined under a microscope it is possible that Cu(DDC)2 could exist as a micro-precipitate.  Consistent with other in vitro studies [42, 292], the primary mechanism of action for Cu(DDC)2  is through proteosome inhibition (see Figure 2). Flow cytometric studies suggested that cell cycle arrest does not contribute to Cu(DDC)2 cytotoxicity. Interestingly, DSF and copper have been reported to be effective against brain tumour initiating cells (BTICs) which are senescent [116, 288, 293]. Thus, the ability of Cu(DDC)2 to 74  be effective regardless of cell cycle stage may contribute to the drug’s ability to kill BTICs. The in vitro studies (see Figure 4.2) indicate that the activity of Cu(DDC)2 is not mediated by the generation of ROS, i.e. no increase in ROS production was observed when cells were incubated with Cu(DDC)2. Previously Tawari et al. noted an increase in ROS when DSF and copper were added to cell media but not when DDC and copper were added [115]. The authors suggested that ROS generation was a by-product produced which occured when DSF and Cu were mixed in vitro. However ROS would not be generated if using pre-formed Cu(DDC)2 complex in liposomes, as done in the studies reported here. This was done to help avoid precipitation of Cu(DDC)2 under conditions where the complex is added as a DMSO solubilised form. The in vitro results suggested that Cu(DDC)2 prepared in DSPC/Chol liposomes was available to the cells in culture, a preliminary indication that Cu(DDC)2 dissociated from the liposomes.  The in vivo studies completed with Cu(DDC)2 produced in DSPC/Chol liposomes indicated that the drug was rapidly eliminated from plasma after i.v. administration (>90% of the injected dose was eliminated with 15 minutes, see Figure 4.3). This was surprising, in part because the in vitro studies shown in Figure 4.1C suggested that the Cu(DDC)2 formulations were stable when prepared in the DSPC/Chol liposomes. The inability for in vitro release assays to predict drug release from liposomes is a widely discussed challenge in liposomology. The result seen herein could be attributed to an insufficient sink to drive Cu(DDC)2 release. Methods such as the use of “acceptor” multilamellar vesicles could be employed to better accurately predict drug release [294].  Cu(DDC)2 prepared in DSPC/Chol liposomes was therapeutically active. When administered at its MTD, the effects resulted in a delay in tumour progression. This was determined in a sc model as well as an intracranial model where Cu(DDC)2 was given by CED directly to the site of tumour cell inoculation (Figure 4.4). The therapeutic activity of Cu(DDC)2 may be limited because of its rapid elimination from the plasma compartment and this would be addressed in this model. An advantage of the technology used here is that the composition of the liposome in which the Cu(DDC)2 is formed can be changed to improve the in vivo stability of the resulting formulation. When Cu(DDC)2 was prepared in DSPC/DSPE-PEG2000 (chol-free) liposomes there was an improvement in plasma Cu(DDC)2 levels which likely equated to an improvement in Cu(DDC)2 retention within the liposome. Cu(DDC)2 prepared in 75  DSPC/DSPG/Chol liposomes also showed some improved stability in vivo. In both of these examples, it could be argued that the anionic surface charge played a role in improved stability of the formulation[269]. Alternatively, because the DSPC/DSPG/Chol liposomes contained only 10% cholesterol (a low cholesterol formulation), improved Cu(DDC)2 retention could be due to the removal of cholesterol[256]. It is important to note that an increase in drug retention was not seen in the chol free SM/DSPE-PEG2000 formulation. For Cu(DDC)2 prepared in DSPC/Chol liposomes, factors such as Cu(DDC)2 to lipid ratio, choice of entrapped copper salt and the internal liposomal pH did not appear to affect the stability of the formulation (see Figure 4.5). The Cu(DDC)2 plasma AUC(0-∞) was 4.2 fold higher for the DSPC/DSPE-PEG2000 formulation when compared to Cu(DDC)2  prepared in DSPC/Chol liposomes. Although the resulting DSPC/DSPE-PEG2000 formulation was better tolerated, its therapeutic activity was not better than the DSPC/Chol formulation (Figure 4.8). It can be concluded that the decreased plasma elimination rate affects the safety profile of the resultant formulation, but more significant improvements in the stability of the formulation will be needed to enhance the activity of Cu(DDC)2 in vivo.  The Cu(DDC)2 formulations described here do provide some therapeutic benefit, but the full therapeutic potential of Cu(DDC)2 may only be achieved through careful selection of the appropriate cancer indication and/or by using it in combination with another drug. For example, Lun et al. have demonstrated that temozolomide (TMZ) can be used in combination with DSF and copper [288]. It would be a natural transition to examine if Cu(DDC)2 could be used in combination with TMZ. Also, given a mechanism that involves proteosome inhibition, Cu(DDC)2  may be useful when combined with other drugs known to enhance the activity of known proteasome inhibitors such as BTZ. In this context, it has been shown that BTZ acts synergistically with CDK9 kinase inhibitors [295]. Cvek at al. have examined the mechanism by which Cu(DDC)2 inhibits the cellular 26S proteosome and have described that this occurs through inhibition of the JAMM domain in the 19S proteasome lid [42], a site distinct to where BTZ acts. For this reason Cu(DDC)2  may be effective when used in combination with drugs that work well with BTZ, or alternatively, Cu(DDC)2 may prove active against BTZ resistant cancers [296].  76  4.4 Conclusion This Chapter disclosed the first studies ever to evaluate the anti-cancer activity of Cu(DDC)2 prepared using a technique which synthesizes Cu(DDC)2 in the core of liposomes. This method solves problems associated with Cu(DDC)2 insolubility and allows for its direct administration. Two formulations were tested in an in vivo MV-4-11 leukemia model and both produced a ~50% reduction in tumour volume at their respective MTDs when compared to control groups. Additional studies are needed to optimize the therapeutic potential of the Cu(DDC)2 formulations described here. Additionally, it would be of particular interest to establish the activity of Cu(DDC)2 in combination with other drugs.                            77  5. Development of a Copper-Clioquinol Formulation Suitable for Intravenous Use * 5.1 Introduction Clioquinol (CQ) was commonly used as an oral antimicrobial agent for treating diarrhea and skin infections [297, 298]. However, in the 1960s its use in Japan was associated with a debilitating neurological disorder referred to as subacute myelo-optic neuropathy (SMON). This eventually led to CQ being withdrawn from the market [297, 299]. Interestingly, epidemiologic reports suggest that CQ was not responsible for SMON and no other population showed a similar adverse response [297, 298]. Today, CQ is commonly used as a topical antibiotic under the trade name Vioform®[298] and more recently this drug has been the focus of repurposing efforts for the treatment of Alzheimer’s disease[136, 300] and cancer [191, 274].  This Chapter focuses on the potential use of CQ as an anti-cancer agent. It has been noted that the anti-cancer effects of CQ are enhanced when it is administered as a copper CQ (Cu(CQ)2) complex[50, 191, 217, 274, 301]. The structure of Cu(CQ)2 has been characterized [190], however the mechanism(s) responsible for its activity have not been fully elucidated. Ding et al. have suggested that CQ may act as a copper ionophore [50, 191]. Alternatively, others have suggested that Cu(CQ)2 may act as a proteasome inhibitor, similar to the postulated mechanism of copper diethyldithiocarbamate [302, 303]. Regardless, there have been no reports assessing the activity of Cu(CQ)2 in vivo. The low aqueous solubility of this complex has hindered its development as an anti-cancer drug candidate. CQ has been tested as a single agent and its use required a mixed solvent system containing DMSO, cremophor and ethanol [274]. Due to the toxicities associated with such formulations, it has not been possible to fully assess the anti-cancer potential of Cu(CQ)2 [64].                                                          * Adapted from: M. Wehbe, A. Malhotra, M. Anantha, C. Lo, N. Dos Santos and M.B. Bally. Repurposing the topical antifungal clioquinol for the treatment of cancer. (2017) Submitted to Drug delivery and translational research 78  The goals of the current study were: (i) to develop and characterize a Cu(CQ)2 formulation suitable for parenteral administration without the use of solvents, (ii) to evaluate the efficacy of Cu(CQ)2 as an anti-cancer agent, (iii) to assess the use of Cu(CQ)2 as a copper ionophore to boost the anti-cancer activity ascribed to disulfiram (DSF). In Chapter 3, it was demonstrated that copper complexes can be synthesized inside liposomes. The resultant formulation remains in solution and is suitable for intravenous administration. The studies presented here are the first to assess the anti-cancer activity of Cu(CQ)2. The results suggest that Cu(CQ)2 is not potent enough to exert meaningful anti-cancer activity in vivo, even when used in combination with DSF. 5.2 Results 5.2.1 Cytotoxicity of CQ and its Copper Complex The activity of CQ and Cu(CQ)2 against A2780-S, A2780-CP, A549, U251 and MV-4-11 cells were determined and the results are summarized in Figure 5.1. Both compounds were solubilised in a final DMSO concentration of 0.5% and viability was measured 72 hrs post-treatment. It should be noted that a visual precipitate was observed when the CQ or Cu(CQ)2 concentration added was >100 µM. The results with A2780-S, A2780-CP and A549 cells suggest that the activity of CQ is enhanced significantly when added as the copper complex (Fig. 5.1A, B, C). The IC50 of Cu(CQ)2 was between 20-60 μM, while CQ alone showed very little toxicity even at concentrations >100 μM. In contrast, the activity of CQ and Cu(CQ)2 were equivalent when added to U251 and MV-4-11 cell lines (Fig. 5.1D and E). The IC50 of CQ was 32 and 46 μM while it was 27 and 32 μM for Cu(CQ)2 in U251 and MV-4-11 cells, respectively.  79   Figure 5. 1: The cytotoxicity of CQ and Cu(CQ)2 against 5 different cancer cell lines. Cytotoxicity curves for CQ (-●-) and Cu(CQ)2 (-■-) were obtained for (A) A2780-S, (B) A2780-CP (C) A549, (D) U251 and (E) MV-4-11 cells. Cells were seeded for 24 hrs and then treated with CQ or Cu(CQ)2 at doses ranging from 0.05 – 400 µM for 72 hrs. Cell viability for the adherent cell lines (A2780-S, A2780-CP, A549, U251) was determined using an INCell analyzer 2200 where viability was assessed based on loss of plasma membrane integrity 72 hours following treatment; i.e. total cell count and dead cell count were determined using Hoechst 33342 and ethidium homodimer staining, respectively. The viability of the non-adherent cell line (MV-4-11) was measured using PrestoBlueTM. Data are presented as mean ± SEM, where the values were determined in triplicate three times.  5.2.2 Copper CQ Liposome Characterization The results summarized above demonstrate that Cu(CQ)2 is active against a number of cancer cell lines in vitro; with an IC50 ranging from 20-60 μM. It is a challenge to develop a drug formulation when the selected drug exhibits activity in the micromolar range and this challenge becomes even greater when the drug is sparingly soluble in aqueous solution. To address this challenge, a formulation method is available where the copper complex is synthesized inside liposomes, as described in Chapter 3. This strategy is illustrated by the data summarized in Figure 5.2, wherein Cu(CQ)2 was synthesized inside DSPC/Chol (55:45, mol ratio) liposomes prepared to contain CuSO4. The liposomes (20 mM final liposomal lipid concentration) were added to 5 mg CQ (as powder) prior to incubating at 40oC. The colour of the solution changed from white to yellow/green within 3 minutes indicative of Cu(CQ)2 formation (Fig. 5.2A). Formation of Cu(CQ)2 was found to be temperature dependant (Fig. 5.2B). When samples were incubated at 4oC there was no 80  observable color change and the measured Cu(CQ)2 to liposomal lipid ratio was less than 0.02 after 60 minutes. The rate of Cu(CQ)2 synthesis was faster as the incubation temperature increased to 25oC, where the measured Cu(CQ)2 to liposomal lipid ratio was 0.1 after 30 minutes. The optimal temperature for complex formation was 40oC, where the measured Cu(CQ)2 to liposomal lipid ratio was 0.2 after 3 minutes. In these studies, CQ was added in excess but as noted in [13], the amount of Cu(CQ)2 formed inside the liposomes is completely dependent on the amount of copper trapped in the liposome. This is illustrated in Fig. 5.2C. Increasing the initial theoretical Cu(CQ)2 to liposomal lipid ratio beyond 0.15 produced no further increase in the measured Cu(CQ)2 to liposomal lipid ratio when using the optimal temperature of 40oC. In this context, the initial theoretical Cu(CQ)2 to liposomal lipid ratio was estimated on the assumption that each mole of copper would complex 2 moles of CQ [190]. When the initial theoretical Cu(CQ)2 to liposomal lipid ratio was 0.2, the measured Cu(CQ)2 to liposomal lipid ratio was 0.17, which was similar to what was measured for formulations prepared with a large excess of CQ. Preliminary studies assessing the stability of the resulting Cu(CQ)2 suggested less than 10% of the associated Cu(CQ)2 was released from the liposomes when incubated in serum (80%) over 24 hr (Fig. 5.2D).  81   Figure 5. 2: Synthesis of Cu(CQ)2 in liposomes prepared with encapsulated 300 mM CuSO4. (A) Photograph of solutions consisting of CQ (5 mg/mL) added to CuSO4-containing liposomes (20 mM liposomal lipid) over a 1 hr time course at 40oC. (B) Formation of Cu(CQ)2 inside DSPC/Chol liposomes (20 mM) as a function of time over 1 hr at 4oC (●), 25 oC (■) 40oC (▲) and 60oC (▼) following addition of CQ (5 mg/mL). (C) Measured Cu(CQ)2 to liposomal lipid as a function of theoretical Cu(CQ)2 to total liposomal lipid ratio estimated based on the amount of CQ added to the liposomes. For these studies the liposomal lipid concentration was fixed at 20 mM and the added CQ amount was varied. (D) In vitro stability of the Cu(CQ)2 formulation over 24 hrs in 80% fetal bovine serum. Cu(CQ)2 was measured using a spectrophotometric assay (B-C) or HPLC (D) and liposomal lipid was measured through use of a radiolabeled lipid (3H-CHE). All data are plotted as mean ± SEM.  82  5.2.3 Tolerability and Pharmacokinetics Following Intravenous Administration of the Cu(CQ)2 Formulation The maximum tolerated dose of Cu(CQ)2 was found to be 30 mg/kg i.v once daily Monday, Wednesday, Friday for two weeks. The formulation was well tolerated at this dose, no weight loss greater than 5% (data not shown) and no notable changes in health status were observed. Following necropsy (14 days after last treatment) there were no gross morphological changes noted. This dose (30mg/kg) and route of administration was used for the pharmacokinetic studies. The Cu(CQ)2 elimination profile was characterized and compared to control liposomes (prepared in 300 mM copper sulfate and exchanged into SH buffer pH 7.4) and the results have been summarized in Figure 5.3. For the analysis of plasma samples, an HPLC assay designed to measure CQ was used (see Methods) as the measurement of Cu(CQ)2 was not possible. At 24 hrs post-injection of Cu(CQ)2, the amount of CQ in the plasma falls below the limit of detection (Fig. 5.3A). Based on these data, approximately 25% of the injected Cu(CQ)2 dose was eliminated within one hour and greater than 90% was eliminated within 8 hrs. As illustrated in Fig. 5.3B, the CQ to liposomal lipid ratio decreases as a function of time after administration. This is indicative of CQ release from the liposomes and into plasma compartment. For example, the measured CQ to liposomal lipid ratio at 4 hrs is 50% less than that of the injected formulation. It is not clear from this data that CQ is being released from the liposomes as CQ or Cu(CQ)2. For this reason, the plasma copper concentrations were also determined (see Methods). The results, shown in Fig. 5.3C and 5.3D, are based on plasma copper levels determined after subtraction of background copper levels determined in plasma obtained from untreated mice. It is assumed, therefore, that the copper being measured is due to the injection of the Cu(CQ)2 formulation. As shown in Fig. 5.3C (filled circles), animals injected with Cu(CQ)2 have plasma copper levels that decrease over time, where >90% of the injected copper dose was eliminated after 8 hrs. The results shown in Fig. 5.3D (filled circles), also suggest that the copper to liposomal lipid ratio is decreasing as a function of time after administration. To gain a better understanding of whether the liposomal formulation using (DSPC/Chol, 55:45 mol ratio) was releasing Cu(CQ)2 as the complex, the results obtained in animals treated with the Cu(CQ)2 formulation were compared to animals treated with control liposomes prepared to contain just copper (see Methods). This 83  data (filled squares in Fig. 5.3C and 5.3D) indicates that copper elimination is significantly reduced following administration of the copper containing liposomes. This is best illustrated by the results in Fig. 5.3D where it appears that the initial copper to liposomal lipid ratio decreases by less the 50% for the copper liposomes, but more than 85% for the Cu(CQ)2 formulation at 24 hrs. As noted in Fig. 5.3E, the elimination of liposomal lipid following administration of Cu(CQ)2 and the copper containing liposomes were comparable. In aggregate, the results suggest that following administration of the Cu(CQ)2 formulation, both copper and CQ are released. The release rates are comparable which suggest that Cu(CQ)2 is being released from the liposomes as a function of time after administration. Since the assays used here were unable to directly measure Cu(CQ)2, it was not possible to assess whether the Cu(CQ)2 complex is stable following release from the liposomes. It can be concluded that the estimated Cu(CQ)2 (assuming CQ is complexed to copper) are potentially sufficient to engender therapeutic effects based on the IC50 of Cu(CQ)2 shown above. 84   Figure 5. 3: Cu(CQ)2 and copper liposome plasma elimination following intravenous injection in CD-1 mice. The Cu(CQ)2 liposomes (30 mg/kg CQ, 3.2 mg/kg Cu, 115.6 mg/kg lipid) were dosed in CD-1 mice. Copper liposomes (liposomes prepared in 300mM CuSO4) were injected at the same copper and liposomal lipid dose of 3.2 and 115.6 mg/kg, respectively. (A) CQ plasma concentration over 24 hrs; where CQ was measured by HPLC methods. (B) CQ to liposomal lipid ratio over 24 hrs following administration of the Cu(CQ)2 formulation. (C) Plasma copper levels following injection of Cu(CQ)2  (●) and copper liposomes (■) over 24 hrs, where Cu2+ was measured using AAS (see Methods). (D) Copper to liposomal lipid ratio measured over 24 hrs following injection of copper liposomes or the Cu(CQ)2 formulation. (E) The liposomal lipid concentration was measured using scintillation counting of 3H-CHE. All data are plotted as mean ± SEM, (n=5). 85  5.2.4 Anti-tumor Efficacy Following Intravenous Administration of Cu(CQ)2 A sc tumor model of A2780-CP (a platinum resistant ovarian cancer cell line) was developed, in these cells the IC50 of Cu(CQ)2 was approximately 20 µM and the IC50 of CQ was >100 µM. The A2780-CP model is fast growing, where control animals must be terminated due to tumor progression (tumors reach a size >800 mm3, see Methods) within 18-22 days following cell injection. For these studies, Cu(CQ)2 treatment was initiated 4 days post cell inoculation. The results, summarized in Fig. 5.4A, indicate that treatment with copper liposomes and Cu(CQ)2 caused a slight, but not significant change in A2780-CP tumor growth rate.  The therapeutic activity of Cu(CQ)2 was then evaluated in NRG mice bearing sc U251 tumours. This cell line was selected because it was sensitive to both CQ and Cu(CQ)2 (Figure 5.1); the IC50 of Cu(CQ)2 and CQ in this glioblastoma cell line was approximately 30 µM. Dosing (30 mg/kg i.v once daily Monday, Wednesday, Friday for two weeks) began when the average tumour size reached 50-100 mm3.The results, summarized in Figure 5.4B, suggest that treatment with Cu(CQ)2 had no impact on the growth rate of the U251 tumors when compared to the growth rate in animals treated with the vehicle (SH buffer) or control copper liposomes. Based on these studies it was concluded that Cu(CQ)2, when administered as a single agent, was not efficacious.  Figure 5.4: Efficacy of Cu(CQ)2 in animals bearing subcutaneous A2780-CP and U251 tumour xenographs. NRG mice with sc injected cell lines (see Methods) where treatment with vehicle (SH buffer, ●), copper liposomes (liposomes prepared in 300 mM CuSO4) (Copper dose of – 3.2 mg/kg, ■) or Cu(CQ)2 (30 mg/kg, ▲). The liposomal lipid dose was 115.6 mg/kg. Treatments were given iv on  Monday, Wednesday and Friday for 2 weeks. (A) A2780-CP tumour growth in NRG mice (n=8), dosing began on day 4 post cell 86  inoculation. (B) U251 tumour growth in NRG mice (n=6), treatment began when tumours reached 50-100 mm3. Data are reported as mean ± SEM.  5.2.5 Efficacy of Cu(CQ)2 in Combination with Disulfiram The studies summarized above suggest that the Cu(CQ)2 formulation was not efficacious when administered as a single agent. Cu(CQ)2 acts as a copper ionophore [50, 217], thus its potential to act in combination with DSF was explored. The anti-cancer effects of DSF are dependent on having high intracellular copper levels which could be achieved using a copper ionophore [259]. These studies were completed using the A2780-CP cell line which exhibits sensitivity to Cu(CQ)2 but not CQ. The results summarized in Fig. 5.3D, are consistent with the published literature suggesting that Cu(CQ)2 can cross lipid bilayers [50]. To illustrate this in a cell model, an assay based on copper dependent quenching of  Phen Green™ fluorescence was used [304, 305]. The results, summarized in Figure 5.5, show that the fluorescent intensity of cells incubated with Phen Green™ decreases following addition of Cu(CQ)2. This decrease in A2780-CP cell associated Phen Green™ was not observed when cells were treated with copper alone. A decrease, albeit not significant, in fluorescence was noted when the cells were treated CQ alone, but this is likely due to CQ binding copper in the serum containing cell culture media.  DSF is known to metabolize to form DDC which can complex with copper to form Cu(DDC)2, a cytotoxic agent [66, 120]. To test whether combinations of Cu(CQ)2 and DSF were cytotoxic, the agents were added alone and in combination to A2780-CP cells. The results, summarized in Figure 5.5B, indicate that cells exposed to Cu(CQ)2 (inverted filled triangles) or DSF (filled triangles) alone exhibited compound IC50 values of 19μM and 1.7μM, respectively. When DSF was combined with cells with Cu(CQ)2 (1:1 ratio) the IC50 of DSF decreased to 110nM. The IC50 of DSF in cells treated with Cu(CQ)2 is essentially equivalent to the IC50 of DSF and CuSO4 (90 nM) which indicates that Cu(CQ)2 does not inhibit the formation of Cu(DDC)2.  The in vivo activity of Cu(CQ)2 when combined with DSF was evaluated in animals bearing sc A2780-CP tumors. For these studies DSF was dosed orally (100 mg/kg) as described elsewhere [288]. DSF treated animals were dosed concurrently with the Cu(CQ)2 formulation or copper liposomes (liposomes prepared in 300 mM CuSO4). The results, 87  summarized in Figure 5.6, suggest that Combinations of DSF with Cu(CQ)2-liposomes resulted in a modest, but not significant, reduction in tumor growth which could not be differentiated from animals treated with combinations of DSF with copper liposomes.    Figure 5. 5: Cu(CQ)2 mediated increase in copper delivery to cells and its in vitro activity when combined with disulfiram (DSF). (A) A2780-CP intracellular copper levels were assessed using the cell permeable dye Phen GreenTM. Cell associated Phen GreenTM fluorescence was measured 1 hr treatment with the vehicle (0.01% DMSO), CQ, copper, or Cu(CQ)2. The cells were then incubated with Phen GreenTM for 30 minutes (see Methods). The fluorescence of the probe is quenched in the presence of Cu and thus a decrease in cell associated fluorescence is indicative of higher intracellular copper levels. Cell associated fluorescence 88  was measured using an INCell Analyzer 2200. Results shown are an average of 3 studies done in triplicate (mean ± SEM). (B) Cytotoxicity curves were generated in A2780-CP cells after 72 hr treatment with DSF (--), Cu(CQ)2 (--) or DSF in combination with Cu(CQ)2 (-■-) or CuSO4 (-▲-). Cell viability was determined using an INCell analyzer 2200 where viability was assessed based on loss of plasma membrane integrity 72 hours following treatment; i.e. total cell count and dead cell count were determined using Hoechst 33342 and ethidium homodimer staining, respectively.  Figure 5.6: Efficacy of DSF in combination with Cu(CQ)2 and copper liposomes (liposomes prepared in 300mM CuSO4) determined in NRG mice with sc A2780-CP tumors. Treatment with CuSO4-liposomes (Cu-lips) (copper does of 3.2 mg/kg ■), DSF (100 mg/kg,), DSF and Cu-liposomes (100 mg/kg DSF and 3.2 mg/kg copper, ○) or DSF and Cu(CQ)2 (100 mg/kg DSF and 30 mg/kg Cu(CQ)2, □) was initiated 4 days after sc inoculation of the A2780-CP cells. Cu(CQ)2 and copper liposomes were dosed iv Monday, Wednesday and Friday for two weeks and DSF was dosed orally (see Methods) Monday to Friday for 2 weeks. (A) A2780-CP tumour growth in NRG mice (n=13) and (B) tumour size on day 20 was determined as described in the Methods Data are reported as mean ± SEM.  5.3 Discussion Recently, efforts have been directed towards repurposing CQ as an anti-cancer drug [303]. The activity of CQ and Cu(CQ)2 against a range of cancer cell lines suggests that Cu(CQ)2 is only effective at concentrations ranging for 20-60 µM (see Figure 5.1). The anti-cancer activity of CQ alone is worse, with IC50 values greater than 100 µM. Interestingly, the activity of CQ is enhanced when administered as a copper complex, although some cell lines show copper independent activity. This may be a consequence of the cellular context in which CQ is presented [299, 306], or it may be due to higher intracellular copper levels in some cell lines when compared to others [307]. Regardless, the preclinical data suggesting CQ anti-cancer effects was compelling enough to lead to a clinical trial wherein CQ was given to 11 patients with hematologic malignancies [17]. This study was designed to test whether the metal ionophore activity of CQ and its associated inhibition of the proteasome could engender therapeutic effects in patients with refractory hematologic malignancies. 89  CQ was given oral in a classic dose escalation phase 1 study. The maximum tolerated dose was determined, however there was minimal activity and no evidence of proteasome inhibition. These authors concluded that the poor activity was due to poor intracellular delivery of CQ [303].  CQ can be administered orally but does suffer from extensive first pass metabolism [298]. This did not affect its utility as an anti-microbial drug but did pose a challenge when attempting to repurpose this drug for cancer, where high plasma concentrations are required. One method to overcome first pass metabolism is through iv injection; owing to the solubility challenges of both CQ and Cu(CQ)2 this was not possible. Herein, a novel formulation of CQ, where the Cu(CQ)2 complex is synthesized inside the core of liposomes that is suitable for development as a pharmaceutical was investigated.    The aqueous core of the liposome is used to carry out a synthesis reaction between copper and CQ, the complex is left in solution (suspended inside the liposome). The amount of Cu inside the liposome is the limiting reagent when forming Cu(CQ)2 (see Figure 5.2) and the complex formed inside the liposome showed no release in vitro over a time course of 24 hrs. The formulation appears stable with respect to particle size, polydispersity and Cu(CQ)2/liposomal lipid ratio. Pharmacokinetic studies completed with the resultant Cu(CQ)2 formulation indicate that blood levels of CQ can be maintained at concentrations well above the Cu(CQ)2 IC50 for at least 8 hrs after iv administration at a dose of 30 mg/kg (see Figure 3). Analysis of the plasma samples strongly suggest that Cu(CQ)2 dissociates from the Cu(CQ)2 formulation following administration (see Figure 5.3D). However because of an inability to measure Cu(CQ)2 in plasma, it was unclear whether the Cu(CQ)2 released from the liposomes remained in a complexed form. The CQ concentration in the plasma was more than sufficient to exert therapeutic effects for at least 8 hours and the formulation addresses the limitation encountered by investigators interested in evaluating CQ activity in patients.  Having overcome the formulation challenges of Cu(CQ)2, it was reasonable to ask whether the resulting formulation was efficacious in vivo. Our results suggest that the Cu(CQ)2 formulation is not effective, even when administered in combination with DSF, an agent that is significant more potent when combined with a copper [66, 116]. The studies 90  with Cu(CQ)2 alone were completed in two subcutaneous tumour models (A2780-CP and U251) representing cell lines in which CQ toxicity was copper dependant (A2780-CP) and copper independent (U251). These studies used a dose intensive schedule (Monday, Wednesday and Friday x 2 weeks) because Cu(CQ)2 is active only when present at µM levels. Despite a multi-dosing schedule and evidence to suggest that the CQ levels in the plasma compartment were above 100 µM for at least 8 hrs, the Cu(CQ)2 formulation did not show any activity. The original studies with CQ were based on its potential to act as a copper ionophore [50] and for this reason it was explored in combination with DSF. The in vitro results (see Figure 5.5) support the fact that DSF/ Cu(CQ)2 combinations are effective and that nanomolar levels of DSF (in the presence of Cu(CQ)2) are sufficient to exert significant cytotoxicity, however the activity of the combination in vivo (see Figure 5.6) indicated otherwise. While the combination did show good activity in vitro, there is an inability to coordinate the pharmacokinetics of both DSF and Cu(CQ)2 formulation such that the two agents reach the tumour site at sufficient levels to achieve effective therapy. It is possible that this may be overcome by assessing sequential combination treatments in the future; as was done by Verreault et al.[308]. Alternatively, studies have suggested that another 8-hydroxyquinoline analogue, 5-nitro-8-hydroxyquinoline, is much more potent than CQ [18] and future studies could investigate formulations of this analogue. The methodology described is broadly applicable to the synthesis of many different metal-complexes inside liposomes and provides the opportunity to select for formulations that will be better suited for clinical development then the Cu(CQ)2 formulation described here.  5.4 Conclusion This work examined whether CQ and Cu(CQ)2 could be formulated in a manner suitable for development as an anti-cancer agent. A liposomal Cu(CQ)2 formulation was described that solves the solubility issues plaguing efforts to assess the activity of the highly water insoluble Cu(CQ)2 complex. Further the resultant formulation ensured that therapeutically effective concentrations of CQ or Cu(CQ)2 could be maintained in the plasma compartment over time. However, the resulting formulation was not efficacious whether used alone or in combination with DSF, a drug that is known to be activated in the 91  presence of copper. While this formulation did not exhibit interesting therapeutic effects in vivo the formulation methods are suitable for other analogues of 8-hydroxyquinoline which exhibit more potent anti-cancer activity.                92  6. Copper Complexes of Bidentate Ligands Exhibit Potent In Vitro Anti-Cancer Activity Regardless of Platinum Sensitivity Status* 6.1 Introduction Pt drugs are the most successful class of inorganic medicinal compounds used to treat cancer [36, 309]. They are a mainstay in cancer therapy, being utilized in approximately 50% of chemotherapeutic regimens [36, 310]. CDDP was first used to treat leukemia in the 1960s, but through several inorganic medicinal chemistry programs, other Pt-based drugs (CBDCA, oxaliplatin, paraplatin etc.) have been produced and approved by the FDA [32, 36]. Mechanistically, these drugs are known to act by forming Pt-DNA complexes that cause DNA damage that accumulate to a point that is beyond repair, ultimately leading to cell death [310, 311]. Pt drugs are currently used as first-line therapy in blood, lung, ovarian, testicular, and head and neck cancers. While some cancers are Pt sensitive and thus respond to Pt drugs [36], many others are Pt-insensitive due to inherent or acquired resistance [310, 311]. There is a need  to define drugs capable of treating Pt insensitive cancers [312]. While Pt drugs have been successful for many patients, they also produce serious side effects including nephrotoxicity, neurotoxicity and ototoxicity [313]. To address these adverse effects, many inorganic medicinal chemistry investigations have focused on developing alternatives to Pt-based drugs by replacing Pt with other divalent metals, such as copper [48]. Copper-based therapeutics are generally less toxic owing to the physiological processes that detoxifying excess of copper [62]. In line with this approach, many groups have demonstrated in vitro that copper complexes of natural compounds have anti-cancer properties; some examples include dithiocarbamates and analogues of 8-HQ [301, 314]. To date, however, no copper complex has transitioned into clinical use for any indication in humans. While the platins that are used in the clinic are water soluble,                                                         * Adapted from: M. Wehbe, C. Lo, A. Leung, W.H. Dragowska, G. Ryan, M.B. Bally, Copper complexes of bidentate ligands exhibit potent anti-cancer activity regardless of platinum sensitivity status, (2017) Submitted to Investigational new drugs 93  one major challenge of copper-based therapeutics is their inherently poor aqueous solubility [66]. This problem has made in vivo testing very challenging and required solvents containing mixtures of DMSO and Cremophor which are not clinically applicable due their high toxicity[214, 315]. Chapter 3 solved the solubility issue of copper-based therapeutics by synthesising  copper complexes within liposomes [66]. This technology, referred herein as Metaplex™, was used to examine the effects of copper DDC (Cu(DDC)2) in animals bearing a xenograft tumours [276]. In the present studies, five bidentate copper ligands (Figure 6.1) were selected and screened both as ligands and the corresponding copper complexes in a panel of eight cancer cells. As indicated above DDC [42, 65], Pyr [214], Plum [316, 317], 8-HQ [65, 216, 219] and CQ [65, 191, 217, 301] have all been shown to have anti-cancer activity but have never been tested directly against Pt-resistant cancers. The activities of these complexes were compared to the activity of CDDP and CBDCA (Figure 6. 2). The screen showed that many copper complexes are more active in vitro than the Pt controls. It was demonstrated that a Metaplex™ formulation of Cu(DDC)2 was active in animals bearing Pt-resistant A2780-CP xenograft tumours. The results highlight the potential of the copper-based therapeutics as candidates to treat Pt resistant cancers.  94   Figure 6.1: Ligands and their respective copper complex structures. (A) DDC and Cu(DDC)2 (B) Pyr and Cu(Pyr)2. (C)Plum and Cu(Plum)2. (D) 8-HQ and Cu(8-HQ)2 and (E) CQ and Cu(CQ)2.   Figure 6. 2: Structures of commonly used platinum drugs. (A) CDDP and (B) CBDCA.  95  6.2 Results 6.2.1 Pt-Resistance Does not Impact Sensitivity of Cancer Cells to Cu(DDC)2 Complex CDDP and CBDCA, two commonly used Pt-based therapeutics were tested in a pair of isogenic ovarian cancer cells: A2780-S cells are the parental cells that are sensitive to CDDP while A2780-CP cells are platinum-resistant. As shown in Figure 6.3A and B, the IC50 of CDDP and CBDCA was 3.7-and 8.5-fold greater, respectively, in the Pt resistant cells when compared to Pt sensitive cells. To determine if Pt resistance impacts activity of copper complexes, A2780-S and A2780-CP cells were treated with Cu(DDC)2. The data in Figure 6.3C show that the IC50 value of the copper complex was not different for Pt sensitive or resistant cells. These data suggest that copper complexes may be effective regardless of Pt sensitivity status.   Figure 6. 3: Cytotoxicity profiles of Pt sensitive A2780-S () and resistant A2780-CP (■) ovarian cancer cells (or) following 72 hr treatment with (A) CDDP, (B) CBDCA and (C) Cu(DDC)2. Fraction affected cells was assessed based on viability data normalized to vehicle controls. (D) The IC50 ± 95% CI for 96  A2780-S (black) and A2780-CP (white) after treatment with CDDP, CBDCA and Cu(DDC)2. Data are presented as the mean of 3 independent experiments ± SEM.  6.2.2 Activity of Copper Complexes in Cancer Cells with Different Pt Sensitivity The IC50 values ± 95% confidence intervals for the ligands and their corresponding copper complexes are shown in Figure 6.4. Cells were treated with the ligand/drug or complex (2:1 ligand:Cu ) for 72 hrs. All cell lines were also exposed to CuSO4 as a control to ensure that copper alone did not impact viability (IC50 > 10µM for all cell lines, data not shown). The results in Figure 6.4A indicate that treatment of different cells with the ligand DDC results in an IC50 ranging from 5-300 μM DDC. In contrast, the IC50 for the Cu(DDC)2 ranged from 0.02-0.15 μM corresponding to differences of 90-11,000 fold (Fig. 6.4A). The IC50 of Pyr ranged from 1-30μM, and when used as a copper complex (Cu(Pyr)2), the IC50 decreased to 0.1-7.4 μM (Fig. 6.4B). The IC50 of Plum ranged from 1.5–11 μM while the IC50 of and Cu(Plum)2 Ranged from 0.8-3 μM in the tested cell lines. In some cell lines (A2780-S, A2780-CP, MV-4-11, Cal-27, FaDu) there was only approximately a 2-fold difference in activity between Plum and Cu(Plum)2 (Fig. 6.4C). Considering the 2:1 (ligand:Cu) complexation ratio, these data indicate that the copper Plum complex is as active as the uncomplexed ligand, suggesting that the cytotoxic effects observed for Plum is copper-independent. 8-HQ was more active as a copper complex (IC50 for Cu(8-HQ)2 ranged between 0.2-4.5μM compared to 1.5-30μM for 8-HQ); however, in A2780-S and FaDu cells, the toxicity of 8-HQ appeared to be copper independent (Fig. 6.4D). The IC50 for CQ and Cu(CQ)2 ranged from 10-350μM and 20-60μM, respectively. Here the dependence on copper complexation for CQ cytotoxicity appears to be cell line-specific: copper-dependent activity was observed in A2780-S, A2780-CP and A549 cells, whereas in MV-4-11, SCC-25 and H1933, the cytotoxicity of CQ was copper-independent. Moreover, CQ appears to be more cytotoxic than Cu(CQ)2 in Cal-27 and FaDu cells (Fig. 6.4E).  The IC50s obtained from cytotoxicity assays for each cell line were arranged as a heat-map in order of Pt sensitivity to CDDP (Fig. 6.5). An IC50 cut-off of 10µM was used to distinguish more potent agents from those that were regarded as pharmaceutically inactive. In general, the ligands appear to be less active in the Pt resistant cell lines whereas the copper complexes augmented anti-cancer activity considerably. The Pyr, Plum and 8-97  HQ ligands showed activity in most cell lines, however, their activity was enhanced in the tested cell lines when used as complexes with copper; where the greatest increases were observed for Cu(Pyr)2. DDC and CQ were the least active ligands. The Cu(CQ)2 complex did not show improvement in cytotoxicity whereas the copper complex of DDC was the most active in all cell lines. In general, except for Cu(Pyr)2, the activity of the copper complexes used in this study show no correlation with Pt CDDP sensitivity in tested cell lines (Table 6.1). The heatmap indicates that (Cu(DDC)2 is the most effective complex tested  against Pt-resistant cancers.    98   Figure 6. 4: Ligand/drug and copper complex cytotoxicity. A panel of 8 cancer cell lines (A2780-S, MV-4-11, A549, SCC-25, Cal-27, A2780-CP, FaDu and H1933) were treated for 72 hrs with  the ligand/drug (■) or copper complex (□): (A) DDC/Cu(DDC)2, (B) Pyr/Cu(Pyr)2 ,(C) Plum/Cu(Plum)2 , (D) 8-HQ/Cu(8-HQ) 2 and  (E) CQ/Cu(CQ) 2. The IC50 values were obtained based on the viability data acquired with IN Cell 2200 or PrestoBlue® (M-V-411). The fold difference between the IC50 of the ligand and respective copper complex is shown above the histogram bars.  99   Figure 6. 5: Screen of copper-based therapeutics in cancer cells with different Pt sensitivities. IC50 values for Pt drugs (CDDP and CBDCA), ligands (DDC, Pyr, Plum, 8-HQ, CQ) and respective copper complexes are shown for cancer cell lines of differing origin arranged in order of sensitivity to CDDP. IC50 values were calculated from viability data (n=3 experiments/each cell line) obtained with IN Cell Analyzer 2200 platform or PrestoBlueTM assay (MV-4-11 cells). The agents with IC50 values >10 µM (black) are considered inactive. Table 6. 1: Correlation between CDDP and copper complex sensitivity based on the IC50 values Drug Cu(DDC)2 Cu(Pyr)2 Cu(Plum)2 Cu(8-HQ)2 Cu(CQ)2 Pearson R2 0.07 0.70 0.10 0.09 0.44 P-value 0.05 0.01 0.44 0.48 0.07 Significant No Yes No No No   100  6.2.3 Efficacy of Liposomal Cu(DDC)2 in a Pt-resistant A2780-CP Tumour Xenograft Model Cu(DDC)2  has represented a formulation challenge because of its poor aqueous solubility. In Chapter 3, Cu(DDC)2 was synthesized inside a liposomal formulation [66]. This formulation (see Methods) was used to assess the activity of the Cu(DDC)2 in a Pt-resistant A2780-CP subcutaneous tumour xenograft model. The data, shown in Figure 6.6, indicate that Cu(DDC)2 engendered a statistically (p<0.05) significant ~50% reduction in tumour burden when compared to the activity of the vehicle control. These data provide proof-of-concept results suggesting that liposomal copper complexes have the potential to be developed as a novel class of therapeutic agents for use in the treatment of Pt-resistant cancers.  Figure 6. 6: In vivo activity of liposomal Cu(DDC)2 in animals bearing Pt resistant A2780-CP tumours. 1 x 106 A2780-CP cells were inoculated sc into immune-compromised NRG mice which were subsequently treated intravenously with vehicle, copper containing liposomes and Cu(DDC)2 liposomes using a 3 x per week for 2 weeks dosing schedule. Mean tumour volume was determined on day 18 (the humane endpoint for vehicle treated animals). Data represents the mean ± SEM (n=7). * indicates statistical difference (p<0.05)    101  6.3 Discussion Pt drugs are standard first-line therapies for the treatment of lung, ovarian, and head and neck cancers [29, 318]. While some cancers are inherently resistant, the widespread use of Pt-based therapies has resulted in the development of resistant cancers, often in the relapse setting where prognosis is poor [312, 318]. In vitro, cell lines can be developed to replicate this effect (e.g. A2780-CP), wherein cells are treated with low dose Pt drugs such as CDDP over extended periods of time until resistance is observed through changes in IC50 values [319, 320]. The two major mechanisms responsible for Pt resistance are DNA repair and Pt drug transport into cancer cells [36, 321]. Through extensive characterization, it was determined that at equivalent levels of drug accumulation, the A2780-CP cell line is 2-fold more efficient at repairing CDDP-DNA lesions when compared to the parental, Pt-sensitive cell line (A2780-S) [321]. The copper transporter CTR1 is the major influx transporter for cisplatin both in vitro and in vivo and has been reported to be reduced in cisplatin resistant cancers [318, 322]. This mechanism was attributed to cisplatin influx resulting in the rapid degradation of CTR1[323]. Identifying new therapeutic entities that are active in Pt resistant cancers is a priority to improve treatment outcome for patients.  The ovarian cancer cell line A2780-CP had a clear resistance to CDDP and CBDCA when compared to the activity of these drugs in A2780-S cells. In order to test the utility of CBTs in platinum-resistant cancer cells, preliminary studies investigated Cu(DDC)2 in Pt sensitive and insensitive cell lines. Cu(DDC)2 was active irrespective of Pt sensitivity at equivalent doses. This Pt sensitivity-independent activity could be explained by the Cu(DDC)2 mechanism of toxicity which overcomes resistance mechanisms commonly seen in Pt-resistant cancers. Cu(DDC)2 is able to accumulate in cancer cells irrespective of CTR1 expression [324] and cytotoxicity is mediated through proteosome inhibition [42]. These results suggest that CBTs use mechanisms of cytotoxicity outside of those used by platinums and warrant further investigations in resistant cancers.  Five copper binding ligands (DDC, Pyr, Plum, 8-HQ and CQ) were tested in a panel of eight cancer cell lines of different origins (blood, lung, ovarian, head and neck) and differing sensitivity to Pt-based drugs exemplified by CDDP/CBDCA activity. The in vitro 102  data suggests that the cytotoxicity of ligands DDC, Pyr and 8-HQ appeared to be copper-dependent whereas the cytotoxicity of Plum and CQ were copper independent. This finding is important as DSF (metabolized to DDC in vivo) + Cu-Gluconate and CQ were involved in clinical trials with no listed benefit [289, 325, 326]. By knowing the relationship between Cu and the activity of the complex, it can be reasoned that ligands such as DDC, Pyr and 8-HQ should be administered as copper complexes to ensure that the active (copper complexed) species reaches the cancer cells. This is supported by work performed by Katano et al which showed that Pt resistant cancers have lower basal levels of copper, suggesting that the uncomplexed ligands would be less active [322]. In the current study, H1933 was the least sensitive to Pt as reflected by a >10 µM IC50 cytotoxicity to all the ligands tested. In contrast, Plum and CQ could be used as single agents and pharmaceutical challenges associated with their solubility may be overcome using the Metaplex™ approach used in Chapter 4 to prepare a nanomedicine formulation of Cu(DDC)2 with no loss in activity[66, 276]. This data supports the utility of copper-based therapeutics in the treatment of Pt-insensitive cancer. To date, not a single copper-based therapeutic has received FDA approval. This is in part due to the solubility challenges associated with these complexes. All copper complexes studied as well as the ligands Plum, 8-HQ and CQ are water insoluble (<0.1 mg/mL); in contrast, DDC and Pyr are water soluble as ligands but their therapeutic activity is significantly enhanced in the presence of Cu. All copper complexes had to be dissolved in DMSO (final concentration 0.5%) to allow for in vitro testing. The ICH guidelines for residual solvents in a product indicate that DMSO cannot be included at concentrations above 0.5% which would be needed to administer the complexes at relevant doses [64]. In Chapter 3 the Metaplex™ technology as a platform approach to formulate copper complexes inside liposomes in order to overcome solubility issues and to allow for parenteral administration of copper complexed ligands was described [66]. Thus, as a proof-of-concept a liposomal formulation of Cu(DDC)2 was tested in a xenograft tumour model of A2780-CP Pt-resistant ovarian cancer. In this study, it was shown that liposomal Cu(DDC)2 (8 mg/kg) but not Cu-liposomes produced a statistically significant ~50% reduction in tumour burden when compared to the vehicle control. This difference is not considered clinically relevant (based on RECIST criteria [287]) but the results does 103  suggest that copper complexes are a class of therapeutic that should be further investigated. Cu(DDC)2 pharmacokinetics for the liposomal product show rapid release from the liposome and degradation in the plasma, and as such would require further development to improve circulation lifetime and stability [66]. Also the use of this formulated Cu(DDC)2 preparation in combination with other agents typically combined with platinums (eg. the taxanes [327]) is warranted. The other complexes discussed are proposed as future developments to identify those which have activity as single agents in Pt resistant cancers and as those which can augment therapy in combination with traditional chemotherapeutics.  6.4 Conclusion Pt drugs have been widely successful in the treatment of cancer. The treatment of Pt-resistant cancers has been a pharmaceutical focus that requires new therapeutics with mechanisms of action differing from those invoked by Pt-based therapies. Copper-based therapeutics represents a new class of drugs that can address many of the challenges associated with Pt-resistant cancers. This data shows that four out of five screened ligands that form copper complexes reached IC50 values below 10 µM in the eight cancer cell lines regardless of their Pt sensitivity. Additionally, one of these complexes (Cu(DDC)2) was tested in vivo in a Pt-resistant ovarian cancer xenograft model and attained a 50% reduction in tumor volume when compared to vehicle-treated control mice. These data not only provide in vivo validation that copper-based therapeutics could be used to treat Pt-resistant cancer but also provides proof-of-concept that the Metaplex™ technology could be used to develop formulations of inherently insoluble copper complexes for pre-clinical and clinical evaluations.     104  7. Summarizing Discussion This thesis describes a method by which poorly soluble copper complexes can be synthesized inside the core of liposomes; an approach that has been referred to as MetaplexTM. The method is simple and scalable.  The technology has been validated for ligands representative of differing functional group classes such as DDC (S-donor), CQ (N,O-donor), Qu (O-donor) and CX-5461 (mixed) (see Figure 1.3 and Table 3.1). Each of these classes can represent a large variety of molecules and I believe that such compounds all have the potential to be formulated using this approach. The synthesis reaction and general approach to preparing such formulations was described in detail in Chapter 3; results which highlight that complexation is solely dependent on the amount of copper entrapped inside the liposome. The copper complex to lipid ratio can thus be estimated by measuring the amount of copper associated with the liposome and the nature of the complexation reactions. The copper complex to lipid ratio can be tuned by changing the concentration of the copper salt solution used to rehydrate the lipids and by the salt form used (i.e. copper sulfate vs copper gluconate) or through changing the lipid composition. With respect to the latter point, it is known that liposomes composed of charged lipids (anionic/cationic) have a higher trapped volumes when compared to liposomes prepared with neutral lipids [240]. This is reflected in the studies summarized in Figure 3.3: as the liposomal lipid composition changed from DSPC/Chol to DSPC/Chol/DSPE-PEG(0.5-5%), the measured copper to lipid ratio increased from 0.2 to 0.45 even though both formulations were prepared using 300mM copper sulfate solutions.  My interest in developing formulations of copper complexes arose fortuitously from an interest to repurpose the anti-alcohol drug DSF for use in cancer. DSF was identified in a screen to be active against brain tumour initiating cells and this activity was enhanced in the presence of copper [116]. An examination of the DSF pharmacokinetic literature showed that DDC is the primary metabolite of DSF and it was well known that DDC is a Cu chelating agent [268, 328]. In chapter 3, it was shown that the activity of DSF + Cu or Cu(DDC)2 are identical (see Figure 3.1). The ability to investigate the anti-cancer potential of Cu(DDC)2 ,the active agent generated when DSF is mixed with copper, is difficult since it 105  is sparingly soluble in aqueous solution and this created the pharmaceutical challenge that I focused on. The Cu(DDC)2 formulation first described in Chapter 3 was suitable for further in vivo studies and these were summarized in Chapter 4. The formulation of Cu(DDC)2 prepared in DSPC/Chol (55:45) liposomes, when administered i.v. using a dosing schedule of 8 mg/kg (M,W,F x2), did effect a ~50% reduction in tumour burden when using a subcutaneous tumour model. However, the pharmacokinetic studies with this formulation suggested (see Figure 4.3) that the encapsulated Cu(DDC)2 was rapidly released from the injected liposomes. One advantage of this formulation approach is based on the well established understanding that liposomal lipid composition can influence the rate of associated drug release from the liposomes following i.v. administration. I believed that the anti-tumor activity of formulated Cu(DDC)2 could be improved if its retention inside liposomes was improved. By changing liposomal lipid composition it was demonstrated that a 4.2-fold increase in plasma AUC(0-∞) could be achieved relative to the DSPC/Chol formulation (see Figure 4.7), this increase in plasma AUC was due to the fact that the optimized liposomal formulation exhibited significantly better retention of Cu(DDC)2 following administration . Despite the improvement in drug retention, it was found that the optimized formulation showed equivalent anti-cancer activity when administered at their respective maximum tolerated dose (MTD). In this context, it should be noted that the MTD of the DSPC/Chol formulation was 1.5 times lower than that for the optimized formulation, suggesting that the improved drug retention did reduce the toxicity of the formulated Cu(DDC)2. A study by Daniel et al. examined the use of pyrrolidone diethyldithiocarbamate (PyDDC) and CQ as copper complexes with proteasome inhibitory activity comparable to what has been shown for Cu(DDC)2  (PyDDC is an analogue of DDC) [301]. CQ is an approved topical anti-fungal agent with anti-cancer activity [191]. In my hands, the anti-cancer activity of CQ was enhanced in the presence of copper, but this was not in all cell lines (see Figure 5.1). I choose to develop a CQ formulation because, unlike DDC which is a water soluble ligand that forms a water insoluble copper complex, CQ is a water insoluble ligand which becomes even less soluble when prepared as a copper complex. The development of the Cu(CQ)2 formulation not only enabled the in vivo evaluation of Cu(CQ)2, but also provided a unique opportunity to test whether the formulation approach could be 106  applied to poorly soluble ligands. To prepare the formulation, CQ was added in powder form to Cu-containing liposomes. It this scenario, some portion of the CQ must first dissolve into solution prior to permeating across the lipid membrane where it can then interact to form Cu(CQ)2. For complexation to occur the solution of CQ and copper containing liposomes required heating to at least 40oC (see Figure 5.2). Pharmacokinetic studies with the resulting Cu(CQ)2 formulation suggested that loss of the associate complex from the liposomes following administrtaion was slower then shown for the formulated Cu(DDC)2, but  similar to the Cu(DDC)2 formulation the results suggest that the complex, rather than the ligand alone, was released from the liposomes (see Figure 5.3). The formulation developed was suitable for use in vivo. Studies were completed to determine whether Cu(CQ)2 exhibited significant anti-cancer effects. Despite using a dose intensive schedule, Cu(CQ)2 failed to cause a significant reduction in tumour burden. The Cu(CQ)2 plasma concentration achieved appeared to reach levels that should have been sufficient to cause anti-cancer effects (seeFigure 5.3), but it should be noted that the levels of Cu(CQ)2 achieved in the plasma are a combination of Cu(CQ)2 inside the liposome and Cu(CQ)2 that has dissociated from the liposomes. In this context, levels of Cu(CQ)2 accessible to tumour cell populations may have been much lower than needed and ultimately tumour levels of Cu(CQ)2 would not be sufficient to cause therapeutic effects. This lack of activity may also be due to the instability Cu(CQ)2 after it was released from the liposomes. I would suggest that other, more potent, analogues of 8-HQ (e.g. 5-amino-8-hydroxyquinoline  [314]) could be developed using the approach described in this thesis.  Based on the results in Chapter 5, further investigation of Cu(CQ)2 as a single agent for treatment of cancer is not warranted. Formulations of Cu(DDC)2 and Cu(CQ)2 could prove more therapeutically interesting if used in combination with other drugs. It is known, for example, that the activity of the anti-cancer drug flavopiridol in combination with the proteasome inhibitor bortezomib is enhanced [329]. Thus combinations of flavopiridol and Cu(DDC)2 or Cu(CQ)2 could prove interesting.  As noted in chapter 5, studies have suggested that Cu(CQ)2 can act as a copper ionophore [50]. I believed that it was reasonable to assess the use of Cu(CQ)2 in combination with DSF, with the goal of generating Cu(DDC)2 within the target cancer cell population. As shown in this thesis, the IC50 of Cu(CQ)2 is >1M while the IC50 of  Cu(DDC)2 107  is typically <200nM. For this reason, I evaluated the combination and demonstrated that the Cu(CQ)2 and DSF combination resulted in significant cytotoxicity. This was not observed in vivo as the Cu(CQ)2 and DSF combination did not result in a statistically significant reductions in tumour growth when compared to vehicle control in a tumour model generated using a sensitive cell line (A2780-CP tumour model; see Figure 5.6). This strategy may require further optimization. For example, Verreault et al. compared the effects of sequential versus concurrent dosing of two anti-cancer agents and the results suggested that the combination may require one to coordinate the pharmacokinetic profiles of both agents [308]. Thus, Cu would need to be delivered first to the tumour cells by treatment with Cu(CQ)2. These Cu-“loaded” cancer cells would then be treated with DSF which would be given orally. This sequential strategy should be investigated in the future.  Finally, while the Cu(DDC)2 and Cu(CQ)2 formulations did not provide a strong therapeutic response, the studies summarized in Chapter 6 suggest that further efforts with this class of agents should focus on platinum resistant cancers.  7.3 Novelty of Metal-based Complexation Reactions to Prepare Liposomal Drug Formulations The use of metals and specifically copper in liposome loading of anti-cancer agents has been reported before. This has largely been developed around the campothecins (topotecan, Irinotecan) but has also included the anthracyclines (doxorubicin). With these agents it is difficult to separate the “remote loading” from the complex synthesis. During the development of remote loading it was found that drugs with a protonizible amine functionality could be encapsulated inside the core of liposomes when an electrochemical or proton gradient was present. Thus, the unencapsulated drug would be neutral in charge on the exterior of the liposome core but would become charged once it enters the liposome resulting in its encapsulation. This process is referred to as drug loading and is applicable to both the anthracyclines and campothecins. Both of these drugs classes also contain copper binding motifs which could contribute to the encapsulation and effect drug release.  It is challenging to distinguish between pH gradient loading and encapsulation due to metal complexation. One reason is that attempts to eliminate the pH gradient by 108  increasing the pH of the metal containing solutions results in formation of insoluble metal hydroxides [330]. This is to say that all unbuffered metal solutions that are used in liposomal drug development are naturally acidic and it is not completely possible to separate the two processes (drug protonization and metal complex formation). Further, much of the work with liposomes containing metal solutions involves the use of metal ionophores which are used to facilitate drug loading. These reduce the amount of metal retained in the liposome and this, in turn, reduces the likelihood that drug-metal complexation can occur. In formulations where the drug has both a protonizable amine and metal binding motif (in the presence of ionophores); one can assume that the main driving force for loading is the pH gradient.  The Cu salts used to prepare liposomes thus far have been CuSO4, CuCl2 and Cu-Gluconate. These salts can be prepared as unbuffered solutions. For example, liposome can be manufactured in 300mM CuSO4 (pH 3.5) [257, 331, 332]. The pH of copper solutions has been increased using appropriate buffers, but these are at best metastable solutions; capable of maintaining the copper in solution for only short periods of time (<1hr). Liposomes with encapsulated buffered Cu-gluconate have been described where triethanolamine (TEA) was used to increase the pH of the copper-gluconate solution to 7 [257]. Irinotecan can be encapsulated via remote loading in these copper gluconate-containing liposomes. However, in these formulations, the amount of TEA entrapped directly correlates to the amount of irinotecan encapsulation. In other words, encapsulated solutions of 90mM TEA would facilitate irinotecan loading to a drug-to-lipid ratio of approximately 0.09 after a 60 minute incubation and 270mM TEA would be able to produce liposomes with a final irinotecan-to-lipid ratio of 0.18 [257]. The copper gluconate concentration for these studies was 100mM and the external buffer used was a sucrose phosphate buffer (300mM sucrose, 40mM phosphate) at pH 7. For these studies there was no evidence to suggest that copper complexation facilitated irinotecan loading and it was proposed that loss of TEA from the inside of the liposome generated a transmembrane pH gradient (interior acidic) that was sufficient to engender irinotecan loading.       Topotecan and irinotecan have been encapsulated in liposomes through the use of entrapped copper in the presence or absence of A23187 [246, 286, 332-335]. It is 109  important to note that campothecins contain an α-hydroxy-δ-lactone ring moiety, a functionality that is unstable at pH greater than 7. The active form of the drug is believed to be the lactone ring closed structure which is stabilized in acidic pH and is essential for therapeutic activity [336, 337]. The hydrolysis of the lactone yields the carboxylate (inactive) form of the drug [338, 339]. Under conditions where the liposome interior is acidic (<5.0), the drug is stabilized in its lactone form [330, 333]. As indicated, irinotecan and topotecan can be encapsulated in liposomes using pH gradient-dependent methods (e.g. pH 4.0 citrate buffer, ammonium sulfate, MnSO4 buffers with added A23187, etc) [16, 242, 332, 340, 341]. pH gradient loading was achievable because these drugs have protonizable amine functions. However, the chemical structure of irinotecan and topotecan suggests several coordination sites capable of complexing copper, with one of the sites potentially located at the lactone ring. Stabilization of topotecan in an active lactone form was previously attributed to the formation of a coordination complex with copper ions at this site [330, 333]. The sites of topotecan-copper complexation structure, however, have not been elucidated but it was confirmed that a complex is present using titration methods [330]. Interestingly, drug loading appears to be driven by two distinct mechanisms in the presence and absence of A23187 as reported by Taggar et al. [330]. In the absence of A23187, the loading occurred both at 40˚C and 60˚C and was driven by Cu-topotecan complexation. In contrast, in the presence of A23187, the loading occurred at higher temperature (60˚C) in response to transmembrane pH gradient. Efficient encapsulation in the presence of Cu2+ did not require utilization of A23187. However, when copper complexation was combined with transmembrane pH gradient, substantial improvement in the drug retention was reported [333].  The results summarized above suggest that there is something unique about copper and its potential to bind selected drug candidates, however the examples above have been limited to drugs that contain a protonizable amine functionality as well as copper binding motifs [13]. As another example, mitoxantrone was encapsulated into Cu2+-containing liposomes unbuffered or buffered to pH 7.5 with TEA [342]. Both CuSO4 and CuCl2 resulted in complete drug encapsulation. Li et al. attempted to account for drug loading mechanism by using CuSO4 solutions pH adjusted with TEA. They concluded, similar to the research described above, that TEA is membrane permeable and the formulation likely behaved 110  similarly to (NH4)2SO4 remote loading methods where loss of TEA from the liposomes created a pH gradient that facilitates loading. They attempted to use nigericin, an ionophore which exchanges H+ from the interior of the liposome in exchange for potassium, and showed that mitoxantrone could be encapsulated into CuSO4 liposomes in the presence of nigericin which would suggest encapsulation in a pH gradient independent manner. The CuCl2 containing liposomes could not be used to stably encapsulate mitoxantrone under these conditions.  To clearly highlight the utility of copper-complexation as a method of candidate drug remote loading into liposomes the focus has shifted to compounds that do not have protonizable amine function but do have donor moieties (O, N and S) that can bind copper. This thesis reports that CBTs can be synthesized in liposomes;[66] a strategy that was defined as the Metaplex™ technology (Figure 7.1). This approach eliminates the use of solvents, addresses solubility issues and results in formulations of CBTs (CLCs and CDCs) that are suitable for parenteral administration. The method utilizes the liposome as a nanoscale reaction vessel in which the copper complex is synthesized. Briefly, liposomes are prepared using extrusion in the presence of copper containing solutions and the external copper is removed using column chromatography.[66, 276] The prepared liposomes with copper encapsulated in the core are mixed with the ligand of interest and complexation can be observed in the form of colour changes and spectrophotometric shifts in absorbance. The resulting CBTs remains in suspension and the external buffer in which the liposomes are suspended can be exchanged to one that is suitable for systemic administration.  CBTs represent a class of compounds with potential activity in a wide range of diseases is promising despite the pharmaceutical challenges presented by the inherent solubility issues of these compounds. There is a wide selection of CBTs that have been created as potential drug candidates. This thesis described the Metaplex™ methodology which provides a simple, transformative solution, enabling for the first time, the development of CBTs as viable candidate drugs; drugs that would represent a new class of therapeutics for patients. 111   Figure 7. 1: Schematic representation of MetaplexTM. The lipid composition of interest is dissolved into chloroform and this is then dried using a rotary evaporator. The lipid film is rehydrated using a Cu containing solution. The resulting multilamellar vesicles (MLVs) undergo five freeze-thaw cycles which is followed by extrusion using 100 nm filters. Size exclusion chromatography was performed using Sucrose/HEPES/EDTA (SHE, 300, 20 and 15 mM) pH 7.4 buffer to remove external unencapsulated copper. The resulting Cu containing liposomes are mixed with the ligand of interest and the unencapsulated ligand was removed using size exclusion chromatography. The liposomes containing the copper complex were concentrated using tangential flow dialysis.   The difference between remote loading with metals (absence of ionophores) and MetaplexTM are three-fold. First, the candidate ligands/drugs do not contain a protonizible 112  amine functionality and thus cannot be loaded using traditional remote loading method that rely on use of a transmembrane pH gradient. As seen in Figure 3.2C, the removal of the proton gradient did not affect the formation of Cu(DDC)2 inside the DSPC/Chol (55:45) liposomes. Second, there is a subset of CBTs that have been described in Chapter 1, known as CLCs, which have copper dependant activity. Thus, the activity of these ligands requires copper complex formation for activity and this is in contrast to other drugs where the metal was used solely to facilitate encapsulation of the added drug/candidate drug. Third, remote loading has been used on hydrophilic drug candidates; this was later adapted to hydrophobic drugs through dissolution of the drug candidate using solvents such as DMSO [343].The approach has been used specifically in the formulation and development of hydrophobic copper complexes with both hydrophilic ligands (DDC, CX-5461) and hydrophobic ligands (CQ, Qu). While the dissolution of the ligands in a solvent was unnecessary, it could be done to reduce the incubation time during the complexation process. One would need to consider solvents such as DMSO as competitive binders of copper and this could affect complex synthesis.  7.4 Future Work Owing to the versatility of the platform technology many other possible research projects can be developed around this formulation approach. Here, I will briefly discuss possible the applications which I find most compelling. 7.4.1 Other Metals While Cu(II) is known to coordinate with N,O and S containing ligands, other metals could be used as alternatives. Specifically, the potential to use Zn(II) as an alternative metal is considerable. Zn complexes may exhibit different formulation attributes (stability, release rates, etc), but more interestingly the activity of the Zn-ligand complex may be different from the Cu-ligand complex.  This is illustrated by the data shown in Figure 7.2 and 7.3. The use of Zn(II) did not increase the activity of DDC when compared to Cu(DDC)2 in platinum sensitive and platinum resistant cancer cells (Fig. 7.2), however the activity of Zn(CQ)2 complexes was 1 to 2 orders of magnitude greater then when compared to the 113  Cu(CQ)2 complexes (Figure 7.3). This is likely related to the specific mechanism of action of each complex formed. The use of Zn or other divalent metals such as Fe, Co, Ni, etc. can be incorporated into liposomes through the addition of unbuffered solutions of these metals and it is reasonable to anticipate that the formation of other divalent metal ion complexes would be comparable to that achieved when using copper loaded liposomes.  Figure 7. 2: The cytotoxicity of DDC (●), Cu(DDC)2  (■) and Zn(DDC)2() in A2780-S (A) and A2780-CP (B) cells. Cytotoxicity curves were generated in A2780-S and A2780-CP cells after 72 hr treatment with DDC, Cu(DDC)2  or Zn(DDC)2. Cell viability was obtained using the INCell analyzer 2200 where viability was assessed based on loss of plasma membrane integrity 72 hours following treatment; i.e. total cell count and dead cell count were determined using Hoechst 33342 and ethidium homodimer staining, respectively. (C) IC50 ± 95% CI were calculated from cytotoxicity curves generated for DDC, Cu(DDC)2  and Zn(DDC)2 in A2780-S and A2780-CP after 72 hr treatment using Graphpad Prism software. Data is presented as the mean of 3 independent experiments ± SEM 114   Figure 7. 3: The cytotoxicity of CQ (●), Cu(CQ)2  (■) and Zn(CQ)2() in A2780-S (A) and A2780-CP (B) cells. Cytotoxicity curves were generated in A2780-S and A2780-CP cells after 72 hr treatment with CQ, Cu(CQ)2  or Zn(CQ)2. Cell viability was obtained using the INCell analyzer 2200 where viability was assessed based on loss of plasma membrane integrity 72 hours following treatment; i.e. total cell count and dead cell count were determined using Hoechst 33342 and ethidium homodimer staining, respectively. (C) IC50 ± 95% CI were calculated from cytotoxicity curves generated for CQ, Cu(CQ)2  and Zn(CQ)2 in A2780-S and A2780-CP after 72 hr treatment using Graphpad Prism software. Data is presented as the mean of 3 independent experiments ± SEM 7.4.2 Other Diseases and Complexes Although the focus of this thesis was on copper-based anti-cancer agents, copper complexes with significant anti-microbial, anti-viral and anti-inflammatory activity have also been identified. Although the pharmaceutical development of metal complexes targeting these indications may not require development of injectable solutions (i.e. topical anti-bacterial agents, oral anti-inflammatory drugs) many will be sparingly soluble and formulations could be adapted for these agents.  115  Additionally, this thesis has focused on bidentate ligands which form dimeric structures which are the simplest case. The method could be altered to develop other types of copper complexes such as NCI-109268 which is a ligand that forms a dichloro-copper complex [39]. The copper complex of NCI-109268 appears to act as a proteasome inhibitor, similar to Cu(DDC)2 and Cu(CQ)2. For a compound like NCI-109268 (Figure 7.3), the liposomes would need to be prepared in CuCl2 solutions instead of CuSO4 solutions and the ligand would be introduced to pre-formed CuCl2 containing liposomes.  This is illustrated in Figure 7.4.   Figure 7. 4: Structures of the copper based proteosome inhibitor NCI-109268 7.5 Conclusion This thesis research was initiated based on an idea related to repurposing DSF for use in cancer. However, after recognizing that the active agent following administration of DSF was Cu(DDC)2 the focus switched to the development of a formulation of Cu(DDC)2  suitable for intravenous administration. I hypothesized that Cu(DDC)2  could be synthesized inside liposomes and that the resulting formulations would be suitable for further pharmaceutical development as a novel class of anti-cancer agent. I have demonstrated that when DDC is added to copper containing liposomes that Cu(DDC)2  formed inside the liposomes. Perhaps more importantly, I have shown that this approach, where CBTs can be synthesized inside liposomes can be applied to other copper binding ligands, thus defining a novel platform approach from which other CBTs or MBTs could be developed.  Of course, addressing the formulation concerns for CBTs represents only one of 116  many hurdles that must be crossed if this novel drug class will be therapeutically successful. More information about mechanism of action and potential for off target effects will need to be obtained for each candidate CBT being developed. Further the safety profile of candidate CBTs will need to be fully understood. For example, the literature surrounding copper in medicine has to date focused on the use of chelators to correct copper dysregulation but as candidate CBTs are identified it will be necessary to examine their effects on copper homeostasis and associated issues. 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