Medicine, Faculty of
DSpace
UBCV
Chen, Kent Tsung Jeng
Doctor of Philosophy - PhD
University of British Columbia
For more than 30 years, treatment of acute myeloid leukemia (AML) has remained largely unchanged and reliant on chemotherapeutic drug combinations. Despite the approval of novel targeted therapies, broad-spectrum chemotherapy drugs have been used to address the genetic heterogeneity of AML. One class of broad-spectrum compounds called flavonoids has shown promise in the treatment of AML. Of the flavonoids, quercetin and flavopiridol were selected for further investigations. Both quercetin and flavopiridol are hindered therapeutically by their poor solubility in aqueous solutions and tendency to bind serum proteins. To overcome these limitations, quercetin and flavopiridol were reformulated in copper-containing liposomes using the flavonoids’ metal-binding properties. Both formulations attained significant increases in apparent solubility (>100-fold). When injected intravenously into animals, both formulations exhibited an increased plasma area under the curve (AUC)) with no acute or chronic toxicities at the doses given. Given quercetin’s general lack of therapeutic activity as a single agent (IC50 of >10µM when tested against cancer cell lines), the research focused on the formulation of flavopiridol. In subcutaneous AML models, the flavopiridol formulation exhibited enhanced activity compared to a low pH free flavopiridol formulation. Despite the significant therapeutic potential uncovered in this thesis, liposomal flavorpidol’s potential is likely best achieved as part of a combination treatment. A combination regimen called FLAM (sequential treatment of flavopiridol, cytarabine, and mitoxantrone) inspired the development of a combination product consisting of liposomal flavopiridol and liposomal mitoxantrone. Free flavopiridol and mitoxantrone combined to produce significant synergistic activity in vitro. However, when administered in vivo, the combination of liposomal flavopiridol and liposomal mitoxantrone (referred to as enFlaM) resulted in an unexpected increase in toxicity and limited the dose of the combination that could be used in vivo. Although the enFlaM combination exhibited therapeutic effects at reduced doses, the effects were significantly less than what could be achieved with a well-tolerated dose of the single agent. Despite that further modifications will be necessary to devise an optimal combination scheme for the administration of enFlaM, the liposome-based Metaplex technology demonstrated the abilities to reformulate poorly soluble drugs to overcome limitations that would otherwise hinder their clinical utilities.
Development of copper-flavonoid complexes for treatment of acute myeloid leukemia (AML) by Kent Tsung Jeng Chen M.Sc., The University of British Columbia, 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 (Interdisciplinary Oncology) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) June 2020 © Kent Tsung Jeng Chen, 2020 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: Development of copper-flavonoid complexes for treatment of acute myeloid leukemia (AML) submitted by Kent Tsung-Jeng Chen in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Interdisciplinary Oncology Examining Committee: Dr. Marcel Bally, Pathology and Laboratory Medicine/Interdisciplinary Oncology Supervisor Dr. Howard Lim, Medical Oncology Supervisory Committee Member Dr. Urs Hefali, Pharmaceutical Sciences University Examiner Dr. Xiaoyan Jiang, Interdisciplinary Oncology University Examiner iii Abstract For more than 30 years, treatment of acute myeloid leukemia (AML) has remained largely unchanged and reliant on chemotherapeutic drug combinations. Despite the approval of novel targeted therapies, broad-spectrum chemotherapy drugs have been used to address the genetic heterogeneity of AML. One class of broad-spectrum compounds called flavonoids has shown promise in the treatment of AML. Of the flavonoids, quercetin and flavopiridol were selected for further investigations. Both quercetin and flavopiridol are hindered therapeutically by their poor solubility in aqueous solutions and tendency to bind serum proteins. To overcome these limitations, quercetin and flavopiridol were reformulated in copper-containing liposomes using the flavonoids’ metal-binding properties. Both formulations attained significant increases in apparent solubility (>100-fold). When injected intravenously into animals, both formulations exhibited an increased plasma area under the curve (AUC)) with no acute or chronic toxicities at the doses given. Given quercetin’s general lack of therapeutic activity as a single agent (IC50 of >10µM when tested against cancer cell lines), the research focused on the formulation of flavopiridol. In subcutaneous AML models, the flavopiridol formulation exhibited enhanced activity compared to a low pH free flavopiridol formulation. Despite the significant therapeutic potential uncovered in this thesis, liposomal flavorpidol’s potential is likely best achieved as part of a combination treatment. A combination regimen called FLAM (sequential treatment of flavopiridol, cytarabine, and mitoxantrone) inspired the development of a combination product consisting of liposomal flavopiridol and liposomal mitoxantrone. Free flavopiridol and mitoxantrone combined to produce significant synergistic activity in vitro. However, when administered in vivo, the combination of liposomal flavopiridol and liposomal mitoxantrone (referred to as enFlaM) resulted in an unexpected increase in toxicity and limited the dose of the combination that could be used in vivo. Although the enFlaM combination exhibited therapeutic effects at reduced doses, the effects were significantly less than what could be achieved with a well-tolerated dose of the single agent. Despite that further modifications will be necessary to devise an optimal combination scheme for the administration of enFlaM, the liposome-based Metaplex technology demonstrated the abilities to reformulate poorly soluble drugs to overcome limitations that would otherwise hinder their clinical utilities. iv Lay Summary Acute myeloid leukemia, or AML, has been challenging to treat especially when the disease relapses. Unfortunately, new AML treatments have been lacking for the past few decades. Thus, there is a clear need for the development of novel therapeutics and in this thesis, flavonoids have been identified as a class of compounds that shows promise in the treatment of AML. These flavonoids, however, face solubility issues that make formulating and administering them in a clinical setting challenging. Through this thesis, new methods to formulate promising drugs were explored in order to enhance the flavonoids’ therapeutic activity and to amplify the benefits they can potentially harness. The results from this thesis demonstrated the robustness of the liposome-based Metaplex technology to reformulate poorly soluble drugs, providing solution to solubility challenges that would otherwise limit the clinical utilities of these drugs. v Preface The research conducted in this thesis was designed and incepted by myself and Dr. Marcel Bally. Chapter 1 includes background information on AML and a review on the current and future therapeutic landscape of AML. A portion of this chapter has been published: Chen, K.T., Gilabert-Oriol, R., Bally, M.B., and Leung, A.W. 2019. Recent treatment advances and the role of nanotechnology, combination products, and immunotherapy in changing the therapeutic landscape of AML. Pharmaceutical Research. 36(9):125. Doi: 10.1007/s11095-019-2654-z This work was completed in collaboration with Dr. Ada Leung and Dr. Roger Gilabert-Oriol. Dr. Leung wrote sections on immunotherapy and Dr. Gilabert-Oriol contributed to the sections on formulations for quercetin and flavopiridol. Dr. Bally edited the manuscript. I organized the manuscript and wrote the vast majority of the text and designed all the figures. The research described in Chapter 2 has been published: Chen, K.T., Anantha, M., Leung, A.W., Kulkarni, J. A., Millitao, G.G.C., Webhe, M., Sutherland, B., Cullis, P. R., Bally, M.B. Characterization of a liposomal copper(II)-quercetin formulation suitable for parenteral use. Drug Delivery and Translational Research (online September 3rd, 2019). Doi: 10.1007/s13346-019-00674-7 I designed and completed all work related to the liposomal quercetin formulation: these include all in vitro characterization studies and formulation preparations for in vivo pharmacokinetic (PK) study. Animal work was conducted by the animal technicians from the Investigational Drug Program (IDP) and plasma quercetin concentrations were determined on the HPLC by Malathi Anantha. Analyses of lipid and copper concentrations from PK study were done by me. In vitro cytotoxicity studies investigating effects of quercetin alone and in combination with irinotecan were completed by myself (A549 lung cancer cell line) and Dr. Gardenia Militao (BxPC3 pancreatic cell line). Cryo-transmission electron microscopy images were obtained by Dr. Jayesh Kulkarni from Dr. Pieter Cullis’ lab at the University of British Columbia. Dr. Militao contributed to the BxPC3 dose response curve. Dr. Webhe provided valuable intellectual inputs. Dr. Sutherland trained me to do tissue culture initially and provided vi intellectual inputs to the project. Dr. Bally edited the manuscript. I analyzed all the data, wrote the entire manuscript, and prepared all figures and tables. The research described in Chapter 3 is under review for a publication: Chen, K.T., Millitao, G.G.C, Anantha, M., Bally, M., Leung, A.W. Development and characterization of a novel flavopiridol formulation for treatment of acute myeloid leukemia. I designed and completed the in vitro cytoxicity study that investigated the effects of exposure time of flavopiridol on AML cells. Dr. Gardenia Militao completed the in vitro characterizations of liposomal flavopiridol with my assistance and guidance and using my experimental designs. I designed the in vivo studies (including model development) and prepared all formulations for in vivo studies. Animal work was conducted by the animal technicians from the Investigational Drug Program (IDP) and plasma drug concentrations were determined on the HPLC by Malathi Anantha. Dr. Bally edited the manuscript. Dr. Leung provided valuable intellectual inputs and also edited the manuscript. I analyzed all the data, wrote the manuscript, and prepared all figures and tables. The research described in Chapter 4 will be submitted for publication in 2020. I designed and completed the in vitro cytotoxicity study investigating the effects of flavopiridol and mitoxantrone as single agents and in combination. I also designed and executed all the in vivo studies, preparing all formulations for the studies. Animal work was conducted by the animal technicians from the IDP and drug concentrations were determined on the HPLC by Malathi Anantha. I wrote the text for the chapter, analyzed all data, and prepared all the figures and tables. Chapter 5 includes a discussion on the role of nanomedicine in combination therapy as well as a review on the immunotherapy landscape of AML. The immunotherapy portion of the discussion is part of the review article also mentioned in Chapter 1. I wrote the text for Chapter 5 and prepared all figures and tables. vii All manuscripts were reviewed and edited by my PhD research supervisor, Dr. Marcel Bally. The scientific research presented in this thesis was completed in accordance with the University of British Columbia Policies and Procedures, Biosafety Practices and Public Health Agency of Canada guidelines (Biohazard Approval Certificate Protocols B08-0113, B10-077, B14-0241). All rodent studies were completed under an animal care protocol (A18-0276) approved by the Institutional Animal Care Committee (IACC). The IACC for studies conducted at BC Cancer’s Vancouver Cancer Research Centre was done in their Animal Resource Center with ethic oversight by the University of British Columbia Animal Care Committee and in accordance to the Canadian Council of Animal Care (CCAC). viii Table of Contents Abstract ........................................................................................................................................... iii Lay Summary ................................................................................................................................... iv Preface ............................................................................................................................................. v Table of Contents .......................................................................................................................... viii List of Tables .................................................................................................................................. xii List of Figures ................................................................................................................................ xiii List of Abbreviations ...................................................................................................................... xv Acknowledgements ..................................................................................................................... xviii Dedication ..................................................................................................................................... xix 1. Introduction ................................................................................................................................ 1 1.1 Project overview, hypothesis, and aims................................................................................ 1 1.2 Acute myeloid leukemia - background ................................................................................. 3 1.3 Challenges of treating AML ................................................................................................... 4 1.4 Novel therapeutic strategies for AML ................................................................................... 5 1.4.1 Midostaurin .................................................................................................................... 9 1.4.2 Enasidenib..................................................................................................................... 10 1.4.3 Venetoclax .................................................................................................................... 10 1.4.4 Glasdegib (DaurismoTM) ................................................................................................ 11 1.4.5 Gemtuzumab ozogamicin ............................................................................................. 12 1.4.6 CPX-351 (Vyxeos®) ........................................................................................................ 12 1.5 Flavonoids – opening new doors in AML ............................................................................ 13 1.6 Liposomes ............................................................................................................................ 14 1.6.1 Phospholipids................................................................................................................ 16 1.6.2 Cholesterol .................................................................................................................... 17 ix 1.6.3 Poly(ethylene glycol) (PEG)- modified lipids ................................................................ 17 1.6.4 Encapsulation of drugs and drug candidates in liposomes .......................................... 18 1.7 Nanoparticles for the treatment of AML ............................................................................ 20 1.8 Nanoparticulate formulations of quercetin and flavopiridol .............................................. 21 1.9 Summary ............................................................................................................................. 23 2. Characterization of a liposomal copper (II)-quercetin formulation suitable for parenteral use....................................................................................................................................................... 25 2.1 Introduction ......................................................................................................................... 25 2.2 Materials and methods ....................................................................................................... 26 2.2.1 Materials ....................................................................................................................... 26 2.2.2 Determination of quercetin solubility .......................................................................... 26 2.2.3 Liposome preparation .................................................................................................. 26 2.2.4 Copper-quercetin formation inside liposomes ............................................................ 27 2.2.5 Characterization of copper quercetin binding ............................................................. 28 2.2.6 In vitro serum stability assay ........................................................................................ 28 2.2.7 Cryogenic electron microscopy (CEM) of copper-quercetin formulations .................. 29 2.2.8 Pharmacokinetics of the copper-quercetin formulations ............................................ 29 2.2.9 In vitro cytotoxicity assays of quercetin alone and in combination with irinotecan ... 30 2.2.10 Statistical analysis ....................................................................................................... 31 2.3 Results ................................................................................................................................. 32 2.3.1 Formation of copper-quercetin within copper containing DSPC/Chol (55:45 mol%) liposomes. .............................................................................................................................. 32 2.3.2. Suitability of the copper-quercetin liposomes as a formulation for intravenous use. 41 2.3.3. Therapeutic potential of quercetin when used in combination with Irinotecan ........ 44 2.4 Discussion ............................................................................................................................ 45 2.5 Conclusions .......................................................................................................................... 49 x 3. Development and characterization of a novel flavopiridol formulation for treatment of acute myeloid leukemia .......................................................................................................................... 51 3.1 Introduction ......................................................................................................................... 51 3.2 Materials and methods ....................................................................................................... 53 3.2.1 Materials ....................................................................................................................... 53 3.2.2 Tissue culture ................................................................................................................ 53 3.2.3 Dose response curves ................................................................................................... 53 3.2.4 Spectrophotometric analysis of flavopiridol and copper-flavopiridol in methanol ..... 54 3.2.5 Liposome preparation .................................................................................................. 54 3.2.6 In vitro characterization of liposomal copper-flavopiridol ........................................... 55 3.2.7 In vivo characterization of flavopiridol ......................................................................... 56 3.2.8 Statistical analysis ......................................................................................................... 59 3.3 Results ................................................................................................................................. 60 3.4 Discussion ............................................................................................................................ 70 3.5 Conclusions .......................................................................................................................... 73 4. Development of a novel combination product of liposomal flavopiridol and liposomal mitoxantrone for treatment of acute myeloid leukemia ............................................................. 74 4.1 Introduction ......................................................................................................................... 74 4.2 Materials and methods ....................................................................................................... 76 4.2.1 Materials ....................................................................................................................... 76 4.2.2 Tissue culture ................................................................................................................ 76 4.2.3 In vitro dose response curves for flavopiridol and mitoxantrone ................................ 76 4.2.4 Liposome preparation .................................................................................................. 77 4.2.5 Preparation of liposomal flavopiridol and liposomal mitoxantrone ............................ 78 4.2.6 In vivo studies ............................................................................................................... 79 4.2.7 Statistical analysis ......................................................................................................... 80 xi 4.3 Results ................................................................................................................................. 81 4.4 Discussion ............................................................................................................................ 93 4.5 Conclusions .......................................................................................................................... 96 5. Summarizing discussion ............................................................................................................ 97 5.1 Summary of results ............................................................................................................. 97 5.2 Nanoparticle-based combination therapy – the road ahead ............................................. 99 5.3 Challenges of developing combination therapy ............................................................... 100 5.4 Small molecule immunotherapy for treatment of AML ................................................... 102 5.4.1 Hematopoietic stem cell transplantation (HSCT) ....................................................... 103 5.4.2 Antibody-drug conjugates .......................................................................................... 104 5.4.3 AML vaccines .............................................................................................................. 104 5.4.4 Chimeric antigen receptor T (CAR-T) cell therapy ...................................................... 105 5.4.5 Immune checkpoint inhibitors (ICPIs) ........................................................................ 106 5.4.6 Small molecules selected to achieve immunogenic cell death and the immunotherapeutic potential of liposomal flavopiridol and/or liposomal mitoxantrone . 106 5.5 Conclusions and future perspectives ................................................................................ 107 6. References .............................................................................................................................. 110 xii List of Tables Table 1.1 Therapeutic landscape of AML 8 Table 2.1 Particle size (nm) of CuSO4-liposomes and CuG-liposomes incubated at 60°C for 60 minutes in the absence or presence of quercetin 34 Table 2.2 Pharmacokinetics parameters of quercetin following intravenous administration (5mg/kg) 44 Table 3.1 Clinical signs of toxicity for preliminary tolerability study 68 Table 4.1 Toxicity study of flavopiridol and mitoxantrone as single agents 85 Table 4.2 Toxicity study of flavopiridol and mitoxantrone in combinations 86 Table 4.3 Toxicity study of flavopiridol and mitoxantrone in combinations 87 Table 4.4 Toxicity study of liposomal flavopiridol and liposomal mitoxantrone in combination 88 Table 4.5 Toxicity study of liposomal flavopiridol and liposomal mitoxantrone in combination 89 xiii List of Figures Figure 1.1 Most commonly found mutations in AML 4 Figure 1.2 Progression of AML therapeutics over the years 6 Figure 2.1 Temperature-dependent formation of copper-quercetin complexes in liposomes 33 Figure 2.2 Copper-dependent quercetin association with liposomes and the impact of intra-liposomal pH 35 Figure 2.3 Quercetin association with liposomes prepared with CuSO4 or Cu-gluconate 37 Figure 2.4 Quercetin association with liposomes prepared using solutions containing different copper gluconate (CuG) concentrations 38 Figure 2.5 Spectrophotometric studies evaluating quercetin-copper interactions 39 Figure 2.6 Representative cryo-transmission electron microscopy images of liposomes 41 Figure 2.7 In vitro stability of copper-quercetin formulations prepared using liposomes prepared in 300 mM CuSO4 or 100 MM copper gluconate 42 Figure 2.8 Pharmacokinetics of copper-quercetin formulations made with liposomes prepared in 300mM CuSO4 or 100mM CuG 43 Figure 2.9 In vitro assay assessing the cytotoxic effects of quercetin alone and in combination with irinotecan 45 Figure 3.1 Effects of exposure time of flavopiridol on acute myeloid leukemia (AML) cells in vitro. 60 Figure 3.2 Effects of temperature on the formation of copper-flavopiridol complexes in liposomes 61 Figure 3.3 Copper-dependent association of flavopiridol with liposomes and the impact of transmembrane pH gradient 62 xiv Figure 3.4 Pharmacokinetics of flavopiridol in vivo following intravenous injection of different flavopiridol formulations 64 Figure 3.5 Copper-flavopiridol prepared in DSPC:DSPE-PEG2000 (95:5 mol:mol) liposomes was compared to the low pH free-drug formulation 66 Figure 3.6 Preliminary tolerability study 67 Figure 3.7 Efficacy of liposomal and free flavopiridol formulations given intravenously to mice with established MV4-11 and MOLM-13 tumours 69 Figure 4.1 In vitro assay assessing the cytotoxic effects of flavopiridol alone and in combination with mitoxantrone 81 Figure 4.2 Efficacy of liposomal and free mitoxantrone given intravenously to mice inoculated with P388 murine leukemia cells 83 Figure 4.3 Efficacy of liposomal flavopiridol and liposomal mitoxantrone in combination given intravenously to mice inoculated with P388 murine leukemia cells 90 Figure 4.4 Efficacy of liposomal and free flavopiridol and mitoxantrone given intravenously to mice with established MOLM-13 tumours 92 Figure 5.1 Immunotherapy strategies for AML treatment 103 xv List of Abbreviations ALL Acute Lymphoblastic Leukemia AML Acute Myeloid Leukemia ANOVA Analysis of Variance AUC Area Under the Curve CAR-T Chimeric Antigen Receptor T Cell CDK Cyclin Dependent Kinase CEM Cryogenic Electron Microscopy CHE 3[H]-Cholesteryl Hexadecyl Ether CLL Chronic Lymphoblastic Leukemia CPT-11 Irinotecan CR Complete Remission CRT Calreticulin DMPC 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine DMSO Dimethyl Sulfoxide DSPC 1,2-Distearoyl-sn-Glycero-3-Phosphocholine DSPE 1,2-Distearoyl-sn-Glycero-3-Phosphoenthanolamine EDTA Ethylenediaminetetraacetic Acid FBS Fetal Bovine Serum FCS Fetal Calf Serum FDA Food and Drug Administration FLT-3 FMS-Like Tyrosine Kinase-3 GAVX Gene-Transduced Tumour Cell Vaccine GM-CSF Granulocyte Macrophage Colony Stimulating Factor GO Gemtuzumab Ozogamicin xvi GVHD Graft-versus-Host Disease HSCT Hematopoietic Stem Cell Transplantation IC50 Inhibitory Concentration of 50% ICD Immunogenic Cell Death ICPI Immune Checkpoint Inhibitors IDH2 Isocitrate Dehydrogenase 2 I.V. Intravenous LSC Leukemic Stem Cell MLV Multilamellar Vesicles NK Natural Killer Cells PACA Poly(alkylcyanoacrylate) PE Phosphatidylethanolamine PEG Poly(Ethylene Glycol) PG Phosphatidylglycerol PK Pharmacokinetics PLGA Poly(Lactic-co-Glycolic Acid) P-TEFb Transcription Elongation Factor B ROS Reactive Oxidative Species SEM Standard Error of the Mean SHE Sucrose HEPES SM Sphingomyelin SMO Transmembrane Protein Smoothened SOC Standard of Care Tc Phase Transition Temperature xvii TFF Tangential Flow Filtration UV Ultraviolet xviii Acknowledgements The thesis work presented here was supported by Dr. Marcel Bally’s grants awarded by the Canadian Institutes of Health Research (CIHR; 153132) and the Canadian Cancer Society Research Institute (CCSRI; 705290). I would like to thank my supervisor, Dr. Marcel Bally, for his continuous guidance, encouragement, and support and for providing me with all the opportunities for me to grow as a researcher throughout this degree. I am also thankful to my supervisory committee (Dr. Thomas Chang, Dr. Howard Lim, and Dr. Emma Guns) for giving me invaluable suggestions and guidance throughout my degree. I would also like to thank the Investigational Drug Program team at the BC Cancer Research Centre, specifically Dr. Nancy Dos Santos, Nicole Wretham, Edan White, Dr. Ioana Popescu, and Charles Nieh for their technical contribution to the in vivo studies presented in this thesis. I would also like to thank my fellow Bally lab mates, past and present, for helping me through the challenges of my studies. Special thanks to Dr. Brent Sutherland, Dr. Ada Leung, Malathi Anantha, Dr. Roger Gilabert Oriol, Dr. Gardenia Militao, Dr. Mohamed Wehbe, Norman Chow, and Visia Dragowska for your expertise and intellectual support. Finally, I would like to thank my family and friends for their immense support and inspiration throughout this degree as well as providing me with moral support when the going got rough. Dedication xix Dedication To my loved ones1 1. Introduction 1.1 Project overview, hypothesis, and aims Acute myeloid leukemia (AML) is the most common myeloid leukemia and it is particularly prevalent amongst adults aged 65 years or older (1, 2). According to Statistics Canada, there was a total of 1115 new AML cases with an incidence rate of 3.9 cases per 100,000 Canadian population in 2017. The current standard of care (SOC) for AML consists of two phases: remission induction therapy followed by consolidation therapy (2, 3). Induction therapy consists of a “7+3” regimen which entails 7 days of continuous infusion of cytarabine in combination with 3 consecutive days of anthracycline treatment (typically daunorubicin) (3). Upon achieving initial remission from induction therapy, patients then undergo consolidation therapy to eradicate residual leukemia and this regimen typically includes high doses of cytarabine with or without hematopoietic stem cell transplantation (3, 4). For most patients who are 60 years of age or younger, a complete remission is achievable with this initial cytotoxic induction therapy (5, 6). However, despite the likelihood of achieving initial remission, AML remains challenging to treat, particularly when the patients are older or when the disease relapses or becomes refractory (5). Despite efforts to improve therapeutic outcomes and to better understand this disease, the standard of care for AML has remained largely unchanged for more than 40 years (2, 3). With the ultimate goal of improving AML treatment outcomes in the first line setting, this research chose to further develop a class of compounds called flavonoids as promising broad spectrum candidates to deliver improved outcomes for AML patients. In particular, quercetin and flavopiridol were selected as the flavonoids to be developed. The development of quercetin and flavopiridol as treatments has been by problems including low solubility and serum protein binding. For this reason, this thesis defined liposomal formulations of quercetin and flavopiridol - a strategy that relied on the ability of flavonoids to complex with metals, such as copper. The hypothesis guiding this research was that copper-flavonoid complexes can be synthesized within liposomes and the resulting formulations will be suitable for parenteral administration and will be therapeutically effective in murine models of AML. 2 The specific aims of the research were: 1. To characterize the use of copper complexation methods to formulate copper-quercetin and copper-flavopiridol within liposomes and establish the suitability of these formulations for use in preclinical studies. 2. To define the pharmacokinetic profile and the maximum tolerated dose of liposomal copper-quercetin and copper-flavopiridol formulations as well as to select a liposomal formulation suitable for use in efficacy studies. 3. To assess the therapeutic activity of the selected liposomal copper-flavonoid formulation in murine models of AML. 4. To evaluate the use of the selected liposomal copper-flavonoid formulation in combination. The flavopiridol formulation developed by utilizing copper-binding to facilitate liposome association resulted in a novel therapeutic candidate that exhibited improved efficacy in the selected AML models. Even though evidence provided in this thesis suggested that the true value of a formulation of flavopiridol would be as a component of a drug combination, the initial research evaluating the combination of the reformulated flavopiridol and a formulation of mitoxantrone demonstrated significant toxicity. Well-tolerated doses could be defined with the combination, but the therapeutic effects at these tolerated doses were significantly less than what could be achieved with well-tolerated doses of the individual agents. To better understand this thesis research, a summary of AML, AML treatment, and lipid based nanomedicines has been provided below. Although the research describes for the first time liposomal formulations of copper-flavonoid complexes, other investigators have described other nanomedicine formulations of quercetin and flavopiridol. For this reason, a brief summary of these formulations has been provided to allow the reader to compare and contrast the formulation methods. 3 1.2 Acute myeloid leukemia - background AML can arise as a de novo malignancy in an otherwise healthy patient or as a secondary malignancy resulting from mutagenic events caused by leukemogenic agents such as alkylating chemotherapy drugs or radiation (7-9). AML was initially considered a monolithic disease that can be treated with a “one-for-all” chemotherapy drug (10). However, studies over the years revealed that AML is actually a genetically pleiomorphic disease, characterized by genetic heterogeneity at diagnosis (6, 10). A summary of the mutational landscape of AML is presented in Figure 1.1. Therefore, in order to capture this diversity to achieve better treatment outcomes, cytogenetic analysis has become a mainstay in the standard diagnosis of AML (10). As such, the treatment landscape is changing due to (i) a better understanding of what is driving malignancy which points towards targeted therapeutics, (ii) the use of immunotherapy strategies, and (iii) the application of nanoformulation technology to prepare combination products. Here, the clinical challenges of AML, the recently developed therapeutic strategies, and emerging treatment designs are reviewed for the treatment of AML in the newly diagnosed as well as the relapsed or refractory (r/r) setting. 4 Figure 1.1. Most commonly found mutations in AML. 1.3 Challenges of treating AML Management of AML in elderly patients is particularly challenging. Patients who are older than 65 years of age often present adverse cytogenetic risk profiles and these patients typically carry more co-morbidities. In a retrospective analysis of 968 adult AML patients, the percentage of patients with unfavourable cytogenetics increased from 35% in patients younger than 56 years of age to 51% in those older than 75 (11). Furthermore, there was also an increase from 33% in younger patients to 57% in older patients when considering the percentage of patients that develop multidrug resistance (11). The increased incidence of unfavourable cytogenetics coupled with other co-morbidities such as lower white blood cell counts make the older patients less tolerant of intense chemotherapy and hematopoietic stem cell transplantation (HSCT) (4, 11, 12). As a result, treatment outcome deteriorates markedly with age and the 5 prognosis is particularly grim for older AML patients, with complete remission rates of less than 55% and a 5-year survival rate of less than 10% (4, 13). In addition to age, further challenges in treating AML arise when the disease relapses, and this applies to the majority of patients who achieved initial remission (5, 14). The complexities and challenges of treating relapsed and refractory (r/r) AML often stem from the genetic heterogeneity presented at the time of diagnosis (15). Particularly, studies have demonstrated that AML features dynamic clonal evolution at relapse, with the appearance of new mutations that may contribute to the pathogenesis of r/r AML (16). In fact, this clonal evolution has been highlighted as the primary driver of chemotherapy resistance and the cause of treatment failure in r/r AML (16, 17). One of the implicated genetic aberrations that has shown to be prominent in chemo-resistance in AML, contributing to relapses, is the elevated expression of proteins belonging to the Bcl-2 protein family, particularly Mcl-1 (18-20). Mcl-1 is a pro-survival, anti-apoptotic protein that primarily functions as an inhibitor of apoptosis effectors Bak and Bax (19, 20). As such, elevated levels of Mcl-1 have been shown to delay apoptosis, leading to chemo-resistance and the ensuing relapse of the disease (18, 20, 21). Over the past four decades, multiple clinical trials were conducted to examine alternative doses, schedule, and even new cytotoxic agents in an attempt to achieve therapeutic improvements (13, 22). Unfortunately, AML patients still face the same therapeutic obstacles and relapse after complete remission (2, 3, 13). AML clearly presents an unmet medical need and new therapeutic strategies to improve treatment outcomes of AML, in particular r/r AML, are urgently required. 1.4 Novel therapeutic strategies for AML For decades, AML has been a therapeutic graveyard of failed drug programs designed to look for new treatment regimens. The vast majority of the therapeutic studies have typically come from tinkering with existing therapeutics such as increasing doses of daunorubicin and cytarabine for induction and consolidation therapy respectively or alternatively assessing daunorubicin replacements such as mitoxantrone or vosaroxin (23-26). Fortunately, there was a change in the therapeutic landscape for AML in 2017 and 2018 with novel therapeutic agents 6 being approved by the Food and Drug Administration (FDA) for the treatment of AML (Figure 1.2). Figure 1.2. AML therapeutics over the years. 2017 saw the approval of four new therapeutic options by the FDA: Midostaurin (Novartis Pharmaceuticals), Enasidenib (Celgene and Agios Pharmaceuticals), Vyxeos® (also known as CPX 351; Celator/Jazz Pharmaceuticals), and Gemtuzumab ozogamicin (Pfizer) (27). Further progress was made in 2018 with the approval of Venetoclax (AbbVie and Genentech) and DaurismoTM (Pfizer). Ivosidenib (Agio Pharmaceuticals) and gilteritinib (Astellas Pharma US) were also approved as single-agent treatment for refractory and relapsed AML in 2018. Nevertheless, given the tumour heterogeneity and the ability of AML cells to activate a plethora of pro-survival signalling pathways, the use of broad-spectrum chemotherapy drugs or combination treatments that target multiple leukemogenic pathways will likely be required to achieve improvements in overall survival in this patent population (28, 29). As such, carefully selected and rational combinations are capable of exhibiting efficacy far greater than single agent activities and new combinations utilizing new and improved single agents should be developed and investigated. The approval of these new therapeutics attempts to address the many 7 challenges of AML treatment and has provided AML patients with alternatives where intensive chemotherapy is not an option. A summary of the therapeutic agents discussed below is presented in Table 1.1. 8 Table 1.1. Therapeutic landscape of AML Drug Structure Use Common toxicities Mechanism of actions Cytarabine Part of the 7+3 standard of care treatment Cerebellar toxicity leading to ataxia, granulocytopenia, thrombocytopenia, GI toxicities Interference and inhibition of DNA replication leading to apoptosis and cell death Daunorubicin Part of the 7+3 standard of care treatment Myelosuppression, alopecia, diarrhea, vomiting, neuropathy, rash Inhibition of topoisomerase II leading to double strand break in DNA and activation of apoptotic pathway Midostaurin For patients with FLT3-positive AML Anemia, febrile neutropenia, neutropenia, thrombocytopenia, diarrhea, vomiting, pneumonia, exfoliative dermatitis Inhibition of FLT3 leads to downstream deactivation of cell proliferation pathways such as PI3K/AKT and MAPK/ERK CPX351 (Vyxeos®) First treatment for patients with two types of AML: newly diagnosed therapy-related AML (t-AML) or AML with myelodysplasia-related changes (AML-MRC) Hemorrhage, febrile neutropenia, rash, edema, diarrhea, vomiting, arrhythmia, nausea Preferential payload delivery of cytarabine and daunorubicin to leukemic cells at fixed synergistic ratio through the use of liposomes Enasidenib For the treatment of adult patients with relapsed or refractory acute myeloid leukemia with an isocitrate dehydrogenase-2 (IDH2) mutation Nausea, vomiting, diarrhea, elevated bilirubin, decreased appetite Inhibition of mutated IDH2, which results in the promotion of the differentiation and proliferation pathways of leukemic cells Gemtuzumab ozogamicin For the treatment of adult patients with newly diagnosed CD33-positive acute myeloid leukemia (AML) Fever, nausea, infection, vomiting, bleeding, thrombocytopenia, stomatitis, neutropenia CD33 tracking leads to targeted delivery of calicheamicin to leukemic cells, which causes DNA double strand breaks, inducing the apoptotic pathway 9 Glasdegib For the treatment of newly-diagnosed acute myeloid leukemia (AML) in adults who are 75 years of age or older or who have other chronic health conditions or diseases (comorbidities) that may preclude the use of intensive chemotherapy Anemia, fatigue, hemorrhage, febrile neutropenia, nausea, edema, thrombocytopenia, dyspnea, decrease appetite, mucositis, constipation, rash Binds to the smoothened receptor, which leads to inhibition of dysregulated Hedgehog signalling pathway that would otherwise prevent differentiation of leukemic stem cells Venetoclax For use in combination with azacitidine or decitabine or low-dose cytarabine for the treatment of newly-diagnosed acute myeloid leukemia (AML) in adults who are age 75 years or older, or who have comorbidities that preclude use of intensive induction chemotherapy Nausea, diarrhea, thrombocytopenia, constipation, neutropenia, febrile neutropenia, fatigue, vomiting, peripheral edema, pneumonia, hemorrhage, myalgia, hypotension, dizziness, oropharyngeal pain Inhibition of the activity of overexpressed anti-apoptotic protein, Bcl-2, thereby promoting the activation of the apoptotic pathway 1.4.1 Midostaurin Some of the most common genetic aberrations found in AML are FLT3 mutations (30). Mutations of the tyrosine kinase FLT3 activate downstream pathways such as the PI3K and RAS/ERK pathways (31). FLT3 mutations can occur as internal tandem duplication (ITD) or can be found in the tyrosine kinase domain (TKD) (30, 31). FLT3 mutations confer a negative prognosis in AML patients and have emerged as important targets for therapy (31). One of these promising inhibitors that specifically act against FLT3-TKD and FLT3-ITD mutants is midostaurin (30, 32). As a single agent, midostaurin has limited utility in AML (30). However, when used in combination with standard of care “7+3” chemotherapy, midostaurin significantly prolongs overall survival as demonstrated in a phase 3 clinical trial with 717 patients, where FLT3-positive AML patients survived 49 months longer based on median survival and had a 5.2 month-improvement in event-free survival. Additionally, these patients had a 24.3% lower risk of death compared to just chemotherapy alone (33). In light of the positive data, FDA approved midostaurin on April 28th, 2017 to be used with the traditional standard of care (cycles of 10 cytarabine and daunorubicin) for the treatment of adult AML patients with newly diagnosed FLT3-mutations (30). This marked the first new FDA approval for AML treatment in decades (32). Additionally, midostaurin is currently under investigation for use in combination with the aforementioned Vyxyeos® to treat patients with FLT3 mutations and preliminary findings revealed robust synergy when the two therapeutic agents were used concurrently (34). 1.4.2 Enasidenib Enasidenib is an isocitrate dehydrogenase 2 (IDH2) inhibitor (35). Isocitrate dehydrogenase mutations are observed in 15-20% of AML patients and cause the reduction of α-ketoglutarate to 2-hydroxyglutarate (36). 2-hydroxyglutarate inhibits the function of histone lysine demethylases, leading to hypermethylation of DNA and histones (36). This, in turn, exerts leukemogenic effects by blocking the differentiation of hematopoietic progenitor cells (36). Enasidenib was approved by the FDA on August 1st, 2017 as a monotherapy drug for the treatment of adult patients with r/r AML with IDH2 mutation. As a single agent, enasidenib is an effective option, with a complete remission (CR) rate of around 20% in AML patients having IDH2 mutations (35, 37). However, as a monotherapy, enasidenib is underutilized as the drug showed marked improvement in CR rate when used in combination with standard induction chemotherapy (37). In a phase 1 clinical study with AML patients possessing IDH2 mutations, combining enasidenib with standard induction chemotherapy resulted in an improvement of CR rates to 67% and 58% in de novo and secondary AML, respectively (37). 1.4.3 Venetoclax Venetoclax is an orally bioavailable Bcl-2 inhibitor (38). As discussed previously, overexpression of Bcl-2 implicates survival of AML cells and is often associated with treatment resistance (38). Inability to completely eradicate leukemic stem cells (LSC) is often believed to be an important cause of treatment failure and disease relapse in AML and high levels of Bcl-2 have recently been identified as a defining characteristic of LSC (39, 40). As a Bcl-2 inhibitor, Venetoclax has shown promising activity when used in combination with hypomethylating agents such as azacitidine or decitabine or with low-dose cytarabine. These venetoclax combinations were 11 granted accelerated approval from the FDA on November 21, 2018 to be used to treat adult AML patients who are aged 75 years or older or patients who have comorbidities that preclude them from being treated with induction chemotherapy (38, 41). Complete remission rates were 37%, 54%, and 21% when venetoclax was used in combination with azacitidine, decitabine, and low-dose cytarabine respectively (38, 41). Phase 3 studies (NCT02993523 and NCT03069352) are currently underway to serve as confirmatory trials to evaluate overall survival when venetoclax is used in combination with azacitidine or low-dose cytarabine. Additionally, venetoclax has shown synergy when used with pan CDK inhibitors such as flavopiridol (alvocidib) and this combination is now being tested as a rational combination in phase I clinical trial (NCT03441555) (42). A novel flavopiridol formulation arising from this thesis is described in detail in Chapter 3. 1.4.4 Glasdegib (DaurismoTM) The Hedgehog signalling pathway is critical to embryonic development by regulating differentiation and proliferation in a time-dependent fashion (43). Dysregulation of the Hedgehog pathway has been detected in a variety of hematopoietic malignancies, predominantly in leukemic stem cells (44, 45). Leukemic stem-like cells are typically quiescent and are thus resistant to conventional chemotherapy treatments (45). Therefore, inhibitors of Hedgehog signalling may be ideal options to target these cells. Glasdegib, or DaurismoTM, is a small molecule inhibitor of the transmembrane protein Smoothened (SMO) (41). As a SMO inhibitor, glasdegib inhibits the activation of downstream Hedgehog targets (41). Based on a randomized phase 2 trial (NCT01546038), the use of glasdegib with low dose cytarabine extended median overall survival by 3.4 months and improved complete response rates by 12.7% relative to using low-dose cytarabine alone (46). Given its promise, glasdegib was approved by the FDA on November 21st, 2018 to be used in combination with low-dose cytarabine to treat AML patients who are 75 years or older or those who have comorbidities that prohibit them from undergoing intensive induction chemotherapy. 12 1.4.5 Gemtuzumab ozogamicin One of the most investigated AML-related targets is CD33 (Siglec-3), an antigen that is highly expressed on blasts of 85-90% patients with AML as well as on normal myeloid cells (47). Expression of CD33 is highly variable with about half of AML patients expressing CD33 on >75% of leukemic blast cells (48). Although anti-CD33 monoclonal antibodies (mAbs) such as lintuzumab are tolerated in human clinical trials, they failed to provide meaningful clinical benefits (49, 50). The only approved CD33-targeting agent is gemtuzumab ozogamicin (GO; Mylotarg™), a humanized CD33 mAb conjugated to a DNA damaging calicheamicin derivative (50). Gemtuzumab ozogamicin was granted FDA approval in 2000. However, based on a phase 3 trial (NCT00085709), the addition of GO to standard chemotherapy not only failed to improve treatment outcomes but was associated with statistically significant increase in treatment-related deaths relative to the standard of care (SOC) arm (48, 51). This led to the voluntary withdrawal of Mylotarg™ by Pfizer in 2010. In September 2017, Mylotarg™ was re-approved by the FDA to be used in combination with daunorubicin and cytarabine for the treatment of newly diagnosed CD33+ AML patients and for r/r CD33+ adult and pediatric patients over the age of two. The approval was based on results from ALFA-0701 (NCT00927498), a multicenter phase III study of 271 newly diagnosed AML patients. Here, the addition of GO to standard chemotherapy extended median event-free survival by 8.2 months (no significant gain in overall survival) with a hazard ratio of 0.66 (50, 52). In light of the success of Mylotarg™ in combination with standard of care chemotherapy, a phase I clinical study was initiated to investigate the potential of using Mylotarg™ with Vyxeos® to treat patients with r/r AML (NCT03672539). 1.4.6 CPX-351 (Vyxeos®) Vyxeos® delivers cytarabine and daunorubicin in liposomes at a fixed synergistic ratio and this demonstrated the ability of liposomes to control ratiometric dosing of anticancer drug combinations (53, 54). Ratio-dependent dosing was based on in vitro data that showed that some drug combinations, but not all, work optimally at a specified drug-to-drug molar ratio. Prior to the inception of Vyxeos®, Mayer et al. examined liposomal formulations of several drug combinations based on the in vitro results showing that drug-drug ratio mattered in achieving synergy (55). For example, CPX-1 demonstrated the importance of ratiometric dosing to 13 synergy maintenance between irinotecan and floxuridine (54). At high irinotecan/floxuridine ratios (10:1), irinotecan may antagonize the activity of floxuridine activity by causing cell cycle arrest in S phase (54, 56). To build on this concept of rationmetric dosing, Mayer et al. experimented with various molar ratios of cytarabine- to-daunorubicin (1:10, 1:5, 1:1, 5:1, and 10:1) and found that only 1:1, 5:1, and 10:1 were synergistic, with the rest being either additive or antagonistic (54). Due to a lack of mechanistic explanations for the interaction, it can only be postulated that given cytarabine’s effects on newly synthesized DNA (therefore S-phase specific), it is possible that at some ratios, daunorubicin may antagonize cytarabine activity by arresting cells in G1 phase (57). Compared to standard of care treatment, Vyxeos® demonstrated superior median overall survival (3.61 months longer), event-free survival (1.22 months longer), and remission rate (14.4% higher) without increasing treatment-related mortality and toxicities (58-60). Liposomes define a well-established nanomedicine technology known to prolong the circulation time of an associated therapeutic in the blood and can decrease toxicities observed at high free drug concentrations (the Cmax) in the blood. The remarkable improvement in treatment outcome by Vyxeos® was attributed to the increased circulation lifetime of the two associated cytotoxic agents as well as maintaining the drug-drug ratio in the plasma compartment at an optimal synergistic drug combination ratio (59, 61, 62). Based on these positive findings, FDA approved Vyxeos® for use in the treatment of adults with newly diagnosed therapy-related AML on August 3, 2017 (63). 1.5 Flavonoids – opening new doors in AML Despite the many promises that the newly approved targeted therapies can deliver, in order to address the genetic heterogeneity of AML, broad-spectrum chemotherapy drugs have been used in the past and and it is argued here that they will continue to be used in the future. One class of broad-spectrum drugs that is of particular interest is a class of compounds called flavonoids. Flavonoids are polyphenolic compounds that are of plant origin and over the years, the interests in flavonoids as anticancer agents have grown as evidence shows that diets consist of flavonoid-rich food items are commonly associated with lowered risk of cancer (64, 65). Preclinically, various flavonoids demonstrated potential for the treatment of haematological malignancies. Flavonoids such as quercetin, tamarixetin, and apigenin are effective at inducing apoptosis in human leukemia cells (66-68). Other natural flavonoids like luteolin, chryslin, 14 naringenin, and baicalein also exhibited antileukemic properties through dysregulation of key cell-cycle specific signalling proteins or induction of macrophage phagocytosis (69, 70). In addition to these naturally derived flavonoids, semi-synthetic flavonoid derives, such as flavopiridol, have displayed potent antileukemic activities (71-73). Acting as a pan-CDK inhibitor, flavopiridol has also been shown to induce apoptosis through the downregulation of anti-apoptotic proteins such as Bcl-2 and Mcl-1 (74, 75). Beyond the preclinical work, only flavopiridol and quercetin progressed to clinical trials against haematological malignancies (76). Despite the remarkable promise that quercetin and flavopiridol displayed as anti-cancer agents, they are both hindered clinically by poor aqueous solubility and activity-crippling limitations such as binding to serum proteins (77-81). One approach to remedy these clinical limitations is through reformulation using nanocarriers, such as liposomes: a technology pursued in this thesis (see Chapters 2 and 3). In this context, the section below provides a brief description of liposomes and their use, particularly in the context of combination therapies such as Vyxeos. 1.6 Liposomes Liposomes were first described in the 1960s by Dr. Alec Bangham when he demonstrated the instantaneous assembly of egg lecithin into multilayered spherical structures when the lipids were introduced and shaken in water (82). Initially, liposomes were used as models for studying properties of cell membranes such as molecule permeability (82). Since then, liposomes demonstrated capabilities to enhance drug candidate solubility, altering toxicity, extending associated drug circulation lifetime, and to deliver and support utilization of macromolecules like proteins (enzymes) and nucleic acids (antisense oligonucleoties, silencing RNA, DNA and mRNA) which ultimately had significant therapeutic potential (83, 84). Over the past six decades, liposomes have firmly established themselves as robust drug delivery systems with a myriad of liposomal formulations approved for various indications from infectious diseases to pain management to cancer (85-89). Liposomes are synthetic vesicles that are spherical, ovoid, or tubular in shape and they are composed primarily of amphipathic lipids like phospholipids (90). 15 There have been many different methods described to prepare liposomes. Similar to what Bangham reported, the simple addition of an aqueous solution to dried lipids results in formation of structures that have multiple concentric lipid bilayers separated by aqueous channels. These multilamellar structures are larger (some greater than 1 micron) and heterogenous exhibiting a broad range of sizes. While multilamellar vesicles (MLVs) can be used for regional applications (for example subcutaneous injection), they are generally not suitable for pharmaceutical development. For this reason, further processing of MLVs is required to make liposomes suitable for parenteral administration. Again a number of different methods have been described, but the method of choice has been extrusion. This method was first described by Olson et al. (91) but modified by Hope et al. (92) in a fashion that allowed higher nitrogen pressures to be used to extrude the liposomes through single channel membranes with a defined pore size. The resulting liposomes exhibited a size comparable to the pore size of the filter used and high lipid concentrations could be used (93). The method was fully scalable and is now used to prepare commercial scale liposomal products. More recently, a mixing methodology involving the use of microfluidic mixing chips to spontaneously form liposomes has been developed. The design of the microfluidic channels, mixing rates, and other parameters result in a one-step method to prepare small lipid structures (94, 95). While this method can be used to make small lipid vesicles, its utility appears to be particularly suitable for preparation of lipid nanoparticles that assemble when cationic lipids are mixed with anionic nucleic acids such as antisense oligonucleotides, siRNA, and mRNA (96-98). For the research described in this thesis, extrusion methods were used to prepare liposomes and for samples that proved difficult to extrude, low percentages of ethanol were used as a solvent to assist in uniform vesicle production. The bilayered structure of liposomes makes them effective delivery systems for both hydrophobic and hydrophilic substances, where hydrophobic molecules can reside within the lipid bilayer and the hydrophilic molecules can be encased in the inner aqueous core of the liposomes (90). As a therapeutic system for treating AML, liposomes also present an interesting angle in that they can be used as delivery vehicles to shuttle compounds of interests to the bone marrow. Specifically, the use of negatively charged liposomes to enhance bone marrow delivery has been demonstrated previously (99, 100). In particular, the recently approved 16 Vyxeos, a negatively charged phosphoglycerol-containing liposomal formulation, showed preferential uptake by the bone marrow (101). This bone-marrow tracking property of liposomes makes them an attractive delivery vessel for therapeutics that target leukemic stem cells. Leukemic stem cells are known to be responsible for relapsed AML and these undifferentiated, quiescent cells are also resistant to cell cycle-dependent cytotoxic therapies (102, 103). Studies have shown that leukemic stem cells reside in the endosteal and vascular niches in the bone marrow, where the cells are further protected from chemotherapy-induced apoptosis (102, 104). As such, targeting bone marrow using liposomes could be effective in successful treatment of AML, particular chemo-resistant and relapsed AML. In the sections below, the methods used to associate compounds of interest with liposomes are described, but first a brief description of the lipid components commonly used in preparing liposomes are provided. 1.6.1 Phospholipids Phospholipids consist of a glycerol backbone with a phosphate headgroup esterified at the C(3) position and fatty acyl chains esterified at the C(1) and C(2) positions (105). Charge of the headgroup and the composition of the fatty acid chains can dictate how the lipid bilayer that these phospholipids form will behave. For instance, liposomes with the anionic lipid phosphatidylserine are eliminated from the blood compartment upon injection as the negatively charged lipids tend to bind serum proteins and be preferentially taken up by phagocytic cells of the mononuclear phagocytic system (106-108). However, other negatively-charged lipids, such as phosphatidylglycerol, have been shown to be useful in preparing liposomal formulations that exhibit improved anti-cancer effects, such as the Vyxeos product (109). In addition to phospholipid head group charge, the composition of the fatty acyl chains can also affect the characteristics of liposomes and their role as nano-carriers. One of the key properties of phospholipids is their phase transition temperature (Tc). Tc is the temperature at which phospholipids changes from a gel to a liquid-crystalline phase and this temperature is a function of fatty acyl chain length and unsaturation with the Tc increasing by 14 to 17°C with the addition of every two methylene units in the fatty acyl chains and decrease with the 17 introduction of double bonds (90). Tc is an important parameter to consider when preparing liposomes for drug delivery. Retention of encapsulated agents can be modulated using lipids of different Tc. In other words, if encapsulated in liposomes prepared with lipids that have high Tc, the encapsulated content tends to be better retained (110). 1.6.2 Cholesterol Despite being composed typically of phospholipids, liposomes also contain cholesterol and this is because of the general belief that liposomes prepared using phospholipids only can be unstable in the blood compartment (90). To mitigate this instability, cholesterol was incorporated into the liposome (111). When incorporated into the lipid bilayer, the bulky steroid rings of cholesterol are thought to modulate the rigidity of the liposomal membrane, thereby increasing its stability at high temperatures and in the presence of destabilizing agents such as proteins. Cholesterol also thought to prevent close “packing” of phospholipids at lower temperature thus maintaining the fluidity of the membrane (111-113). Much of the evidence to support this is now in question, as low cholesterol or cholesterol-free liposomes can be prepared and are suitable for drug delivery applications. The key appears to be prevention of liposome-liposome aggregation and this can be achieved with other lipids, such as the PEG-modified lipids described below (114). 1.6.3 Poly(ethylene glycol) (PEG)- modified lipids PEG is a neutral, non-toxic polymer that is water-soluble and can be chemically linked to lipids. This is typically done through use of phosphatidylethanolamine (PE) where PEG is conjugated to this phospholipid’s amine group. While it has been reported that addition of PEG-modified lipids to the composition of liposomes will enhance liposome circulation lifetime (115), it is also reported that the use of PEG-lipids can enhance drug release rates (116) and for this reason, its use must be done judiciously. In drug delivery systems, it is most likely that PEG association reduces particle- particle interactions by providing a steric barrier (114). For the purpose of this thesis research, PEG modified PE was used to surface-stabilize cholesterol free liposomes (see Reference 108 and Chapter 3). In addition to PEG, other lipids such as phosphatidylglycerol (PG), has been used as an alternative. For example, Vyxeos is a low cholesterol liposome that 18 has been stabilized with PG. However, PEG modified PE has been shown to be most chemically stable in a liposome when exposed to biological systems (117). 1.6.4 Encapsulation of drugs and drug candidates in liposomes In general, there are three ways in which a compound can become associated with a liposome. The compound can be added to the liposomes as they are being prepared: a method referred to as passive encapsulation because the compound becomes associated with the liposome either in the lipid bilayer (a hydrophobic compound) or in the aqueous interior (a hydrophilic compound) (118). In reality, the compound is likely associated with both compartments, but the distribution between the fatty acyl chains and the aqueous interior is likely reflected in the compounds’ water or lipid solubility. This methodology has limitations as it requires processing of the liposomes in the presence of the compound and if the compound is very toxic, there will be manufacturing risks. In addition, the process can be very inefficient, particularly for preparations that do not involve organic solvents or detergents; trapping efficiencies are typically less than 50%, while often approaching 10% (118). Additionally, if the compound used associates with the lipid bilayer, it can alter the properties of the formulation and this in turn can modulate the utility of the liposomal formulation as a drug carrier. Remote loading offers a highly efficient process to associate a candidate compound with pre-formed liposomes (118). One of the most employed remote loading methods involves associating drug candidates with liposomes that have an established transmembrane pH gradient (119-121). Using the pH gradient method, drug candidates with weakly basic amine functions can be added to the exterior of liposomes that have a well-buffered low interior pH (for example encapsulated 300 mM citric acid). The candidate drug redistributes across the lipid bilayer and becomes protonated in the interior of the liposomes, which is at a low pH. The protonated form of the drug is much less membrane-permeable and can thus be trapped within the liposomes (119). Despite the robustness of the pH gradient method, it comes with its limitations as many drug candidates do not possess the necessary protonizable amine function to facilitate pH gradient-mediated loading (122). As such, an alternative encapsulation method was established that relied on the ability of the compound of interest to react with a compound 19 inside the liposome. One example would include the use of transition metals to complex the compound of interest (123). This was originally applied to encapsulate compounds that had both metal complexation moieties as well as amine functions. This methodology was essential for the development of Vyxeos where the two drugs were associated with the liposomes using two methods. Cytarabine was associated via passive encapsulation and daunorubicin was associated with the liposomes through copper-complexation and a pH gradient (54). Importantly, over the past decade, it has now been established that metal-complexation can facilitate the association of compounds that do not have amine functions and those that are sparingly soluble in aqueous solution (solubility of less than 1 mg/mL) (124, 125). This methodology has been referred to as the Metaplex technology. The Metaplex technology is a remote loading method that utilizes a transition metal ion gradient and this ion gradient is used as the main driving force to encapsulate compounds of interest within liposomes (124, 125). As indicated, the candidate drugs would typically not have a protonizable amine, have limited aqueous solubility, and contain metal-binding moieties (124, 125). By using liposomes as nano-scale reaction vessels, the Metaplex technology can be used to formulate sparingly soluble compounds (122). Due to a larger chemical space that would satisfy the requirement for metal-ligand binding to occur, Metaplex has the potential to expand and be used for a more diverse group of compounds than the pH gradient method (126, 127). It is important to note that recent studies from the laboratory of Dr. S. Li have demonstrated that permeability enhancers can be combined with compounds suitable for remote loading. Compounds can be added to liposomes in the presence of a permeability enhancer (such as dimethyl sulfoxide (DMSO) and/or increased temperature) (128, 129). These compounds can then equilibrate across the liposomal membrane and once the permeability enhancer (DMSO) is removed, the compound becomes trapped. This methodology was similar to that developed by Dos Santos et al. who first disclosed the use of ethanol to facilitate pH gradient loading of an anthracycline into liposomes that lacked cholesterol (130). These liposomes were less permeable to the compound of interest and for this reason a permeability enhancer was used. At an appropriate concentration, ethanol could be added to the cholesterol-free liposomes 20 (prepared with PEG-modified lipids as stabilizers) such that the physical properties of the liposomes did not change, but remote loading could be achieved (130). The work of Dos Santos et al. focused on compounds that exhibited aqueous solubility of greater than 1 mg/mL. In contrast, the research described by the research team led by Li selected compound that were sparingly soluble (128, 129). It was argued that in order to get encapsulation via remote loading that the rate of loading was dependent on achieving solutions of the compound being encapsulated (128, 129). If the solvent used was at a concentration that was not disruptive to the liposome structure and the solvent could be removed, these permeability enhancing methods proved useful in preparing liposomal formulations of compounds that had never been prepared before. However, it is worth noting that the research described in this thesis highlighted that when using metal complexation to remotely load a compound, solubility was not a significant concern. For example, as shown in Chapters 2 and 3, solid quercetin or flavopiridol was mixed with liposomes and as the complex formed inside the liposome, the precipitated drug was “brought” into solution. The methodology increased the apparent solubility of these flavonoids up to 500-fold. 1.7 Nanoparticles for the treatment of AML In addition to liposomes, other nanoparticles have been studied to assist in the development of novel AML treatments. Importantly, Vyxeos®, a liposomal nanomedicine, represents the most recent success stories for nanomedicines in general and highlights the use of liposomes as carriers for drug combinations. With Vyxeos® helping to pave the way for other nanomedicines and drug delivery systems, studies have been developed to evaluate the use of various types of nanocarriers as means to improve the overall efficacy of anti-leukemic agents and to decrease the toxicities presented by these agents of interest. Of the various drug delivery platforms investigated, liposomes remain one of the most widely utilized systems (131, 132). In a study published in 2014, Tan et al. developed a method to co-encapsulate safingol and C2-ceramide, which are both bioactive sphingolipids that have displayed efficacy against AML but are limited clinically due to hemolytic toxicities (133). The resulting formulation significantly reduced the toxicities presented by the sphingolipids when compared to the free drug and the co-encapsulated formulation extended median survival time from 24 to 37 days compared to 21 the singly encapsulated C2-ceramide (133). Other liposomal formulations such as liposomal daunorubicin-emetine (a protein synthesis inhibitor) and liposomal GTI-2040 (ribonucleotide reductase-targeting inhibitor) were also developed and examined preclinically against AML cells (134, 135). Co-encapsulation of daunorubicin and emetine resulted in enhanced activity against MOLM-13 cells in vitro, with an increase in the induction of apoptosis by a factor of six (134). Similarly, liposomal GTI-2040 demonstrated significantly improved anti-tumour activity (decreased tumour size and prolonged overall survival of mice) when compared to the free drug (135). In addition to liposomal formulations, polymer-based nanoparticle delivery systems have been explored for AML indications, including polymeric nanoparticles. In 2012, Simon et al. encapsulated ATRA (all-trans retinoic acid), a chemotherapy drug used to treat acute promyelocytic leukemia (an AML subtype), in polymeric nanoparticles made with poly-D,L-lactic-co-glycolide (PLGA) (136). This polymer-based formulations allowed ATRA, an otherwise orally administered drug, to be available for intravenous injections, which would be beneficial for patients who are unable to swallow (136). Varshosaz et al. synthesized folate and retinoic acid grafted/dextran (FA-RA/DEX) polymeric micelles for targeted delivery of doxorubicin in AML (137). The doxorubicin-loaded micelles showed enhanced in vitro cytotoxicity against KG-1 cells when compared to the free drug (137). Another example of a nanocarrier demonstrating therapeutic improvements in AML includes dendrimer-based formulations. Dendrimers are nano-scale polymers that are globular in shape with branch-like configurations (138). Szulc et al. used dendrimers to formulate cytarabine triphosphate and the resulting cytarabine-complexed dendrimers significantly enhanced the cytotoxicity of cytarabine against 1301 cells (a T cell leukemia cell line) (138). 1.8 Nanoparticulate formulations of quercetin and flavopiridol The research described in this thesis focuses primarily on quercetin and flavopiridol. As indicated in Chapters 2 and 3, quercetin and flavopiridol require reformulation to maximize their therapeutic potential. In the literature, efforts have already been made to reformulate these two compounds using various nanoparticle carriers. For example, there are some publications that describe quercetin nanoparticles designed for the treatment of cancer. 22 Quercetin, for example, was entrapped in HSPC/Chol/ PE-PEG2000 liposomes (139), soy lecithin/ cholesterol/ PEG4000 liposomes (140), egg sphingomyelin/ cholesterol/ ceramide-PEG2000 liposomes (141), and lipoid S75/ oleic acid liposomes (142). In these instances, quercetin was entrapped by passive encapsulation when lipid films were being made. Additionally, polymers like poly(lactic acid) nanoparticles (143), superparamagnetic iron oxide nanoparticles (144), and mesoporous silica nanoparticles (145) have all been used to formulate quercetin. As shown in Chapter 2, a quercetin-encapsulated liposomal product was successfully developed using a metal complexation technology that enhanced quercetin’s circulation longevity and improved its apparent solubility (146). As indicated above, flavopiridol is an active drug under investigation for many different types of cancer including AML. However, clinical use of flavopiridol is largely hindered by its relatively low aqueous solubility (78) and high binding affinity to plasma proteins (147). In order to solubilize flavopiridol, it has been dissolved in 30% hydroxypropyl beta-cyclodextrin/0.1 M citrate buffer (pH 4.52) to form a complex that can be clinically administered to patients (148). However, this formulation was reported to induce several side effects such as nausea/vomiting, diarrhea, fatigue, and neutropenia (149). Nanoparticles such as liposomes are well-documented to improve solubility and can protect the associated drug from protein binding and/or enzymatic degradation (90, 150, 151). Therefore, the use of well-designed nanoparticle formulations should help increase the solubility of the drug, prevent its interaction with plasma proteins, and perhaps reduce toxicities associated with the current low pH formulation. Two different types of nanoparticles have been used to formulate flavopiridol. In one study, flavopiridol was loaded by a transmembrane pH-gradient into liposomes composed of different lipids: HSPC/Chol, HSPC/Chol/ Tween-80, and HSPC/Chol/ DSPE-PEG2000 (152). HSPC/Chol/ DSPE-PEG2000, with a mean diameter of 120.7 nm and entrapment efficiency of 70.4%, was selected for pharmacokinetic studies in mice. Liposomal flavopiridol increased the elimination phase half-life from 57.0 min for free drug to 339.7 min for the liposomal drug, decreased clearance from 0.036 L/min to 0.012 L/min, and augmented the AUC from 3.4 min μmol/L to 10.8 min μmol/L (152). These results highlighted the capacity of liposomes to modulate the pharmacokinetic profile of flavopiridol. As shown in Chapter 3 of this thesis, the metal complexation methods used here resulted in more efficient flavopiridol loading into the 23 liposome, did not require the use of permeability enhancers (e.g., flavopiridol powder was mixed directly with the liposomes), and the application of cholesterol free liposomes resulted in significant improvements in the ability of the liposomes to extend the drug’s circulation lifetime. In another study, flavopiridol was encapsulated into poly(lactic-co-glycolic acid) (PLGA) nanoparticles to produce a formulation for local delivery and sustained release of the drug (153). Drug release assays in vitro showed that the nanoparticles had a sustained release of flavopiridol for up to 3 days. This formulation was prepared to inhibit astrocyte growth and inflammatory factor synthesis during the reparation of spinal cord injuries (153). In this context, flavopiridol nanoparticles decreased cell-cycle activation, reduced glial scarring, and facilitated survival and regeneration of neurons in vivo. Presumably, the use of PLGA nanoparticles of flavopiridol could be extrapolated to other diseases such as cancer, where modified pharmacokinetics resulting in extended sustained release of flavopiridol would be of advantage. The development of flavopiridol nanoparticles is limited to the formulation methods described above. 1.9 Summary Liposomes are versatile drug delivery platforms. As both quercetin and flavopiridol contain metal-binding moieties, the research described in this thesis used Metaplex technology, specifically copper-complexation, to prepare liposomal formulations of quercetin and flavopiridol. The formulations exhibited significant increases in apparent solubility and the results demonstrate that liposomal flavopiridol was therapeutically superior to a currently described low pH formulation. The research investigated the use of liposomal flavopiridol as a component of combination treatment in murine models of AML, in hopes of defining an improved combination product for AML- one that would be therapeutically superior to Vyxeos. The latter research show that the combination exhibited 4-fold or greater toxicity when compared to the single agents. Further, at well tolerated doses, the combination was less effective in AML models when compared to single agent therapy. While disappointing, the 24 flavopiridol formulation method is novel and appears superior to other formulation methods. This formulation has significant potential to be developed. 25 2. Characterization of a liposomal copper (II)-quercetin formulation suitable for parenteral use 2.1 Introduction One of the most common phenolics found in fruits and vegetables are the flavonoids. These phenolic compounds are believed to be one reason why fruit and vegetable-rich diets are commonly associated with lowered risk for cancer (64). The most common, naturally-occurring flavonoid is quercetin (3,3′,4′,5,7-pentahydroxyflavone) and it has garnered much attention as a potential anti-cancer agent (154, 155). Quercetin is well-established as an effective antioxidant, protecting against damage associated with oxidative stress from free radicals or reactive oxidative species (ROS) (156, 157). Specifically, quercetin’s antioxidant activity may be due to catalase’s ability to protect cells from oxidative stress and damage by scavenging H2O2 (156-158). It has also been suggested that quercetin exhibits anti-cancer activity through mechanisms that involve induction of apoptosis. In particular, studies with A549 lung cancer cells, human glioma cells, and human hepatoma cells (HepG2) have shown that treatment with quercetin can concurrently downregulate anti-apoptotic proteins such as Bcl-2, AKT and upregulate pro-apoptotic proteins such as Bax (155, 159, 160). Quercetin’s potential as an anticancer agent is further demonstrated in combination therapy. When used in a combination setting, quercetin appears to enhance cisplatin cytotoxicity against ovarian cancer in various murine models of cancer (154). The use of quercetin may also reduce cisplatin induced ototoxicity as it was shown to protect zebrafish embryos against sensory hair cell damage engendered by cisplatin (161). Due to its poor aqueous solubility, most applications of quercetin, whether in the context of clinical trials or as a supplement to therapy, rely on oral administration. However, poor dissolution in gastrointestinal fluids leads to poor gastrointestinal absorption of quercetin, resulting in low bioavailability (162). Therefore, despite its potential chemotherapeutic benefit, the clinical use of quercetin as an anti-cancer agent has remained essentially irrelevant (163, 164). To fully explore the therapeutic potential of quercetin, there is a need to improve its solubility and various strategies have been used to achieve this including the development of 26 more water soluble pro-quercetin compounds that biologically convert to quercetin (165) as well as various formulation approaches involving the use of liposomes (166), polymers (167), or milling to produce nanocrystals (168). In this study, quercetin’s propensity to bind copper (169-171) was used to develop for the first time a liposomal formulation of copper-quercetin suitable for intravenous use. The resulting formulation exhibits at least a 100-fold improvement in the apparent solubility of quercetin and is suitable for intravenous administration. 2.2 Materials and methods 2.2.1 Materials Quercetin, Sephadex G-50, Copper (II) sulfate, HEPES, ethylenediaminetetraacetic acid (EDTA), and sodium chloride (NaCl) were purchased from Sigma-Aldrich (St. Louis, MO). 1,2-Disteoroyl-sn-glycero-3-phosphatidylcholine (DSPC) and cholesterol were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). 3[H]-Cholesteryl hexadecyl ether (CHE) and picofluor-15 scintillation fluid were obtained from Perkin-Elmer, Inc. (Boston, MA, USA). Copper (II) gluconate powder was purchased from Alfa Aesar (Ward Hill, MA, USA). Sucrose was obtained from EMD Chemicals Inc. (Gibbstown, NJ, USA). Citric acid monohydrate crystals were purchased from EmScience (USA). 2.2.2 Determination of quercetin solubility 2 mL of MilliQ water, 300 mM sucrose/20 mM HEPES (pH7.4) (SH) or 150 mM NaCl/20 mM HEPES (pH7.4) (HBS) was added to 10 mg of quercetin and the solution was subsequently mixed for 60 minutes at either 60⁰C or room temperature (RT). Samples at 60⁰C were cooled to room temperature prior to filtration through 0.45 µm filters to remove undissolved quercetin. The resulting clear solutions were then assayed for quercetin via UV-Vis spectroscopy (absorbance at 372 nm) on the Hewlett Packard/Agilent 8453 G1103A UV-Visible Spectrophotometer (Agilent Technologies, Santa Clara, CA, USA). 2.2.3 Liposome preparation DSPC and cholesterol were dissolved in chloroform at a 55:45 mol:mol ratio. 3[H]-CHE was added as a non-exchangeable and non-metabolizable lipid marker lipid marker (172) prior to 27 drying. 3H-CHE was incorporated into the chloroform mixture at a concentration of 5.0 - 12.5 nanocurie per μmol lipid. Chloroform was removed by drying the samples under a steady stream of nitrogen gas until the samples became thick and syrup-like. The samples were then placed under high vacuum to create a “puff” of dried lipid which was kept under vacuum for at least 3 hours to remove residual chloroform. Unless stated differently, the dried lipid was hydrated in 300mM copper sulfate (CuSO4) or 100mM copper gluconate (CuG) (both at pH3.5) (1 mL per 50 µmol of total lipid). To study the role of pH, lipids were hydrated in 100 mM copper gluconate (pH adjusted to 7.4 with trietholamine (TEA)). Lipid hydration results in the formation of multilamellar liposomes and the resulting multilamellar liposomes were then extruded 10 times through two stacked 0.1 µm polycarbonate filters in a 10 mL thermobarrel extruder (ExtruderTM, Evonik Transferra Nanosciences, Burnaby, BC, Canada). The extruded unilamellar liposomes had a mean diameter of 110±20 nm as determined by Phase Analysis Light Scattering methods (ZetaPALS, Brookhaven Instruments Corp., Holtsville, NY). To remove external copper, the sample was passed through a Sephadex G50 column pre-equilibrated with SHE buffer (300 mM sucrose, 20mM HEPES, 15 mM EDTA) adjusted to pH 7.4. The liposome-containing fractions were subsequently passed through a second Sephadex G50 column pre-equilibrated with HBS. The final lipid concentration was determined by measuring 3[H]-CHE using liquid scintillation counting (Packard 1900 TR Liquid Scintillation analyzer). To gain an understanding of quercetin-copper stoichiometry, liposomes were also prepared with different concentrations of either CuSO4 or CuG which were adjusted to control for osmolarity using sodium sulfate or sodium chloride, respectively. Copper-free liposomes were prepared by hydrating lipids in 300 mM citric acid (pH 3.5) or SH buffer (pH 7.4). In all cases, the external buffer was exchanged to HBS (pH 7.4) prior to initiating studies with added quercetin. 2.2.4 Copper-quercetin formation inside liposomes Unless otherwise specified, quercetin was added to liposomes at an initial quercetin-to-lipid ratio of 0.4 (mol/mol). The mixture was then continuously mixed with a mini stir-bar at specified temperatures ranging from room temperature (RT) to 60 ⁰C and the amount of liposome-associated quercetin was measured. For these studies, 80 µL aliquots from the quercetin-liposome mixture were taken at the indicated time points and passed through 1mL 28 Sephadex G-50 mini-spin columns (680xg; 3 minutes) to separate free quercetin from liposome-associated quercetin. The excluded fraction, which appears yellow due to formation of the copper-quercetin complex, was then analyzed for lipid, quercetin, and copper concentrations. Liposomal lipid (3[H]-CHE - tagged) was analyzed by liquid scintillation counting (10 µL of sample to 5 mL of scintillation fluid). Quercetin was measured using absorbance at 372nm on the Hewlett Packard/Agilent 8453 G1103A UV-Visible Spectrophotometer (Agilent Technologies, Santa Clara, CA, USA) after the samples (100 µL) were diluted in acidified methanol (methanol with 3% glacial acetic acid). Copper was measured using atomic absorption spectroscopy where samples were diluted with 0.1% HNO3 (v/v with MilliQ water) and analyzed using the AAnalyst 600 Atomic Absorbance Spectrometer (Perkin Elmer, Inc. Woodbridge, ON, Canada): the samples were dried at 110 oC for 30 s, heated (45 s) to 13 0oC, ashed at 1200 oC for 30 s, and atomized and cleaned at 2000 oC (5 s) and 2450 oC (4s), respectively. 2.2.5 Characterization of copper quercetin binding 5mg of quercetin powder was dissolved in 10mL of methanol and the resulting solution was diluted with methanol to a final quercetin concentration of 5µg/mL. Varying concentrations of CuSO4 and CuG were then added (10 µL) to achieve copper-to-quercetin ratios of 1:8, 1:4, 1:2, 1:1, 2:1, 4:1, and 8:1. These samples were then analyzed spectrophotometrically (Hewlett Packard/Agilent 8453 G1103A UV-Visible Spectrophotometer). 2.2.6 In vitro serum stability assay The indicated liposomal copper-quercetin formulations were prepared at 60oC as described above and concentrated by tangential flow filtration (TFF) using an ultrafiltration hollow fibre column (molecular weight cutoff 500 kDa) (GE Healthcare) to achieve a final quercetin concentration of 16.5 mM (lipid concentration was 100 mM). 400 µL of this sample was added to 1.6 mL of Fetal Bovine Serum (FBS; Gibco, Burlington, ON, Canada) and mixed at 37 ⁰C. At various time points (0, 1, 4, 8, and 24 hours), 80 µL aliquots from the mixture were passed through mini-spin columns as described above. The excluded fraction was then diluted in acidified methanol, and centrifuged at 10,000 rpm at 4⁰C for 10 minutes to remove precipitated 29 proteins. The resulting supernatant was collected and used to determine quercetin, copper, and liposomal lipid concentrations as described above. 2.2.7 Cryogenic electron microscopy (CEM) of copper-quercetin formulations CEM was performed as previously described (173). Particles were concentrated to a final concentration of ~20 mg/mL lipid and added to TEM grids, and vitreous ice was generated using an FEI Mark IV Vitrobot (FEI, Hillsboro, OR). Prepared grids were stored in liquid nitrogen until imaged. A Gatan 70° cryo-tilt transfer system pre-equilibrated to at least -180 °C prior to insertion into the microscope was used as the sample holder. An FEI LaB6 G2 TEM (FEI, Hillsboro, OR) operating at 200 kV under low-dose conditions was used to image all samples. A bottom-mount FEI Eagle 4K CCD camera was used to capture all images. All samples (unless otherwise stated) were imaged at a 55,000x magnification with a nominal under-focus of 1-2 µm to enhance contrast. Sample preparation and imaging was performed at the UBC Bioimaging Facility (Vancouver, BC). 2.2.8 Pharmacokinetics of the copper-quercetin formulations Preliminary pharmacokinetic studies were done in Rag2-M mice obtained from the Animal Resource Centre (ARC) at BC Cancer’s Vancouver Research Centre (Vancouver, BC, Canada). All studies were conducted in accordance with guidelines set by the Canadian Council on Animal Care, as overseen by the University of British Columbia’s Animal Care Committee (protocols A10-0171, A10-0206, and A14-0290). All mice were placed in Optimice cages (up to 5 mice per cage) and cages were equipped with nesting materials and plastic hiding structures (“huts”) for environmental enrichment. Animals were provided food and water ad libitum. The health status of all animals used in these studies was monitored at least once daily following an established standard operating procedure designed to assess animal health status. 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 signs of toxicity. 30 For injected samples, the liposomes were prepared as described above and filter-sterilized through the sterile 50 mL vacuum-driven Steriflip filtration system (Millipore, Billerica, MA, USA). Quercetin was then added to the liposomes incubated for 30 minutes at 60 oC then cooled to room temperature, filtered and exchanged into HBS buffer as described above. The resulting liposomal quercetin formulation was concentrated to 5 mg/mL via TFF as described above. The filter-sterilized formulation was then injected intravenously at a quercetin dose of 50 mg/kg (injection volume was 10 µL/gm body weight). Following iv administration, animals (4 per time point) were sacrificed (isoflurane overdose followed by CO2 asphyxiation) at the indicated time points and blood was collected via cardiac puncture. The blood was then centrifuged (2500 rpm; 15 minutes) and plasma was collected and placed in a tube. The plasma was kept at 4oC until all analyses were completed. Lipid and copper concentrations in plasma were determined as described above. Quercetin concentrations were measured via high performance liquid chromatography (HPLC; Waters, Milford, MA) at an absorbance wavelength of 368 nm. Specifically, plasma samples were mixed with chilled 3% acidified methanol, then centrifuged at 14,000 rpm for 10 minutes to pellet precipitated plasma proteins. 25µL of the resulting supernatant was injected onto a Symmetry C18 column (particle size 3.5µm; 4.6x75mm, Waters). The mobile phase consisted of 70% mobile phase “A” (1% Triethyleneamine in water, pH 6.4 adjusted with glacial acetic acid) and 30% mobile phase “B” (100% acetonitrile). The sample temperature was maintained at 4 °C whilst the column temperature was set at 30 °C. Run time was 5 minutes per sample with a flow rate of 1mL/min using a gradient method with the amount of organic phase increasing from 12 to 40% over 8 minutes. 2.2.9 In vitro cytotoxicity assays of quercetin alone and in combination with irinotecan A549 lung adenocarcinoma cells were kindly provided by Dr. John Minna’s Laboratory (Dallas, TX) while BxPC3 pancreatic adenocarcinoma cells were purchased from ATCC (Manassas, VA). Both cell lines were cultured in RPMI-1640 medium (Life Technologies, Carlsbad, CA) supplemented with 10% fetal bovine serum (Life Technologies) and 2 mM L-glutamine (Life 31 Technologies). All in vitro experiments were performed between the third and eighteenth passages. All cell lines were tested negative for mycoplasma. Dose-dependent viability assays were completed using quercetin and irinotecan (CPT-11) added alone and in combinations to A549 and BxPC3 cells. Briefly, cells were seeded in 384-well plates. 24 hours post seeding, cells were treated with quercetin (Q) or CPT-11(C) alone and in combination at a ratio of 1:2.5 (C:Q) on A549 cells and 1:18 (C:Q) on BxPC3 cells. These ratios were selected based on the ratio of the IC50 of each drug when used alone. Cell viability was determined 72 hours post-treatment using the IN Cell Analyzer 2200 (GE Healthcare, Washington, USA) as described previously (174). In brief, cells were stained with Hoechst for total nuclei count and ethidium homodimer I (Biotium) for dead cells at 72 hours following treatment and imaged with the INCell Analyzer after a 20-minute incubation at 37oC. Cells were classified as “dead” if they showed >30% overlap of the two stains. Dose response curves were constructed using Prism 6.0 (GraphPad Software, La Jolla, CA, USA) by plotting mean ± SEM from at least three independent experiments. IC50 values were interpolated from fitted curves. Combination indices (CI) were derived from the dose response curves using CompuSyn software (ComboSyn, Inc, Paramus, New Jersey, USA). CI values >2 were considered as antagonistic, CI Values <0.8 were considered synergistic. All other CI values were considered additive. 2.2.10 Statistical analysis All data are plotted using the Prism 6.0 software (Graphpad) as mean ± standard error of the mean (SEM). Pharmacokinetics data were analyzed for the elimination half-life and total drug exposure (area under the curve; AUC) using the PK Solutions software (Summit Research Services, USA). 32 2.3 Results 2.3.1 Formation of copper-quercetin within copper containing DSPC/Chol (55:45 mol%) liposomes. Quercetin is a triple-ringed flavonoid with capacity to coordinate copper at three domains: 3’,4’-dihydroxy group on the B ring, 3-hydroxy and 4-carbonyl group in the C ring, and the 5-hydroxy and 4-carbonyl group that spans across the A and C rings (Figure 2.1A). Quercetin exhibits limited aqueous solubility despite heating the mixtures to 60⁰C (solubility is 12.33µg/mL in water (60⁰C) and 38 µg/mL in HBS (60oC); data not shown). As described previously, the ability to form copper-quercetin within copper-containing liposomes is dependent on quercetin permeation across the liposomal lipid bilayer and this process is influenced by temperature (175). Further, the rate of copper-quercetin complex formation is also likely temperature-dependent. Thus, the ability to prepare copper-quercetin complexes within liposomes containing copper sulfate was measured at 22⁰C, 40⁰C, 50⁰C, and 60⁰C. The results (Figure 2.1B) indicate that quercetin association with the liposomes was greatest when the samples were incubated at 60oC, where a 0.18 quercetin-to-lipid ratio (mol/mol) was achieved within 10 minutes and the maximum achievable ratio (0.20) was reached within 60 minutes. At 60 minutes, measured quercetin-to-lipid ratios (mol/mol) of 0.16, 0.12, and 0.07 were achieved when the samples were incubated at 50⁰C, 40⁰C, and 22⁰C, respectively. During the incubation period, there was a colour change from white to yellow (Figure 2.1C), consistent with formation of copper-quercetin following addition of the copper-containing liposomes to quercetin powder. As shown in Table 2.1, incubating the liposomes in the absence of quercetin at temperatures as high as 60°C for as long as 60 minutes had no effects on the stability of the liposomes as indicated by the absence of particle size change. 33 Figure 2.1. Temperature-dependent formation of copper-quercetin complexes in liposomes. A. Quercetin (3,3′,4′,5,7-pentahydroxyflavone) is a three-ringed flavonoid. B. the association of quercetin with 300mM CuSO4 - containing liposomes was assessed as a function of time (5, 10, 30, and 60 minutes) and temperature (22⁰C, 40⁰C, 50⁰C, and 60⁰C) after addition of the liposomes to quercetin as a powder. The results, plotted as the quercetin-to-lipid ratio (mol/mol), were determined after the mixture was eluded through a Sephadex G-50 column (see Methods). Data points represent the mean ± SEM (n=3). C. Photograph of liposome solutions collected following addition of quercetin to 300mM CuSO4 - containing liposomes, which were then subsequently mixed at 60°C for 60 minutes and eluded through a Sephadex G-50 column. The colour change is consistent with formation of copper-quercetin complexes. The samples were cooled to room temperature prior to the pictures being taken. 34 Table 2.1. Particle size (nm) of CuSO4-liposomes and CuG-liposomes incubated at 60°C for 60 minutes in the absence or presence of quercetin Before Incubation After Incubation Lipo-CuSO4-Q 100±10 140±20 Lipo-CuG-Q 100±10 160±20 Lipo-CuSO4 100±10 100±10 Lipo-CuG 100±10 100±10 Other liposomal formulations of quercetin have been described where quercetin is added during liposome formation and quercetin is solubilised by partitioning into the liposomal membrane (140, 176). Therefore, to demonstrate that quercetin association with the copper-containing liposomes was actually copper-dependent, several experiments were completed and these are summarized in Figure 2.2. When quercetin powder was mixed with liposomes prepared using 50, 100, 200, 300, or 400mM copper sulfate and incubated at 60oC for 60 minutes, there was a copper concentration dependent association of quercetin with the liposomes (Figures 2.2A, B and C). The quercetin-to-lipid ratio increased with increasing CuSO4 concentrations and the maximum quercetin-to-lipid ratio of 0.2 was achieved using liposomes prepared in 300mM and 400mM copper sulfate. The results in Fig. 2.2B showed the calculated copper-to-lipid ratios measured for the various formulations used before and after addition of quercetin (Fig. 2.2B) and these results demonstrate that no copper was released from the liposomes following quercetin addition. As suggested by the results in Fig. 2.2C, there was a linear relationship (r=0.95) between the calculated copper-to-lipid ratio and the calculated quercetin-to-lipid ratio, with a slope of 0.52 and a y-axis intercept of 0.044 quercetin-to-lipid ratio. The slope suggests that a 1:2 quercetin-to-copper complex was formed within the liposomes and it can be suggested that the y-intercept represents the amount of quercetin partitioning into the liposomal lipid bilayer under the conditions described. Additional studies were completed to assess the use of liposomes prepared in copper-free solutions and the role of using the low pH (pH 3.5) of the internal copper sulfate solution. The results, summarized in Fig. 2.2D, indicate low levels of quercetin association with copper-free liposome prepared using 300mM citric acid (pH 3.5) or HBS (pH 7.4), the quercetin-to-lipid ratio 35 was less than 0.025 which was lower than predicted based on the extrapolation made using the data in Figure 2.2A. However, these data indicate that the amount of quercetin partitioning into the liposomal membrane accounts for less than 15% of the level obtained when using liposomes prepared in copper sulfate. The results also demonstrate that the level of partitioning into the lipid bilayer was not influence by the low pH within the liposomes. Figure 2.2. Copper-dependent quercetin association with liposomes and the impact of intra-liposomal pH. A. Liposomes prepared in solutions containing various CuSO4 concentrations (0, 50, 100, 200, 300, and 400mM CuSO4) were mixed with powdered quercetin (see Methods) at 60oC and the samples were incubated for 60 minutes. The results were plotted as quercetin-to-lipid ratios. B. Measured copper-to-lipid ratio was plotted against various CuSO4 concentrations. Copper was measured by AAS following separation of the quercetin liposomes on a Sephadex G-50 column. C. The level of liposome associated quercetin was plotted against the measured copper-to-lipid ratio. D. Quercetin association with liposomes prepared in 100mM copper gluconate adjusted to pH 3.5 (filled circles) or 7.4 (inverted triangles). Copper-free liposome controls were prepared in solutions containing 300mM citric acid (adjusted to pH3.5) or HBS (7.4) All liposomes were exchanged into HBS (pH7.4) as described in the Methods. Data points represent mean ± SEM (n=3, where n=number of technical replicates). When using liposomes prepared in a pH 7.4 copper-containing solution (100 mM copper gluconate adjusted to pH 7.4 with TEA, see methods), the rate of quercetin association was lower and the maximum quercetin-to-lipid ratio achieved was lower when compared to the 36 quercetin-to-lipid ratio achieved when using a pH 3.5 copper-containing solutions, even when adjusting for the lower copper concentrations (100mM). However, the results, summarized in Figure 2.2D, suggest that the rate of quercetin association with the CuG liposomes was influenced by the internal pH; the rate of association was faster at pH 3.5. Furthermore, Figure 2.2D reiterates that quercetin association with the liposomes is dependent on the presence of encapsulated copper, but shows that the copper salt used inside the liposome affects quercetin association levels. The measured quercetin-to-lipid ratios (mol/mol) for the liposomes prepared using 100mM CuG were as high as 0.2 and this was 2-times higher than that predicted based on liposomes prepared in CuSO4 solutions. To gain a better understanding of the copper salt effects, liposomes were prepared with 100mM CuG, 100mM CuSO4, or 300mM CuSO4 and the level of quercetin association was determined as a function of time. The results, summarized in Figure 2.3, indicate that the maximum quercetin-to-lipid ratios achieved were 0.18mol/mol and 0.17mol/mol when using liposomes prepared in 100mM CuG and 300mM CuSO4, respectively. The quercetin-to-lipid ratio achieved with liposomes prepared in 100mM CuG was almost double that achieved with liposomes prepared in 100mM CuSO4 (0.18 versus 0.10 mol/mol, respectively) (Figure 2.3A). As shown in Figure 2.3B, liposomes prepared in 300mM CuSO4 had almost 3-times the amount of associated copper when compared to liposomes prepared in 100mM copper solutions, regardless of the salt form. Furthermore, the amount of liposome-associated copper was essentially the same for liposomes prepared in CuSO4 or CuG. Finally, in all formulations studied there was no loss of associated copper following addition of the liposomes to powdered quercetin. 37 Figure 2.3. Quercetin association with liposomes prepared with CuSO4 or Cu-gluconate. A. Liposomes prepared in 100mM CuSO4 (filled circles), 300mM CuSO4 (filled squares) and 100mM copper gluconate (filled triangles) (all pH 3.5) were mixed with powdered quercetin at 60°C and at the indicated time points liposomes were passed through Sephadex G-50 spin columns (see Methods) and the concentration of quercetin and liposomal lipid were measured (see Methods) and plotted as the quercetin to lipid ratio. B. At the same time points shown in Panel A, the eluted liposomes were also assayed for copper by AAS (see Methods) and the results plotted as the copper-to-lipid ratio. Data points represent mean ± SEM (n=3, where n=number of technical replicates). Similar to the results obtained with copper sulfate-containing liposomes, there was a linear correlation between the quercetin association with liposomes and the amount of CuG used when preparing the liposomes (Figure 2.4). The slope of the calculated quercetin-to-lipid ratio 38 plot versus the calculated copper-to-lipid ratio (see Fig. 2.4B) was 2.45, suggesting that the quercetin-to-copper ratio was likely 2:1 when using liposomes prepared with CuG. In the absence of copper, the level of quercetin association with the liposomes was comparable to that measured with the CuSO4 control liposomes (Fig. 2.2A). Figure 2.4. Quercetin association with liposomes prepared using solutions containing different copper gluconate (CuG) concentrations. A. Liposomes prepared in solutions of 0, 10, 25, 75, and 100mM CuG were mixed with powdered quercetin at 60⁰C for 60 minutes. At that time liposomes were passed through Sephadex G-50 spin columns (see Methods) and the concentration of quercetin and liposomal lipid were measured (see Methods) and plotted as the quercetin to lipid ratio. B. The level of liposome associated quercetin was plotted against the measured copper to lipid ratio. Copper was measured by AAS following separation of the quercetin liposomes on a Sephadex G-50 column. Data points represent mean ± SEM (n=3, where n=number of technical replicates). 39 The studies summarized above suggest that the copper-to-quercetin ratio within the formulations differed when using CuSO4 and CuG. The nature of this interaction was further characterized by UV absorption spectrophotometry. As shown in Figure 2.5, quercetin alone exhibits an absorption UV peak at 372nm while complexation with copper shifts the maximal absorbance to 441 nm. Interestingly, complexation with CuSO4 resulted in a more distinct peak at 441 nm (Figure 2.5A). The increase in absorbance at 441nm was measured when a fixed quercetin concentration was incubated with varying concentrations of copper (see Methods). The maximum increase in absorbance at 441nm was achieved at copper-to-quercetin ratio of 2 (mol/mol) when CuSO4 was used and 0.5 (mol/mol) when CuG was used (Figure 2.5B). These findings support the idea that quercetin forms a 2:1 (quercetin:copper) complex in the presence of CuG and a 1:2 (quercetin:copper) complex in the presence of CuSO4 (Figure 2.5C). Figure 2.5. Spectrophotometric studies evaluating quercetin-copper interactions. A. Quercetin (at a fixed quercetin concentration of 5µg/mL in methanol) and the complexes it forms with copper can be studied spectrophotometrically, where quercetin exhibits an absorption 40 maximum at 372nm and the quercetin-copper complex has an absorption maximum at 441nm, indicative of formation of a yellow complex. The shift in the absorption maximum from 372nm to 441nm was most dramatic when using copper sulfate. If copper gluconate was used there was a reduction in absorbance at 372nm and an increase in absorbance at 441nm, suggestive of a more complicated interaction. B. The absorbance 441nm was measured in methanol solutions of quercetin (5µg/mL) at copper-to-quercetin ratios of 1:8, 1:4, 1:2, 1:1, 2:1, 4:1, and 8:1, where copper was added as copper sulfate (filled squares) or copper gluconate (filled circles). C. Proposed molecular structures of copper-quercetin complex when the complex is formed using copper gluconate (left panel) or CuSO4 (right panel). To gain a better understanding of the morphology of the liposome quercetin formulations, liposomes with encapsulated CuG, quercetin associated with CuG containing liposomes, liposomes with encapsulated CuSO4, and quercetin associated with CuSO4 containing liposomes were imaged by cryo-electron microscopy (CEM) (Figure 2.6). The results illustrated two points: i) the liposomes with associated quercetin in liposomes prepared using CuG or CuSO4 did not exhibit an electron dense core, suggesting that if precipitates were formed they were not electron dense, and ii) the association of quercetin in the CuG - containing liposomes exhibited greater particle size - consistent with the particle size analysis obtained with this formulation (Table 2.1). 41 Figure 2.6. Representative cryo-transmission electron microscopy images of liposomes. The images correspond to copper sulfate (CuSO4) – containing liposomes (A), copper gluconate (CuG) – containing liposomes (B), CuSO4 – containing liposomes with quercetin (CuSO4-Q) (C), and CuG-containing liposomes with quercetin (CuG-Q). The quercetin-containing liposomes were prepared at 60°C, incubated for 60 minutes. Please refer to Methods Section 2.7 for the preparation of cryo-TEM images. 2.3.2. Suitability of the copper-quercetin liposomes as a formulation for intravenous use. The stability of the copper-quercetin formulations (one prepared with CuSO4 (300mM) containing liposomes and one prepared with CuG (100mM) containing liposomes) was assessed in the presence of fetal bovine serum (FBS) when incubated at 37⁰C over 24 hours (see Methods). Stability was determined by measuring time-dependent changes in the quercetin-to-42 lipid ratio. The results, shown in Figure 2.7, indicate that there was a decrease in the quercetin-to-lipid ratio over the first 8 hours under these conditions. A 17%, 25%, and 44% decrease was noted at 1, 4 and 8 hours, respectively. There was not a significant change in quercetin-to-lipid ratio after 8 hours and the two formulations appeared to behave similarly. Figure 2.7. In vitro stability of copper-quercetin formulations prepared using liposomes prepared in 300 mM CuSO4 or 100 MM copper gluconate. The formulations were incubated in 80% FBS at 37⁰C over 24 hours with time points taken at 0, 1, 4, 8, and 24 hours (See Methods). At the indicated time points the liposomes were passed through Sephadex G-50 columns and the liposomes (in the excluded fraction) were assessed for liposomal lipid and quercetin as described in the Methods. Data points represent mean ± SEM (n=3, where n=number of technical replicates). Pharmacokinetic studies were completed following injection of the formulations into RAG2m mice (injected iv using a dose of 50 mg/kg) with the liposomal copper quercetin formulations prepared using liposomes made with CuSO4 or CuG. At 1, 4, 8 and 24 hours plasma was collected and then analyzed for liposomal lipid, quercetin, and copper as described in the Methods. The results, summarized in Figure 2.8, indicate that the formulation prepared with CuG-containing liposomes was eliminated more rapidly than formulations prepared using CuSO4 and this was consistent with the increase in particle size and polydispersity observed when using quercetin formulations prepared using CuG - containing liposomes. Analyses of the pharmacokinetics data with PK Solutions software (Summit Research Services, USA) revealed that the area under the curve (AUC) is 8382µg-hr/mL for the quercetin formulations prepared using liposomes made with copper sulfate and 1577µg-hr/mL for quercetin formulations 43 prepared using liposomes made with CuG (Table 2.2). These results highlight two important points: i) the rate of quercetin release from the liposomes in the plasma compartment is comparable for the two different formulations (Figure 2.8C), albeit a more substantial initial loss of associated quercetin was observed when using the formulation prepared with copper gluconate containing liposomes and ii) the results suggest that encapsulated copper is retained within the circulating liposomes for both formulations (Figure 2.8D), suggesting that quercetin is released from the liposomes rather than a copper-quercetin complex. Figure 2.8. Pharmacokinetics of copper-quercetin formulations made with liposomes prepared in 300mM CuSO4 or 100mM CuG. Female RAG2m mice were injected intravenously with a single bolus dose of the indicated liposomal quercetin formulation at a quercetin dose of 50 mg/kg. Plasma concentrations of quercetin (A) and lipids (B) were measured at the indicated time points following administration. Each point represents the mean ± SEM; n=4. C. Measured quercetin and liposomal lipid concentrations were used to calculate the quercetin to lipid ratio as a function of time; an assessment that estimates how fast quercetin is released from the liposomes in the plasma compartment. D. Plasma copper levels were measured by AAS (see Methods) and these values were used to calculate the copper to liposomal lipid concentration in the plasma compartment; an assessment that estimates whether copper is released from liposomes in the plasma compartment. Data points represent mean ± SEM (n≥4, where n=number of technical replicates). 44 Table 2.2. Pharmacokinetics parameters of quercetin following intravenous administration (5mg/kg) T1/2 (hours) AUC (µg*hr/mL) Vd (mL) CL (mL/hr) Lipo-CuSO4-Q 6.91 8382 1.76 0.18 Lipo-CuG-Q 2.47 1577 3.73 0.91 2.3.3. Therapeutic potential of quercetin when used in combination with Irinotecan Based on previous studies it was concluded that quercetin combined with irinotecan (CPT-11) could produce synergistic cytotoxic effects (177-179). Our group has developed a liposomal CPT-11 formulation (Irinophore C) with significant therapeutic activity (180, 181) and this gives a favourable position to develop a combination product comprising CPT-11 and quercetin. For this reason, the combination effects achieved when using these drugs in combination was assessed. Consistent with previous reports, quercetin-induced cytotoxic effects were only achieved when using µM concentrations of the agent: the IC50 of quercetin was 22 and 37µM when added to A549 and BxPC3 cells, respectively (Figure 2.9A and 2.9B). When used in combination with CPT-11, quercetin and CPT11 displayed minimal synergy at high effect levels (>60% cell kill) for A549 but the two agents acted additively or antagonistically at all effect levels when used to treat BxPC3 cells (Figure 2.9C and 2.9D). These data did not provide sufficient justification to further pursue quercetin as a single agent or in combinations in vivo. 45 Figure 2.9. In vitro assay assessing the cytotoxic effects of quercetin alone and in combination with irinotecan. The quercetin (Quer) and/or irinotecan (CPT11) dose response curves determined for A549 lung cancer cells (A) and BxPC3 pancreatic cancer cells (B) using a imaging method reliant on the IN Cell Analyzer as described in the Methods. For combination effects, quercetin and CPT11 were added at ratios of 1:2.5 (CPT11:Quer) for A549 and 1:18 (CPT11:Quer) for BxPC3. The dose response curve for the combination was plotted based on CPT11 concentrations (A, B). Combination Indices (CI) derived from the dose response of the combination treatments are plotted against the fraction of affected (Fa) cells, where a Fa of 1 indicated 100% cell kill. “Fraction affected” values here indicate cell viability. CI>2: antagonistic 2>CI>0.8: additive, and CI<0.8: synergistic. All data are plotted as mean ± SEM (n≥3) (C, D). Data points represent mean ± SEM (n≥3, where n=number of technical replicates). 2.4 Discussion Others have highlighted that quercetin has anti-cancer activity and it has been argued that it can act as an apoptosis promoter through modulations of proteins such as Bcl-2 and Akt-1 (155, 159, 160). Unfortunately, since the water solubility of quercetin is poor, its oral bioavailability is limited (162, 182) and its therapeutic utility is limited because of this. For example, a single 4g oral dose of quercetin in a human resulted in no detectable plasma or urine concentrations of 46 quercetin and less than 1% of the quercetin was absorbed unaltered in the gastrointestinal tract (183). Importantly, this is not the only concern when considering the development of quercetin as a therapeutic to treat cancer. Most studies have shown that quercetin can only induce anticancer effects when present in high µM (10-20µM) concentrations (184, 185). If a suitable intravenous formulation of quercetin was developed, issues related to bioavailability would then be addressed and perhaps suitable concentrations of quercetin in the blood could be obtained. A formulation method for poorly soluble drugs, including flavonoids like quercetin, that considers the use of compounds that can coordinate metals and nanotechnology was recently described (175). In this report, liposomal formulations prepared based on quercetin-copper binding have been characterized. The results suggest that the encapsulated copper salt can influence the copper-quercetin complex that is formed. Importantly, the formulations made exhibit more than 100-fold improvements in apparent solubility and are suitable for intravenous administration. Quercetin is difficult to prepare as an i.v.-injectable formulation. When described, these formulations rely on organic solvents such as dimethyl sulfoxide (DMSO) which, when used, have been shown to be toxic at many levels (186). Therefore, it is not surprising that efforts have been invested to develop alternative quercetin formulations. For example, when compared to free quercetin, liposomal quercetin formulations often displayed improved blood circulation and decreased rates of elimination from the plasma compartment (140, 176, 187). In these previous studies, quercetin was formulated into liposomes passively when the liposomes are first made. Passive encapsulation into liposomes is very inefficient and requires preparation of liposomes at high lipid concentrations (118). In contrast, the novel copper-based methodology described here was extremely efficient, where encapsulation amount was dependent on the copper-to-quercetin ratio formed. The copper-based technology resulted in a more stable and consistent formulations of quercetin. The CuSO4-based formulation showed improved pharmacokinetics compared to liposomal formulations previously described in literature with a 3-fold increase in circulation half-life (2 hours versus 0.5 hours) and an 18 fold increase in total drug exposure (AUC) (1.94 versus 0.106 µmol/mL) (140). This copper-based formulation was well-tolerated at the current maximum feasible injected dose in RAG2m mice 47 with no acute or chronic (14 days) toxicities observed (data not shown). Additionally, necropsy reports of the animals used to assess toxicity studies were unremarkable. In addition to liposomes, bi-functional metal-phenolic films have also been previously investigated to overcome the solubility challenges of quercetin (188). Bertleff-Zieschang et al. described a one-step, pH dependent coordination of quercetin with FeIII ions to form thin films and hollow capsules on solid surfaces (188). The formation of these capsules and films helped address the poor aqueous solubility of free flavonoids like quercetin (188). In this regard, the hollow iron-quercetin capsules are similar to our liposomes as they also provide a solution to solubilize quercetin. However, our liposomes present a few advantages, most notable of which is the physical shielding effects that the phospholipids of the liposomes are able to provide for the encapsulated content. As nanocarriers, liposomes are well known to act as steric barriers that may discourage interactions between entrapped compounds and plasma proteins (189). This property is particularly attractive for quercetin as this flavonoid is known to extensively bind human plasma protein and it has been suggested that this interaction results in poor cellular availability of quercetin (81). By encapsulating quercetin in liposomes, this activity-hindering interaction with plasma proteins can be curbed. Additionally, although Bertleff-Zieschang et al. did not evaluate the pharmacokinetic profile of the hollow capsules, I believe that by shielding quercetin within liposomes, our formulation would also have improved pharmacokinetics, namely circulation time and total drug exposure, when compared to the hollow capsules of quercetin. This improved pharmacokinetic profile could enhance quercetin’s clinical value as quercetin is known to be a substrate of P-glycoprotein and its absorption rate is limited by high efflux rate (190). Metal-based therapeutics made a major leap in the 1970s with the emergence of platinum-based complexes such as cisplatin, carboplatin, and oxaliplatin as widely successful, and potent therapeutics (127). However, efforts have been made to identify alternatives to replace platinum-based therapeutics primarily due to the many crippling toxicities that these drugs engender and copper-based therapeutics emerged as a potential replacement candidate (191). One of the primary reasons that copper was selected as the metal of choice was its role as an essential metal. Unlike platinum, which is not native to the human body, copper is an important trace element in the growth and development of all aerobic organisms and plays a crucial role in a number of biological pathways (127, 192). As such, copper is tightly regulated and its 48 homeostasis is rigorously maintained to avoid copper-related toxicities (192, 193). In the blood, plasma protein carriers such as albumin and ceruloplasmin transport the copper absorbed into the blood stream to the liver and the kidney for excretion and distribution (194). On the cellular level, copper transporters such as CTR1 and copper efflux pumps like ATP7A/B aid in the regulation of cytosolic copper levels (127). The presence of these intricate regulatory mechanisms helps attenuate toxicities that copper-based therapeutics may have otherwise engendered, allowing copper-based drugs to be better tolerated. In addition to having natural regulatory biological pathways, copper was also chosen as the metal of preference due to its potential to enhance anticancer activities. Disulfiram, an FDA-approved drug for treating alcoholism, paints a perfect picture of a copper-activated agent. As an anticancer agent, disulfiram has shown to have minimal anticancer activity in the absence of copper, but is active in the nano-molar range against various cancer cell lines in the presence of copper (195, 196). In the case of quercetin, forming a complex with copper did not enhance the activity of quercetin (data not shown). However, the use of copper still solved the low solubility problem that has long prevented the use of flavonoids such as quercetin in a clinical setting. Here, I have provided proof-of-concept data using copper, but I do not preclude the possibility of using other metals, such as zinc. In agreement with numerous published studies (169, 197, 198), our data suggested that quercetin forms a 2:1 ligand-to-metal complex with copper (II) ions in the presence of CuSO4 and a 1:2 ligand-to-metal complex with copper (II) ions in the presence of CuG (Figure 2.5B). The different complexes formed in the presence of CuSO4 versus CuG help explain the different encapsulation efficiencies observed. Gluconic acid forms strong complexes with many transition metals including copper (199). The presence of a competitive copper chelator would result in the formation of a different copper complex than what is seen with CuSO4. Thus, in the presence of CuG, two quercetin molecules were needed to bind with copper ions to engender dissociation of copper ions away from gluconic acid. In contrast, sulfate does not form a complex with copper ions and the quercetin molecule can bind to two copper (II) ions. Therefore, when considering equivalent Cu (II) concentrations, CuG-containing liposomes appear to facilitate improved quercetin association compared to CuSO4-containing liposomes. However, CuG-quercetin liposomes were cleared from the plasma compartment much more rapidly compared to CuSO4-quercetin liposomes. This may have been the result of the greater variation in particle size as shown in the CEM images of CuG liposomes (Figure 6) and the 49 increase in particle size seen when using CuG – containing liposomes with encapsulated quercetin. The rate of liposome elimination is increased as the size of the liposome increases (200). Regardless of the binding interactions, quercetin and copper-quercetin complexes are poorly soluble in aqueous solutions and this is one factor that limits quercetin’s therapeutic potential. Many studies have demonstrated that as a single agent, quercetin is not a potent chemotherapeutic agent, with effective doses at micromolar concentrations (73, 157, 201, 202). Therefore, it can be argued that quercetin’s therapeutic value may lie in combination therapy. In fact, it has previously been shown that when used in combination, quercetin potentiates existing chemotherapeutic agents such as cytosine arabinoside, doxorubicin, and irinotecan for treatment against leukemia, breast cancer, and gastrointestinal cancer, respectively (203-205). In particular, quercetin has been shown to work well in combination with irinotecan, demonstrating promise in the treatment of advanced non-small-cell lung cancer, with overall response rate of up to 32% (206). In an initial in vitro screen, when quercetin was added concurrently with irinotecan, the interaction between the two drugs suggested additively (Figure 2.9). The difference in the tissue origin and the genetic profiles between the cell lines may have accounted for this. Regardless, further studies to elucidate the therapeutic utility of quercetin combinations and to identify suitable candidates to use in combination with quercetin are needed. More studies are needed to define how a liposomal copper-quercetin formulation would be used in combination in preclinical animal models. Additionally, the use of copper complexation methods to encapsulate quercetin acts as a proof-of-concept and will now allow us to further explore utility of this copper-based technology with other promising flavonoids or flavonoid-like candidates that share similar copper-chelating properties as quercetin. 2.5 Conclusions In summary, I have successfully addressed the solubility issues associated with quercetin by generation of a novel liposomal formulation of copper-quercetin. Compared to other quercetin-based formulations previously described, my formulation takes advantage of the well-50 characterized propensity of liposomes to enhance the circulation longevity of an associated drug. Continued efforts will be made to explore and evaluate quercetin’s value as a component in combinations for treatment of cancer. 51 3. Development and characterization of a novel flavopiridol formulation for treatment of acute myeloid leukemia 3.1 Introduction As described in the Introduction, AML patients still face the same therapeutic obstacles and relapse after complete remission (2, 3, 13). The prognosis is particularly grim for AML patients older than 65 years of age, with a 5-year survival rate of less than 10% (4, 13). As such, new therapeutic strategies to improve treatment outcomes of AML patients are urgently required. One candidate that has shown promising therapeutic potential against AML is flavopiridol (73). Flavopiridol is a semisynthetic flavone derived from the plant alkaloid rohitukine (207). Rohitukine was initially isolated from stems and leaves of Amoora rohituka and later from Dysoxylum binectariferum (208, 209). Preclinically, flavopiridol is well-established as an effective anti-cancer agent against various types of cancer such as leukemia, prostate carcinoma, breast carcinoma, and bladder cancer (72, 210-213). Mechanistically, a number of studies have demonstrated flavopiridol is active against multiple CDKs (210, 211, 214-217). CDKs are important regulators of the cell cycle and hence cell proliferation (218, 219). By inhibiting CDK1, CDK2, CDK4 and the transcription factor CDK9, flavopiridol effectively causes G1 and G2 phase arrests, preventing cells from progressing into the S and M phases, respectively (207, 218, 220). In addition to initiating cellular growth arrests, flavopiridol has also been reported to induce apoptosis in cells such as chronic lymphocytic leukemia and lymphoma (71, 207, 221). As a CDK inhibitor, flavopiridol effectively suppresses the catalytic subunit (CDK9) of the transcription elongation factor b (P-TEFb) (222). This in turn prevents P-TEFb-mediated phosphorylation of the C-terminal domain of RNA polymerase II and promotes subsequent inhibition of transcriptional elongation (222). Among the many CDK inhibitors (19), flavopiridol was the first CDK inhibitor to be tested in humans (223-225). However, despite promising preclinical data, flavopiridol was therapeutically disappointing in phase II clinical trials due in part to toxicities including GI toxicity and tumour lysis syndrome (77, 225-229). Some have postulated the lack of activity was due to the high level of flavopiridol binding to plasma proteins upon intravenous injections (77). Flavopiridol’s 52 propensity to bind proteins was demonstrated in vitro using fetal calf serum (FCS) (230). When incubated with FCS, the free flavopiridol level dropped from 63-100% to 5-8% and the LC50 increased by almost five fold (230). In addition, as with other flavonoids, flavopiridol is poorly soluble in aqueous solutions with a water solubility of approximately 0.025mg/mL (78). Thus, injectable flavopiridol can only be formulated in acidic buffers such as 30% hydroxypropyl β-cyclodextrin/0.1M citrate buffer (231). This formulation does not prevent flavopiridol-protein interactions. As such, there is a need to improve flavopiridol formulation in hopes of making it more clinically relevant. In Chapter 2, I described methods to formulate quercetin through the ability of flavonoids to bind divalent metals inside liposomes to enhance solubility and create a formulation suitable for intravenous use. Similar to quercetin, flavopiridol is defined as an aqueous-insoluble compound (< 1 mg/mL in water) and the same metal-based method can be used to enhance solubility to develop a novel formulation of flavopiridol. Recently, my laboratory developed a novel method of encapsulating drugs that can coordinate with metals, such as copper, held within liposomes (124). Flavones such as flavopiridol contained a copper-binding site that can be used to encapsulate the drug within copper-containing liposomes. This copper-flavopiridol coordination drives the encapsulation of the drug. Once inside the liposomes, the drug can be protected against premature degradation that may otherwise occur in vivo (90). This method also addresses the solubility issue associated with flavopiridol and protects it from protein binding upon administration. Further, if the liposomes can be designed appropriately, they can engender significant increases in circulating drug levels over extended time periods, which in the case of CDK inhibitors should enhance therapeutic activity. In the current study, I have generated a novel, injectable, liposome-based formulation of flavopiridol that exhibits extended circulation lifetimes and enhanced efficacy. 53 3.2 Materials and methods 3.2.1 Materials Flavopiridol was purchased from Selleckchem (Houston, Texas, USA). Copper (II) sulfate, Sephadex G-50, HEPES, ethylenediaminetetraacetic acid (EDTA), and sodium chloride (NaCl) were purchased from Sigma-Aldrich (St. Louis, MO). Sucrose was obtained from EMD Chemicals Inc. (Gibbstown, NJ, USA). 1,2-disteoroyl-sn-glycero-3-phosphatidylcholine (DSPC), sphingomyelin (SM), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-n-[carboxy(polyethylene glycol)-2000] (DSPE-PEG2000), and cholesterol (chol) were obtained from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). 3[H]-cholesteryl hexadecyl ether (CHE) and picofluor-15 scintillation fluid were purchased from Perkin-Elmer, Inc. (Boston, MA, USA). 3.2.2 Tissue culture MV4-11 human biphenotypic B myelomonocytic leukemia cell line was obtained from ATCC (Cat # CRL-9591) in 2015 with Mycoplasma negative certificate issued by ATCC. MOLM-13 human acute myeloid leukemia cell line was obtained from DSMZ (DSMZ # ACC 554) in 2017 with Mycoplasma negative certificate issued by DSMZ. MV4-11 and MOLM-13 were cultured in IMDM (1X) (Life Technologies, Carlsbad, CA) and RPMI-1640 medium (Life Technologies) supplemented with 10% fetal bovine serum (Life Technologies) and 2mM L-glutamine (Life Technologies). The cells were grown and maintained at 37°C in 5% CO2 environment. 3.2.3 Dose response curves Dose-dependent time course in vitro assays were conducted in 48-well plates. 400µL of cells per well were plated at concentrations of 80 cells/µL for MV4-11 and 60 cells/µL for MOLM-13. After plating (24 hrs), the cells were treated with flavopiridol at increasing concentrations (0.025µM to 5µM). At specified time points, the cells were washed and then resuspended in flavorpiridol-free media. The resuspended cells were then re-plated. At 72 hours post-plating, all cells were incubated with PrestoBlue cell viability reagent (ThermoFisher, Waltham, Massachusetts, USA) (10% v/v) for 3 hours. The incubated plates were then imaged in the FLUOstar OPTIMA Microplate Reader (BMG Labtech, Offenburg, Germany). Dose response 54 curves were constructed by plotting mean ± SEM from at least three independent experimental trials using Prism 7.0 (GraphPad Software, La Jolla, CA, USA). 3.2.4 Spectrophotometric analysis of flavopiridol and copper-flavopiridol in methanol 1mg of flavopiridol powder was dissolved in 1mL of methanol. The resulting 1mg/mL solution was then diluted with methanol to 10µg/mL (25µM). The UV absorption spectrum for the diluted flavopiridol solution was then measured on the Hewlett Packard/Agilent 8453 G1103A UV-Visible Spectrophotometer (Agilent Technologies, Santa Clara, CA, USA). To determine the UV absorption spectrum of copper-flavopiridol, copper sulfate was added to 25µM flavopiridol in methanol at 1:1 molar ratio and the resulting mixture was analyzed on the Hewlett Packard/Agilent 8453 G1103A UV-Visible Spectrophotometer (Agilent Technologies, Santa Clara, CA, USA). 3.2.5 Liposome preparation Three different types of liposomes were used in this study: DSPC:Chol (55:45 mol:mol ratio), SM:Chol (55:45 mol:mol ratio), and DSPC:DSPE-PEG 2000 (95:5 mol:mol ratio). For DSPC:Chol liposomes, DSPC and Chol were first weighed and dissolved in chloroform at a 55:45 mol:mol ratio. 3[H]-CHE was added as a lipid marker prior to drying. Specific activity was then determined by adding 10µL of the sample to 5mL of picofluor scintillation fluid. All samples were measured in quadruplicates. The radioactivity was measured on a Packard 1900 TR Liquid Scintillation analyzer. For the bulk sample, chloroform was removed by drying with a stream of nitrogen gas until the samples became thick and syrup-like. To completely remove the choloroform, the samples were then placed under vacuum for at least 3 hours, resulting in the formation of a bubbly lipid film. The dried lipid film was then hydrated in various solutions (300mM copper sulfate, 250mM ammonium sulfate, and 300mM citric acid); 1mL solution per 50µmol of lipids. The sample was hydrated at 65⁰C with constant stirring. The hydrated lipids were then subjected to five cycles of freezing (snap-frozen in liquid nitrogen) and thawing (in 65⁰C water bath). The resulting multilamelar liposomes were then extruded ten times through stacked 0.1µm and 0.08µm filters in a 10mL or 100mL thermobarrel extruder (LIPEX® Extruder, Evonik Industries, Vancouver, BC, Canada) at 65⁰C. The resultant unilamellar liposomes had a 55 mean diameter of 100±20 nm as determined by dynamic light scattering (ZetaPALS, Brookhaven Instruments Corp., Holtsville, NY). For copper-containing liposomes, the extruded liposomes were run through a Sephadex G50 column pre-equilibrated with sucrose/HEPES/EDTA (SHE) buffer pH 7.4 (300mM sucrose, 20mM HEPES, 15mM EDTA) to remove unencapsulated copper. The external SHE buffer was further exchanged with the desired loading buffers through Sephadex chromatography where the matrix was pre-equilibrated with the desired buffer. The loading buffers used in this study include HEPES buffered saline (HBS) pH 7.4 (150mM NaCl, 20mM HEPES), citric acid saline pH 3.5 (150mM NaCl, 20mM citric acid), and HEPES buffer potassium pH 7.4 (155mM KCl, 20mM HEPES). The final lipid concentration was determined by measuring 3[H]-CHE using liquid scintillation counting as described above. For preparation of SM:Chol and DSPC:DSPE-PEG2000 liposomes, an ethanol-based protocol was used. The lipids were weighed and dissolved in ethanol at 65⁰C (1mL of ethanol per 100µmol of lipids). 3[H]-CHE was added as marker and specific activity was measured as previously described. Dissolved lipids were kept warm at 65⁰C for the following steps. The lipids in ethanol were added drop-wise to 300mM copper sulfate (1mL of ethanol per every 6mL of copper sulfate) in 65⁰C with rapid mixing for a minimum of 30 minutes. The multilamelar liposomes which formed were then extruded as previously described. Ethanol was immediately removed from the extruded product by tangential flow filtration (TFF) using an ultrafiltration hollow fibre column (molecular weight cutoff 500kDa) (GE Healthcare). Extra-liposomal copper removal and buffer exchanges were done as described above. Like when preparing DSPC:Chol liposomes, SHE buffer was first used as the external buffer to remove unencapsulated copper and this was subsequently changed to a buffer of choice through Sephadex column chromatography. 3.2.6 In vitro characterization of liposomal copper-flavopiridol DSPC:Chol liposomes were added to pre-weighed flavopiridol powder at a drug-to-lipid ratio of 0.2 (mol/mol). The mixture was then continuously mixed with a mini stir-bar in water baths at temperatures of 30⁰C, 40⁰C, 50⁰C, and 60⁰C. 80µL aliquots from the flavopiridol-liposome mixture were then taken at various time points (5, 10, 15, 30, and 60 minutes) and centrifuged 56 through 1mL Sephadex G-50 mini-spin columns (680xg for 3 minutes) to separate the unencapsulated drug from liposome-associated drug. Flavopiridol concentrations in the liposome fraction were determined by measuring absorbance at 275nm on the Hewlett Packard/Agilent 8453 G1103A UV-Visible Spectrophotometer (Agilent Technologies, Santa Clara, CA, USA) and the lipid concentrations were measured by liquid scintillation counting. Samples visualized on the UV-visible spectrophotometer were diluted in 100% HPLC-grade methanol. To examine the role of copper in trapping flavopiridol in liposomes, DSPC:Chol lipid films were hydrated in different entrapment solutions (300mM copper sulfate pH 3.5, 250mM ammonium sulfate pH 5.0, and 300mM citric acid pH 4.0). The resulting DSPC:Chol liposomes were then added to pre-weighed flavopiridol powder and mixed and incubated in a 40⁰C water bath. Flavopiridol and lipid concentrations were subsequently measured at various time points over 60 minutes. The role of a pH gradient in associating flavopiridol with liposomes was examined by using different buffers outside the liposomes. Buffers used included HBS pH 7.4, citric acid saline pH 3.5 (150mM NaCl, 20mM citric acid), and HEPES buffered potassium pH 7.4 (155mM KCl, 20mM HEPES). DSPC:Chol liposomes with KCl as the external buffer were first incubated with ionophore, Valinomycin, at 30°C for 10 minutes before being added to the flavopiridol powder to collapse the pH gradient that may have existed across the liposomal membrane. The ionophore was dissolved in ethanol at concentrations of 1mg/mL and was added to the liposomes at a ratio of 0.5µg ionophore per µmol of lipids. These liposomes with different external buffers were then mixed and incubated with pre-weighed flavopiridol powder (0.2 drug-to-lipid mole ratio) in 40⁰C water bath. After separating free flavopiridol from liposomal flavopiridol, the lipid and flavopiridol concentrations were measured as described above. 3.2.7 In vivo characterization of flavopiridol In vivo experiments were conducted using female (NOD)-Rag1null-IL2rγnull (NRG) mice from the Animal Resource Centre (ARC) at BC Cancer’s Vancouver Research Centre (Vancouver, BC, Canada). All studies adhered to the guidelines of the Canadian Council on Animal Care and through protocols approved by the University of British Columbia’s Animal Care Committee 57 (IACC) (protocol A18-0276). The IACC provided regular monitoring of work conducted under this protocol through a post-approval monitoring program. All mice were housed in microisolators and were provided enrichment. Temperature and humidity were monitored and animals (in groups of up to 5) were provided free access to water and food. The liposomes were prepared at approximately a 0.2 mol:mol (drug:total lipid) ratio. Prior to injection into an animal, drug-loaded and “empty” DSPC:Chol, SM:Chol, and DSPC:DSPE-PEG2000 liposomes were filter-sterilized through sterile 50mL vacuum-driven Steriflip filtration system (Millipore, Billerica, MA, USA). Prior to filtration, the liposomal formulations were concentrated to 0.5 mg flavopiridol/mL via TFF and the samples were injected intravenously (i.v.) at 5 mg/kg. For pharmacokinetic studies, blood was collected via cardiac puncture and placed into EDTA-containing microtainers. The plasma was separated from blood cells by centrifugation (2500 rpm; 15 minutes). The lipid and flavopiridol concentrations in the plasma were then determined. Lipid concentrations were measured as described previously. Flavopiridol concentrations were measured via high performance liquid chromatography (HPLC; Waters, Milford, MA) at an absorbance wavelength of 264nm. Briefly, plasma samples were mixed with chilled 3% acidified (glacial acetic acid) methanol. The samples were then centrifuged at 14000rpm for 15 minutes at 4⁰C to remove precipitated plasma proteins. 25µL of the resulting supernatant was subsequently injected into a Symmetry C18 column (particle size 3.5µm; 4.6x75mm, Waters). The mobile phase consisted of 71% mobile phase “A” (0.1% Trifluoroacetic acid in water) and 29% mobile phase “B” (100% acetonitrile). Standard curves (ranged from 0.625µg/mL to 10µg/mL) were prepared where 3% acidified methanol was used as the dilution solvent. All standards and samples were kept at 4±0.5 ºC prior to injections and the column temperature was set at 35±5ºC. Run time was 5 minutes per sample with a flow rate of 1mL/min. Pharmacokinetic data were analyzed to determine flavopiridol and lipid elimination half-life and total drug exposure (area under the curve; AUC0-24hrs) using the PK Solutions software (Summit Research Services, USA). Plasma from animals injected with flavopiridol-containing liposomes were further analyzed for copper content. Samples diluted with 0.1%HNO3 (v/v with MilliQ water) were analyzed using the AAnalyst 600 Atomic Absorbance Spectrometer (Perkin Elmer, Inc. Woodbridge, ON, Canada): samples were dried at 110oC for 58 30s, heated (45s) to 130oC, ashed at 1200oC for 30s, and atomized and cleaned at 2000oC (5s) and 2450oC (4s), respectively. To define tolerated doses of the different flavopiridol formulations, mice (n=3) were injected i.v. (lateral tail vein) with the test article using a Monday, Wednesday, and Friday dosing schedule for 2 weeks (M, W, F x2). Following injection, the health of the animals was evaluated through monitoring of body weight loss, appetite changes, and other behavioural changes such as lethargy and signs of stress. When severe toxicity was apparent, the animals were terminated for humane reasons using isoflurane overdose followed by CO2 asphyxiation. Necropsy was performed to further assess signs of toxicity. If the animals survived treatment, they were monitored for 14 days after the administration of the last dose and full necropsies were then completed. Once the tolerated dose was defined based on the treatment schedule defined above, efficacy studies were initiated. Two models were used: the MV4-11 and MOLM-13 acute myeloid leukemia models. Cells were grown and maintained in media as described above in the Tissue Culture section. For in vivo studies, cells were harvested from passage #3 to #10 when they reached a confluence of 80-90%. Passage 0 was defined as when the cells were received from the ATCC and each passage was defined when the cells needed to be split in culture, which was approximately 2 times per week. Cell counts and cell viability were determined using Cellometer Auto T4 (Nexcelom, Lawrence, MA). Cells were then centrifuged at 200xg for 7 minutes. After removal of supernatant, cells were resuspended in growth medium to achieve a final cell concentration of 20x106 cells/mL for MV4-11 and 10x106 cells/mL for MOLM-13. The cell number for inoculation is 1x106 cells and 0.5x106 cells for MV4-11 and MOLM-13, respectively. The cells were then injected subcutaneously into the right flank of mice in a volume of 50 µL using a 28-gauge needle. For MV4-11 studies, treatment started once the average tumour volume reached 100mm3. For MOLM-13 studies, treatment started seven days post cell inoculation. Liposomal flavopiridol formulations were prepared as described above. Free flavopiridol test articles were prepared by dissolving flavopiridol powder in acidified PBS (pH 3.5). Mice were injected i.v. with the indicated dose (mg/kg) in a volume of 200µL per 20g mouse. Mice would not be injected a volume that exceeded 250 µL. On the basis of tolerability studies (see above), flavopiridol doses of 2.5 and 5.0 mg/kg were used when giving liposomal flavopiridol 59 formulations and 5.0 mg/kg when the free flavopiridol formulation was used. Mice were dosed using the M, W, F x 2 schedule and tumour size was determined by measuring tumour dimensions with callipers starting on first day of treatment. Tumour volumes were calculated according to the equation L x W2 /2 with the length (mm) being the longer axis of the tumour. Tumours were allowed to grow to a maximum size of 800mm3 before the animals were euthanized for humane reasons and as a result of tumour progression. 3.2.8 Statistical analysis GraphPad Prism 7.0 software was used to plot all data as mean ± standard error of the mean (SEM) as described in the Figure legends. Statistical comparisons of tumour growth were performed using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test to correct for multiple comparisons (P-value < 0.05 indicates statistical significance). Statistical analysis of survival study was performed using the log rank test, with a P-value < 0.05 indicating statistical significance. 60 3.3 Results Figure 3.1. Effects of exposure time of flavopiridol on acute myeloid leukemia (AML) cells in vitro. The time-dependent cytotoxic effects of flavopiridol on MV4-11 (A) and MOLM-13 (B) human AML cell lines were examined. Cells were exposed to flavopiridol for varying lengths of time (1, 4, 8, 24, 48, and 72 hours) and fraction of cells affected were plotted as shown. Data points represent the mean ± SEM (n=3) (B). “Fraction affected” values here indicate cell viability. As shown in Figure 3.1, flavopiridol inhibits the proliferation of the AML cell lines MV4-11 (Figure 3.1A) and MOLM-13 (Figure 3.1B). The potency of the drug (as judged by the concentration of the drug required to prevent 50% cell viability/proliferation) is dependent on exposure time (see Methods). As the flavopiridol exposure time increased from one hour to 72 61 hours, the IC50 of the drug decreased 30-fold and 40-fold for MV4-11 and MOLM-13, respectively. Figure 3.2. Effects of temperature on the formation of copper-flavopiridol complexes in liposomes. Flavopiridol is a semi-synthetic flavonoid, containing a binding site for copper chelation (red filled circle) (A). Spectrophotometric analysis of flavopiridol (10 µg/mL) in methanol in the absence (thin solid line) and presence of equimolar copper (dashed line; copper was added as copper sulfate) (B).The association of flavopiridol with 300mM CuSO4-containing DSPC:Cholesterol (55:45 mol:mol) liposomes was assessed as function of time (5, 10, 15, 30, and 60 minutes) and temperature (30⁰C, 40⁰C, 50⁰C, and 60⁰C) after addition of the liposomes to flavopiridol powder. The results were determined and plotted as % flavopiridol encapsulated over time. Data points represent the mean ± SEM (n=3) (C). A liposomal formulation was developed to improve flavopiridol’s exposure to these cancer cells in vivo. Studies were initiated based on a method that used coordination chemistry and liposomes to encapsulate flavopiridol. Flavopiridol has the capacity to bind copper at the 4,5-dihydroxyl group (Figure 3.2A) and as shown in Figure 2B, the association of flavopiridol with copper at a 1:1 mole ratio led to a change in the drug’s absorption spectrum and this is also reflected in a change in colour (solution turns yellow) as flavopiridol becomes associated with the liposomes (data not shown). As indicated in Figure 3.2C, the encapsulation of flavopiridol 62 into copper sulfate-containing liposomes is rapid with more than 98% of the flavopiridol added to copper-containing DSPC/Chol (55:45 mol%) liposomes becoming associated with the liposomes within 30 minutes regardless of temperature. It should be noted that temperature did have a minor effect on the rate of flavopiridol association with liposomes. The rate of association was reduced as the temperature decreased from 60oC to 30oC, as evident in the decreased flavopiridol association at 30oC compared to 60oC (60% versus >98%, respectively) after 5 minutes of incubation (Figure 3.2C). Figure 3.3. Copper-dependent association of flavopiridol with liposomes and the impact of transmembrane pH gradient. Powdered flavopiridol was mixed with DSPC:cholesterol liposomes prepared in 300mM CuSO4 (pH 3.5), 300mM citric acid (pH 4.0), and 250mM (NH4)2SO4 (pH 5.0) at 40⁰C. At indicated time points, liposomes were passed through Sephadex G-50 spin columns (see Methods) and % flavopiridol encapsulated was measured (A). DSPC:Chol (55:45 mol:mol) liposomes containing 300mM CuSO4 were exchanged against HBS pH 7.4 (HEPES buffered saline (150mM NaCl, 20mM HEPES)), citric acid saline pH 3.5 (150mM NaCl, 63 20mM citric acid), or HEPES potassium (HEPES buffered potassium (155mM KCl, 20mM HEPES)) and then incubated at 40⁰C. For the latter sample, valinomycin was added (see Methods) in an effort to collapse the pH gradient that may have existed across the liposomal membrane. At time zero, powdered flavopiridol was added and at the indicated time points, liposomes were passed through Sephadex G-50 spin columns (see Methods) and % flavopiridol encapsulated was measured (B). Data points represent the mean ± SEM (n=3). To further understand the role of copper in flavopiridol encapsulation, flavopiridol was added to liposomes prepared with 300mM CuSO4 (an unbuffered salt solution at pH 3.5), comparable to the formulation used to generate the data in Figure 3.2B, 300mM citric acid (pH 4.0) (pH gradient loading), or 250mM (NH4)2SO4 (pH 5.0) (a form of pH gradient loading). The results, summarized in Figure 3.3A, indicate that efficient flavopiridol association with the liposomes was only achieved with the copper-containing liposomes (filled circles). When using copper-free liposomes containing 300mM citric acid (pH 4.0, filled triangle) or 250mM (NH4)2SO4 (filled square), the levels of flavopiridol association were less than 2%. Flavopiridol, like all flavonoids, are sparingly soluble in water (<1mg/mL) but aqueous solutions of flavopiridol can be prepared in acidic buffers (78). Therefore, since the unbuffered copper sulfate solution is pH 3.5, it was important to determine if flavopiridol could still become associated with copper-containing liposomes in the absence of a trans-membrane pH gradient where the external pH was equal to the internal pH (e.g. pH 3.5). To investigate this, liposomes (DSPC:Chol (55:45 mol:mol)) were prepared in copper sulfate solutions and the external buffers were changed (see Methods) to HBS (pH 7.4) or citrate buffered saline (pH 3.5). As indicated in Figure 3.3B, >90% of the added flavopiridol became associated with the liposomes by 30 minutes when the external pH was 3.5 (filled triangles). The rate of loading at this lower external pH was less than that observed at pH 7.4 (filled circles). In formulations where the outside buffer contained potassium, valinomycin was added to collapse the pH gradient. Despite the absence of a pH gradient with these formulations, there was still efficient flavopiridol association with the liposome (Figure 3.3B, solid squares). 64 Figure 3.4. Pharmacokinetics of flavopiridol in vivo following intravenous injection of different flavopiridol formulations. Female CD1 mice were injected intravenously with a single bolus dose of free flavopiridol (low pH formulation) or liposomal formulations of flavopiridol at 5mg/kg. In an attempt to determine if a pH gradient decreased the elimination rate of DSPC/Chol (55:45 mol:mol) liposomal flavopiridol, the divalent cation ionophore A23187 was added (see Methods). Plasma concentrations of flavopiridol were measured by HPLC (see Methods) at the indicated time points following administration. Where appropriate, the plasma level of liposomal lipid (scintillation counting) and copper (AAS) were measured (see Methods). Each point represents the mean ± SEM; n=4. The level of plasma flavopiridol is summarized in panel A. The relative flavopiridol-to-lipid (F/L) ratio or the relative copper-to-lipid (C/L) ratio as a function time could be calculated, normalizing to the F/L and C/L ratios at time 0. Relative F/L and C/L ratios as a function of time are plotted in panels B and C respectively. To assess if liposomal lipid composition enhanced flavopiridol levels in the plasma compartment, the liposomal lipid composition was changed from DSPC/Chol (with and without A23187) to Spingomyelin (SM)/Chol (55:45 mol:mol) or DSPC:DSPE-PEG2000 (95:5 mol:mol).Thirty minutes following i.v. administration, the plasma concentration of flavopiridol was measured. The results are plotted in Panel D. 65 Pharmacokinetic studies were used to characterize the elimination of flavopiridol after injecting mice i.v. (5mg/kg) with the low pH “free” flavopiridol formulation and the DSPC/Chol (55:45 mol:mol) formulation. The results are summarized in Figure 3.4. When DSPC:Chol formulations of copper flavopiridol were injected, flavopiridol was eliminated rapidly from the plasma compartment (Figure 3.4A filled squares) with an elimination half-life (t1/2) of 0.5 hours. Free flavopiridol was eliminated more rapidly but the estimated AUC(0-120min) was only about 2-fold greater for the liposomal formulation (0.804µg-hr/mL for free and 1.607µg-hr/mL for liposomal). As shown in Figure 3.4B, following administration of the DSPC:Chol copper flavopiridol formulation, the relative flavopiridol-to-liposomal lipid ratio decreased rapidly upon injection. The rapid elimination of flavopiridol following administration of the DSPC:Chol formulation of copper flavopiridol was due to release of the encapsulated drug. Interestingly, copper was retained relatively well in these formulations, with approximately 40% copper retention 2 hrs after administration (Figure 3.4C) (compared to < 1% flavopiridol retention at the same time point). Previously, our team demonstrated that the use of encapsulated copper plus a divalent ionophore A23187 engendered significant improvements in drug retention for some drugs like the camptothecins (232). This was explained primarily on the basis of A23187-mediated exchange of entrapped copper for protons, generating a pH gradient while also retaining sufficient copper to decrease liposomal membrane permeability. The addition of A23187 to the preparation of copper-complexed flavopiridol was therefore assessed. The results, also shown in Figure 3.4 (panels A, B and C), indicated that the in vivo stability of the resulting formulation was essentially identical to the formulation prepared without A23187. In an attempt to define a formulation with improved flavopiridol retention, two changes in liposomal lipid composition were made. It has been shown previously that for some encapsulated drugs, the use of sphingomyelin (SM):Chol (55:45 mol:mol ratio) or cholesterol free liposomes (DSPC:DSPE-PEG 2000 (95:5 mol:mol ratio) improved drug retention following iv administration (114, 116). In the initial screening studies, the level of flavopiridol in the plasma compartment 30 minutes after intravenous administration was determined. As shown in Figure 3.4D, both formulations increased plasma levels after administration, but the most significant effect was achieved when using the DSPC:DSPE-PEG 2000 (95:5 mol:mol ratio) liposomes where the flavopiridol levels in the plasma at 30 minutes after injection were 90- and 30-fold higher 66 when compared to the low pH flavopiridol formulation and the DSPC:Chol formulations of copper flavopiridol, respectively. Figure 3.5. Copper-flavopiridol prepared in DSPC:DSPE-PEG2000 (95:5 mol:mol) liposomes was compared to the low pH free-drug formulation. Female CD1 mice were injected intravenously with a single bolus dose of free flavopiridol (in acidified PBS, pH 4.0) or the liposomal flavopiridol at 5mg flavopiridol/kg. Plasma concentrations of flavopiridol (A) and plasma concentrations of lipids (B) over 240 minutes were measured (time points = 15, 30, 60, 120, and 240 minutes) (A). For the liposomal formulation, relative flavopiridol-to-lipid (F/L) ratios or the relative copper-to-lipid (C/L) ratios as a function time were then calculated, normalizing to the F/L and C/L ratios at time 0. Relative F/L and C/L ratios as a function of time are plotted in panels C and D respectively. All data are plotted as mean ± SEM (n=3). These results are better illustrated in Figure 3.5, where the elimination of flavopiridol following administration of the DSPC:DSPE-PEG 2000 liposome formulation of copper flavopiridol in vivo was evaluated over 4 hrs. This liposomal flavopiridol formulation demonstrated a 30-fold increase in AUC0-4hrs (0.804µg-hr/mL versus 26.92µg-hr/mL) compared to free flavopiridol. The plasma elimination of flavopiridol was significantly reduced (Figure 3.5A). However, 75% of the 67 encapsulated drug was still release from the circulating liposomes within 30 minutes (Fig. 3.5C). Similar to the previous liposomal formulations, there was no significant elimination of liposomal lipid over 4hrs following administration (Figure 3.5B) and the copper-to-lipid ratio of the liposomes was maintained. The copper-to-lipid ratio was only 20% lower than that of the original copper-to-lipid ratio after 4hrs (Figure 3.5D), suggesting that flavopiridol is being released in an uncomplexed form. The DSPC:DSPE-PEG 2000 liposome formulation of copper flavopiridol was selected as an optimized formulation and efficacy studies were initiated to determine whether the longer exposure time achieved was sufficient to improve efficacy in murine models of leukemia. Figure 3.6. Preliminary tolerability study. For the preliminary tolerability study, CD1 mice were dosed i.v. with the low pH formulation of flavopiridol or the DSPC/DSPE-PEG200 (95:5 mole%) liposomal formulation of flavopiridol. Percent change in weight loss was collected. 68 Table 3.1. Clinical signs of toxicity for preliminary tolerability study. Prior to initiation of the efficacy studies, dose-range finding tolerability studies were conducted and the results are summarized in Figure 3.6 and Table 3.1. At 5mg/kg, there was no decrease in body weight over time but minor changes in clinical signs were present and these signs appeared slightly worse in the animals treated with the liposomal drug. The signs included mild scruffiness in both the free and liposomal groups as well as dehydration (skin tenting) and hunching in the liposomal group. When the drug dose was escalated to 10 mg/kg, both the free and liposomal formulations caused toxicity; but the toxicities were much more severe in the liposomal group where significant weight loss resulted in termination of all the animals in the group. 1 out of 3 animals needed to be euthanized in the free 10 mg/kg group and these animals showed mild to moderate scruffiness, dehydration, and signs of pain (i.e. squinty eyes). For this reason, the ensuing efficacy studies were conducted at 5 mg/kg with the free drug or 2.5 and 5 mg/kg with the liposomal drug. Formulation Dose Dosing Schedule Clinical Observations Free Flavopiridol 5mg/kg Monday, Wednesday, Friday x 2 weeks Mild scruffiness Free Flavopiridol 10mg/kg Monday, Wednesday, Friday x 2 weeks Mild to moderate scruffiness, skin tenting, squinty eyes, one mouse euthanized due to high cumulative clinical score Liposomal Flavopiridol 5mg/kg Monday, Wednesday, Friday x 2 weeks Mild scruffiness, mild skin tenting, mild hunching Liposomal Flavopiridol 10mg/kg Monday, Wednesday, Friday x 2 weeks Skin tenting, scruffed coat, diarrhea, excessive weight loss (more than 15%), entire group terminated due to dose-related toxicities 69 Figure 3.7. Efficacy of liposomal and free flavopiridol formulations given intravenously to mice with established MV4-11 and MOLM-13 tumours. Female NRG mice were inoculated subcutaneously with MV4-11 (left panels) and MOLM-13 (right panels) acute myeloid leukemia cells with 50µL of 1x106 cells/mL and 0.5x106 cells/mL, respectively. Tumour-bearing mice were then injected intravenously with DSPC:DSPE-PEG2000 (95:5 mol:mol) liposomal and free flavopiridol formulations (Free flavopiridol - 5mg/kg; Liposomal flavopiridol – 2.5mg/kg, and 5mg/kg) every Monday, Wednesday, and Friday for two weeks. Mean tumour volumes were measured three times per week and the results have been plotted as mean ± SEM (A). Red triangles on x-axis indicate days where treatment occurred. “**” indicates statistical significant difference (P<0.05) as determined by one-way analysis of variance with Tukey’s post hoc test for multiple comparisons (see Methods). Percent survival (as defined by when the animals had to be terminated for humane reasons as defined by the IACC animal care protocol) was also plotted for each of the indicated treatment and control groups (B). Survival statistical analysis was performed using the log rank test. “*” indicates statistically significant difference as indicated by a p-value < 0.05. Immuno-compromised mice were inoculated subcutaneously with MV4-11 cells (see Methods) and when tumours were established, the mice were treated i.v. with the liposomal formulation or free flavopiridol formulations at 2.5/5.0 mg/kg and 5mg/kg (see Methods), respectively. The efficacy results are summarized in Figure 3.7. For mice bearing established MV4-11 tumours, treatment with free or liposomal flavopiridol (5 mg/kg respectively) resulted in significant delays in tumour growth (Figure 3.7A – left panel). However the 5 mg/kg liposomal dose 70 resulted in tumour regression and was significantly more active then free flavopiridol. At 31 days post-inoculation, animals treated with 5mg/kg of liposomal flavopiridol showed a 92% reduction (est. 19.2mm3 versus 254.4mm3) in mean tumour volume when compared to animals treated with free flavopiridol at 5mg/kg (Figure 3.7A). In this model the effects of 5mg/kg free flavopiridol and 2.5 mg/kg liposomal flavopiridol were essentially identical. On the basis of these data it appears that the potency of flavopiridol increased at least 2-fold when using the liposomal formulation. Similar results were obtained when using the faster-growing MOLM-13 model (Figure 3.7, right hand panel). Treatment with 5mg/kg of liposomal flavopiridol (7 days after tumour cell inoculation) resulted in significantly longer delays in tumour progression compared to free flavopiridol at equivalent doses. At the 5mg/kg dose of liposomal flavopiridol, there was a delay in the time to reach 800mm3 of 39 days versus 22 days for the equivalent free flavopiridol dose. In both in vivo tumour models, treatment with liposomal flavopiridol at 5mg/kg significantly (p<0.05) prolonged the overall survival of the animals (Figure 3.7B). When compared to free flavopiridol at an equivalent dose of 5mg/kg, liposomal flavopiridol prolonged median survival by 11 days (26% increase) in the MV4-11 murine model and 17 days (74% increase) in the MOLM-13 model (Figure 3.7B). 3.4 Discussion Flavopiridol (also known as Alvocidib) is thought of as a pan-CDK inhibitor with clinical promise. However, the clinical results have been disappointing (233). The benefits that were expected for flavopiridol as a single agent were largely hindered by the drug’s protein binding and the fact that it is a cell cycle specific agent requiring extended exposure to achieve maximum therapeutic effects (234). It is possible that the limitations could be resolved through use of nano-scaled drug delivery systems which can protect the drug from protein binding and extend circulation lifetime to ultimately increase tumor cell exposure to the drug (90). Our efforts to define an improved liposomal formulation of flavopiridol are driven by the fact that this formulation is ideally suited for development of drug combination products, as discussed below. Ten years ago, Yang et al. reported on the development of a liposomal formulation of flavopiridol (235). Despite using what is referred to as “stealth liposomes” (PEGylated liposomal 71 carriers), this group demonstrated only modest increases in the plasma flavopiridol AUC when using the liposomal formulation (the liposomal formulation exhibited an AUC0-24hr that was 3.2-fold greater than the free drug) (235, 236). This work concluded that “further preclinical studies are warranted to define the toxicity and therapeutic efficacy of [the] novel formulation” (235). To date, there has been no further information about this formulation and this may be due to the fact that the changes obtained in pharmacokinetics were essentially modest. The results that are summarized here for a DSPC/Chol flavopiridol formulation prepared using a novel technology involving the formation of flavopiridol-copper complexes are similar to that reported by Yang et al. The results suggest a 2-fold increase in plasma AUC and definitively demonstrate that flavopiridol rapidly dissociates from liposomes following intravenous administration (see Figure 3.3). Our results differ from the previous results in that no pH gradient-mediated encapsulation of flavopiridol was noted, regardless of whether the liposomes were prepared in 300mM (pH 4.0) citrate buffer or ammonium sulfate (see Figure 3.4). This is likely due to differences in methodology since the previous studies required flavopiridol to be pre-dissolved in a low pH buffer prior to addition to the liposomes. Flavopiridol is water-insoluble and for our studies, the powdered drug was added directly to the liposomes. Interestingly, the powder eventually “dissolved” as the drug became associated with the liposomes. This method enhanced the apparent solubility of flavopiridol by at least 50 fold from about 0.02 mg/mL to more than 1mg/mL. Our results clearly reiterate the fact that flavopiridol is a cell cycle-specific agent that exhibits remarkable increases in potency when the exposure time to the drug is increased (Figure 3.1). Although I have defined a new method to prepare a liposomal formulation of flavopiridol based on its ability to complex with copper (Figure 3.2 and 3.3), the formulation rapidly released flavopiridol (but not copper) after it was administered i.v. (Figure 3.4). Under the conditions described here, the efficient loading of flavopiridol is copper-dependent and not pH gradient-dependent (Figure 3.4). Furthermore, the use of metal complexation to prepare a liposomal formulation of flavopiridol did not require pre-solubilization of the drug as the powder form of the drug can be added directly to copper-containing liposomes. Liposomes are well-documented to provide a physical barrier between an encapsulated drug and serum proteins, thereby reducing the level of protein binding whilst prolonging the drug’s circulation time (90, 150). The results in this study demonstrated that flavopiridol retention in 72 the liposome is highly dependent on lipid composition. Specifically, DSPC:DSPE-PEG2000 liposomes appeared most stable, resulting in significant increases in flavopiridol levels in the plasma over extended time periods. As flavopiridol’s therapeutic activity clearly increases with increased exposure time (Figure 3.1), the therapeutic results obtained in two models of AML were considerably better when using the DSPC:DSPE-PEG2000 liposomal formulation (see Figure 3.7). The liposomal formulation was at least 2-times more potent than the low pH free flavopiridol formulation and it is clear that an appropriately selected liposomal formulation will have significant clinical potential. As such, continued efforts are being invested to develop formulations that can further improve the retention of flavopiridol. This effort represents only the first step towards defining an improved formulation for the treatment of elderly patients with refractory AML. One of the biggest challenges of treating AML is to address the poor prognosis of relapsed or refractory AML (RR-AML). Currently, treatment options are quite limited and they typically entail aggressive high-dose chemotherapy followed by allogeneic hematopoietic stem cell transplantation or palliative care for patients unable to tolerate the rigours of intense chemotherapy (237). As such, efforts have been made to improve the outlook for RR-AML patients by treating patients with novel pharmaceuticals (237). With improved pharmacokinetics that led to positive results in preclinical leukemia model studies, the liposomal flavopiridol formulation described in this study demonstrates promise to be a candidate as another treatment option for RR-AML patients. In particular, recent clinical trials have been using flavopiridol as a component of a combination treatment regimen called FLAM (238). The FLAM treatment program consists of administering flavopiridol as 1-hour i.v. bolus injection at 50mg/m2 daily for three days, followed by continuous infusion of cytarabine at 667mg/m2/day for three days and a single dose of mitoxantrone at 40mg/m2 (239). Compared to the conventional “7+3” induction regimen, FLAM has displayed improved induction rate of complete remission in poor-risk (mainly RR-AML) patients (70% versus 46%) with no added toxicity (239). The success of FLAM has led to a Phase 3 clinical trial currently underway (239). In light of FLAM’s recent success, I argue that flavopiridol’s therapeutic value lies in combination therapy. Therefore, to further elucidate the clinical utility of flavopiridol, I plan to use an optimized liposomal flavopiridol formulation in combination therapies. As such, additional studies will be completed to better 73 define the use of liposomal copper-flavopiridol formulations in combination settings using preclinical animal models. 3.5 Conclusions In summary, a novel liposomal formulation of flavopiridol was described by leveraging the drug’s ability to coordinate with copper. The resulting formulation rapidly released flavopiridol from the liposomes following i.v. administration, with the copper staying associated with the liposomes. The liposomal formulation tested engendered a significant increase in plasma AUC following administration and at equivalent doses, this liposomal flavopiridol was more active than the free drug in two animal models of AML. The focus is now on defining liposomal formulations with even better drug retention and to assess the activity of the formulation described here when it is used as a component of a combination product for the treatment of RR-AML. 74 4. Development of a novel combination product of liposomal flavopiridol and liposomal mitoxantrone for treatment of acute myeloid leukemia 4.1 Introduction As discussed in Chapter 3 and the Introduction, acute myeloid leukemia patients continued to face poor prognosis after more than 40 years of stagnant progress in treatment advancements. In particular, patients with adverse risk disease, including patients with secondary AML, have grim outlooks with long-term survival remaining at a mere 10% (240). In order to improve therapeutic responses in AML patients, studies have employed “timed sequential therapy” (TST), which are treatment strategies that rely on opportune timing of cell-cycle specific chemotherapeutic agents to maximize their therapeutic effects on leukemic cells (238). In other words, TST involves selecting leukemia cells into the proliferative cell cycle using one drug and administer a second, cell-cycle specific in close succession to target cells going into cell cycle kinetics (241). Early proof-of-concept preclinical models of TST in leukemia demonstrated increased sensitivity to cycle-dependent chemotherapy in response to drug-induced proliferation (242-246). Furthermore, one of the first attempts to cure AML involved two complete courses of TST and this treatment regimen was shown to result in durable disease-free survival in patients (247, 248). To date, the most promising results for TST are in childhood AML where repetitive DCTER (daunorubicin, cytarabine, thioguanine, etoposide, and dexamethasone) administered in sequential cycles (10 days apart between cycles) demonstrated improved 3-year event-free and disease-free survival rates when compared to standard induction therapy (249). However, despite the success of TST, the regimen has yet to become incorporated into mainstream AML therapy (238). Recently, a variant of TST was proposed: the FLAM regimen (239). FLAM delivers flavopiridol, cytarabine, and mitoxantrone in a sequential manner and the regimen is targeted towards patients with poor-risk acute myeloid leukemia (239). In a multi-centre, randomized clinical trial, the investigators evaluated the FLAM regimen against conventional “7+3” (see Introduction) in 165 poor-risk patients at 10 centres (239). The findings from the study demonstrated a greater number of secondary AML patients (60% versus 35%) achieving 75 complete remission with FLAM than with “7+3” (239). However, despite the promising improvement in response rates, FLAM did not yield significant differences in overall survival (239). As such, there is clearly room for improvements for the FLAM regimen and for this reason the combination of encapsulated flavopiridol (see Chapter 3) and encapsulated mitoxantrone (250) was considered here. As described in the Introduction, liposomes are nano-scaled drug delivery systems that have demonstrated valuable clinical utility through their abilities to modulate pharmacokinetics and attenuate toxicities for drugs they encapsulate (84). The robustness of liposomes was recently showcased in the recently approved combination product for the treatment of AML, Vyxeos (CPX-351) (58). Vyxeos comprises cytarabine and daunorubicin encapsulated into bilamellar liposomes at a 5:1 molar ratio: a ratio that engender the maximum synergistic activity of these two drugs when tested in vitro against multiple cancer cell lines (53). The liposomes were designed to ensure that following intravenous administration that the 5:1 drug ratio was maintained in the plasma compartment and in the tumour for at least 24 hours (53). Vyxeos provided significantly better therapeutic results in clinical trials when compared to the same two drugs administered using the “7+3” regimen (58, 251). As such, Vyxeos was approved in 2017 for use in the treatment of older refractory AML patients (252). The success of Vyxeos highlighted the potential of liposomes to serve as a platform for delivery of drug combinations and inspired studies to assess the combination of liposomal flavopiridol and liposomal mitoxantrone. As described in Chapter 3, a liposomal formulation of flavopiridol was developed and this formulation was about 2 times more potent than unencapsulated, low pH, flavopiridol. Further, a liposomal mitoxantrone formulation was developed in this lab in 1997 - one that exhibited significant activity when tested in syngeneic murine models of leukemia (253). For this reason, it was relatively easy to consider the use of these two formulations in combination: research that was similar to what was completed using optimized formulations of vincristine and doxorubicin (254). In the latter example, the combination proved to be highly antagonistic when used in vivo: a result that was predicted based on in vitro data demonstrating 76 antagonism. As shown below, flavopiridol and mitoxantrone, when used in vitro in combination, were highly synergistic. 4.2 Materials and methods 4.2.1 Materials Flavopiridol was purchased from Selleckchem (Houston, Texas, USA). Mitoxantrone was purchased from the BC Cancer pharmacy (Vancouver, BC, Canada). Sephadex G-50, copper (II) sulfate, HEPES, ethylenediaminetetraacetic acid (EDTA), citric acid, and sodium chloride (NaCl) were purchased from Sigma-Aldrich (St. Louis, MO). Sucrose was obtained from EMD Chemicals Inc. (Gibbstown, NJ, USA). 1,2-disteoroyl-sn-glycero-3-phosphatidylcholine (DSPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-n-[carboxy(polyethylene glycol)-2000] (DSPE-PEG2000), and cholesterol (chol) were obtained from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). 3[H]-cholesteryl hexadecyl ether (CHE) and picofluor-15 scintillation fluid were purchased from Perkin-Elmer, Inc. (Boston, MA, USA). 4.2.2 Tissue culture MV4-11 human biphenotypic B myelomonocytic leukemia cell line was obtained from ATCC (Cat # CRL-9591) in 2015 with Mycoplasma negative certificate issued by ATCC. MV4-11 and MOLM-13 were cultured in IMDM (1X) (Life Technologies, Carlsbad, CA) and RPMI-1640 medium (Life Technologies) supplemented with 10% fetal bovine serum (Life Technologies) and 2mM L-glutamine (Life Technologies). The cells were grown and maintained at 37°C in 5% CO2 environment. P388 murine leukemia ascites were obtained from the NCI Repository at the Frederick National Laboratory in 2018 as frozen vials. The stock vials were suspended in freezing medium (80:10:10 culture medium : DMSO : FBS) containing approximately 106 – 107 cells/mL. 4.2.3 In vitro dose response curves for flavopiridol and mitoxantrone In vitro cytotoxicity assays were conducted using flavopiridol and mitoxantrone alone or in combinations with MOLM-13 and MV4-11 cells. The cells were seeded in 384-well plates. 24 77 hours after seeding, cells were treated with flavopiridol (F) or mitoxantrone (M) either alone or in combination at M:F ratios of 1:2, 1:3, and 1:4 for MOLM-13 and 1:5, 1:10, and 1:15 for MV4-11. The ratios were selected based on the ratio of the IC10, IC50, and IC90 of each drug when used alone. At 72 hours post-plating, cells were incubated with PrestoBlue cell viability reagent (ThermoFisher, Waltham, Massachusetts, USA) (10% v/v) for either 2 hours (MV4-11) or 3 hours (MOLM13) at 37°C. The incubated plates were then imaged in the FLUOstar OPTIMA Microplate Reader (BMG Labtech, Offenburg, Germany). Dose response curves were constructed by plotting mean ± SEM from at least three independent experimental trials using Prism 8.0 (GraphPad Software, La Jolla, CA, USA). IC50 values were interpolated from fitted curves. Combination indices (CI) were derived from the dose response curves using CompuSyn software (ComboSyn, Inc, Paramus, New Jersey, USA). CI values >2 were considered as antagonistic, CI Values <0.8 were considered synergistic. All other CI values were considered additive. 4.2.4 Liposome preparation For DMPC:Chol (55:45 mol:mol), DMPC and Chol were dissolved in chloroform at a 55:45 mol:mol ratio. 3[H]-CHE was added as a non-exchangeable and non-metabolizable lipid marker lipid marker (255). prior to drying. 3H-CHE was incorporated into the chloroform mixture at a concentration of 5.0 - 12.5 nanocurie per μmol lipid. Specific activity was then determined by adding 10µL of the sample to 5mL of picofluor scintillation fluid. All samples were measured in quadruplicates. The radioactivity was measured on a Packard 1900 TR Liquid Scintillation analyzer. Chloroform was then removed by drying the samples under a steady stream of nitrogen gas until the samples became thick and syrup-like. The samples were then placed under high vacuum for at least 3 hours to create a bubbly lipid film in order to completely remove residual chloroform. The dried lipid was then hydrated in 300mM citric acid (pH adjusted to 4) at a concentration of 1mL solution per 50µmol of lipids. The samples were hydrated at 50°C with constant stirring. The hydrated lipids were then subjected to five cycles of freezing (snap-frozen in liquid nitrogen) and thawing (in 50⁰C water bath). The resulting multilamelar liposomes were then extruded ten times through stacked 0.1µm and 0.08µm filters in a 10mL or 100mL thermobarrel extruder (LIPEX® Extruder, Evonik Industries, 78 Vancouver, BC, Canada) at 50⁰C. The resultant unilamellar liposomes had a mean diameter of 100±20 nm as determined by dynamic light scattering (ZetaPALS, Brookhaven Instruments Corp., Holtsville, NY). The extruded liposomes were then run through a Sephadex G50 column pre-equilibrated with HEPES buffered saline (HBS) pH 7.4 (150mM NaCl, 20mM HEPES) to exchange the external loading buffer to HBS. For DSPC:DSPE-PEG2000 (95:5 mol:mol) liposomes an ethanol-based protocol was used. The lipids were weighed and dissolved in ethanol at 65⁰C (1mL of ethanol per 100µmol of lipids). 3[H]-CHE was added as marker and specific activity was measured as previously described. Dissolved lipids were kept warm at 65⁰C for the following steps. The lipids in ethanol were added drop-wise to 300mM copper sulfate (1mL of ethanol per every 6mL of copper sulfate) in 65⁰C with rapid mixing for a minimum of 30 minutes. The multilamelar liposomes which formed were then extruded as previously described. Ethanol was immediately removed from the extruded product by tangential flow filtration (TFF) using an ultrafiltration hollow fibre column (molecular weight cutoff 500kDa) (GE Healthcare). Extra-liposomal copper removal and buffer exchanges were done as described above. Sucrose/HEPES/EDTA (SHE) buffer pH 7.4 (300mM sucrose, 20mM HEPES, 15mM EDTA) was first used as the external buffer to remove unencapsulated copper and this was subsequently changed to HBS through Sephadex chromatography column. The final lipid concentrations for both liposomes were determined by measuring 3[H]-CHE using liquid scintillation counting as described above. 4.2.5 Preparation of liposomal flavopiridol and liposomal mitoxantrone Liposomal flavopiridol was prepared by adding copper sulphate- containing DSPC:DSPE-PEG2000 liposomes to pre-weighed flavopiridol powder as described in Chapter 3. Flavopiridol concentrations were measured via high performance liquid chromatography (HPLC; Waters, Milford, MA) at an absorbance wavelength of 264nm as described in Chapter 3. Liposomal mitoxantrone was prepared using a transmembrane pH gradient-driven loading method. Briefly, mitoxantrone was added to 300mM citric acid DMPC:Chol liposomes. 75µl of 0.1N NaOH was added to 8mL of the mitoxantrone/liposome mixture to bring the pH to 7. The resulting mixture was then mixed with constant stirring at 50°C for one hour. Incubated samples were then passed down an HBS-equilibrated Sephadex column to remove 79 unencapsulated mitoxantrone. The formulation was then concentrated using TFF as described above. Concentration of mitoxantrone was determined using HPLC at an absorbance wavelength of 610nm. Briefly, samples were diluted with chilled 3% acidified (glacial acetic acid) methanol. 20µL of the samples was subsequently injected into C18 column (particle size 4µm; 3.9x150mm, Nova-Pak). The mobile phase consisted of DEA in water (premix 300µl of DEA in 1L of acidified water (pH to 2.5 with H3PO4)) and acetonitrile at a ratio of 80:20. Standard curves (ranged from 1nmol/ml to 25nmol/ml) were prepared where 3% acidified methanol was used as the dilution solvent. All standards and samples were kept at 4±0.5 ºC prior to injections and the column temperature was set at 55±5ºC. Run time was 6 minutes per sample with a flow rate of 1mL/min. 4.2.6 In vivo studies Tolerability/toxicity studies: To define tolerated doses of the different flavopiridol formulations, mice (n=4) were injected intravenously (i.v.) (lateral tail vein) with the test article using specified dosing schedules (Monday, Wednesday, Friday dosing schedule for 2 weeks (M, W, F x2) or Days 1, 3, and 5). Following injection, the health of the animals was evaluated through monitoring of body weight loss, appetite changes, and other behavioural changes such as lethargy and signs of stress. When severe toxicity was apparent, the animals were terminated for humane reasons using isoflurane overdose followed by CO2 asphyxiation. Necropsy was performed to further assess signs of toxicity. If the animals survived treatment, they were monitored for 14 days after the administration of the last dose and full necropsies were then completed. Efficacy studies: Once the tolerated dose was defined based on the treatment schedule defined above, efficacy studies were initiated. Two models were used: MOLM-13 AML model and P388 murine leukemia model. For MOLM-13, cells were cultured and prepared for inoculation as described in Chapter 3. Treatment started seven days post cell inoculation. For P388, cells were collected from ascites from donor animal. Briefly, donor animal was disinfected. The skin was removed from the abdominal area of the animals and the amount of ascites within the abdominal cavity was observed to ensure there is enough to withdraw into 80 syringe. The fluid from the syringe was then transferred to a sterile centrifuge tube placed on ice. P388 cells are counted and placed into a T175 tissue culture flask in 30ml RPMI + 10% FBS. The cells were then incubated at 37°C for 3-4 hours. Suspended P388 cells from the flask were then pulled and resuspended. The cell suspension was then diluted to 5 x 104 cells/mL in Hank’s Balanced Salt Solution (HBSS) and injected intravenously to new mice at 0.5 x 106 cells/mL. The cell number for inoculation was 1x105 cells in an injection volume of 200l per animal. On day 0, tumour cells were implanted intravenously into mice in a volume of 200l using a 28-gauge needle. Liposomal flavopiridol and liposomal mitoxantrone were prepared as described above. Free flavopiridol test articles were prepared by dissolving flavopiridol powder in acidified PBS (pH 3.5). Mice were injected i.v. with the indicated dose (mg/kg) in a volume of 200µL per 20g mouse. Mice would not be injected a volume that exceeded 250 µL. Mice were dosed based on tolerability studies (see above). For MOLM-13 efficacy studies, tumour size was determined by measuring tumour dimensions with callipers starting on first day of treatment. Tumour volumes will be calculated according to the equation L x W2 /2 with the length (mm) being the longer axis of the tumour. Tumours were allowed to grow to a maximum size of 800mm3 before the animals were euthanized for humane reasons and as a result of tumour progression. For P388 efficacy studies, percent survival was determined and the health of the animals was evaluated through monitoring of body weight loss, appetite changes, and other behavioural changes such as lethargy and signs of stress. When severe toxicity was apparent, the animals were terminated for humane reasons using isoflurane overdose followed by CO2 asphyxiation. 4.2.7 Statistical analysis GraphPad Prism 7.0 software was used to plot all data as mean ± standard error of the mean (SEM) as described in the Figure legends. Statistical comparisons of tumour growth were performed using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test to correct for multiple comparisons (P-value < 0.05 indicates statistical significance). Statistical analysis of survival study was performed using the log rank test, with a P-value < 0.05 indicating statistical significance. 81 4.3 Results It was important to demonstrate that flavopiridol and mitoxantrone interacted synergistically and to determine if synergy was observed that it was or was not ratio-dependent, like daunorubicin and cytarabine (53). As shown in Figure 4.1A, when compared to single-agent treatments, the combination of flavopiridol and mitoxantrone exhibited enhanced cytotoxic effects in vitro at all effect levels for MOLM-13 (left panel) and at high effects levels (>60% cell kill) for MV4-11 (right panel). This was observed as a leftward shift in the cytotoxicity curves. Combination index (CI) (see Methods) analysis confirmed the synergistic relationship of flavopiridol and mitoxantrone. As seen in Figure 4.1B, CI values were below 0.8 at all effect levels for MOLM-13 (left panel) and at high effect levels (>60% cell kill) for MV4-11 (right panel). Three different drug-to-drug ratios were evaluated for each cell line and the results indicate that the synergy between flavopiridol and mitoxantrone was ratio-independent. This result made it reasonable to evaluate the therapeutic activity of liposomal formulations of the drugs which were not designed to control for drug-to-drug ratio. Figure 4.1. In vitro assay assessing the cytotoxic effects of flavopiridol alone and in combination with mitoxantrone. The dose response curves for flavopiridol (Fla) and/or mitoxantrone (Mito) were determined for MOLM-13 (left panel) and MV4-11 (right panel) acute myeloid leukemia cells using PrestoBlue cell viability assay as described in the Methods. For combination effects, flavopiridol and 82 mitoxantrone were added at Mito:Fla ratios of 1:2, 1:3, and 1:4 for MOLM-13 and 1:5, 1:10, and 1:15 for MV4-11. The dose response curve for the combination was plotted based on mitoxantrone concentrations (A). Combination Indices (CI) derived from the dose response of the combination treatments are plotted against the fraction of affected (Fa) cells, where a Fa of 1 indicated 100% cell kill. CI>2: antagonistic 2>CI>0.8: additive, and CI<0.8: synergistic (B). “Fraction affected” values here indicate cell viability. Data points represent mean ± SEM (n≥3). When it was first reported in 1997, an optimized DMPC/Chol liposomal mitoxantrone was administered as a single bolus injection at 10mg/kg; a dose that was well tolerated (253). It was important to assess whether the therapeutic activity of this formulation could be reproduced more than 20 years later. Figure 4.2A is adapted from the study published by Lim et al. (1997) and the panel shows that at equivalent doses of 10mg/kg, liposomal mitoxantrone was significantly more effective than free mitoxantrone, with 100% of the treated animals surviving past 60 days. The therapeutic effects of the DMPC/Chol formulation was assessed in the same P388 model and as shown in Figure 4.2B liposomal mitoxantrone administered as a single bolus at 10mg/kg prolonged median survival by 24 days (two-fold increase) - an effect that was less than that observed previously. A survival rate past 60-days of greater than 40% was still observed. A multi-dose schedule with liposomal mitoxantrone at 2mg/kg was significantly less active than liposomal mitoxantrone at 10mg/kg administered as a single bolus injection (Figure 4.2). There were no discernable differences between the toxicity profiles of the two different liposomal mitoxantrone dosing strategies. But in the more recent studies, free mitoxantrone (10mg/kg) was toxic, with the animals having to be euthanized for humane reasons and due to severe (>20%) weight loss. The efficacy result obtained for liposomal mitoxantrone at 10mg/kg were less than what had been reported by Lim et al. 83 Figure 4.2. Efficacy of liposomal and free mitoxantrone given intravenously to mice inoculated with P388 murine leukemia cells. Panel A is survival data adapted from Lim et al. 1997. In Panel B, female BDF-1 mice were inoculated intravenously with P388 murine leukemia cells in a volume of 200µL of 0.5x106 cells/mL. 24 hours after inoculation, the mice were then treated with free mitoxantrone at doses of 0.5, 1, and 10mg/kg and liposomal mitoxantrone at doses of 1, 2, and 10mg/kg. 10mg/kg of free and liposomal mitoxantrone were administered as a single bolus injection, whilst the other doses were administered using the schedule of Monday, Wednesday, and Friday for two weeks. Prior to initiation of efficacy studies using the combination, tolerability studies were conducted and the results are summarized in Tables 4.1, 4.2, and 4.3. Two dosing strategies were considered. The first was a multidose strategy where both drugs were administered Monday, Wednesday and Friday for two weeks. The second evaluated liposomal mitoxantrone given one time followed by liposomal flavopiridol Monday, Wednesday, and Friday for two weeks. As single agents (Table 4.1), free mitoxantrone, liposomal mitoxantrone, free flavopiridol, and liposomal flavopiridol were tolerated with minimal adverse effects at the doses selected. As summarized in Table 4.2, when given as combinations (at 75% the selected tolerated doses of 84 the individual agents), all combinations caused surprising toxicity. Animals showed signs of severe weight loss, GI toxicity (diarrhea), pain (squinty eyes, orbital tightening), and dehydration (skin tenting) (see Table 4.2). By further lowering the doses of the combinations given in the multi-dose schedule, the toxicity issues were reduced and the animals showed minimal clinical signs (Table 4.3). When considering the second strategy, where liposomal mitoxantrone was given one time followed by multiple doses of liposomal flavopiridol (given at a fixed, well-tolerated, dose of 5 mg/kg), severe toxicity was observed even when the liposomal mitoxantrone dose was reduced 4-fold below the well tolerated dose of 10 mg/kg when given as a single agent in the absence of liposomal flavopiridol (see Table 4.4). For example, when animals were given a single i.v. dose of liposomal mitoxantrone at 2.5 mg/kg and multidose liposomal flavopiridol at 5 mg/kg, 50% of the animals had to be terminated for humane reasons and there was a 6% loss in body weight. The tolerability of the combination was improved when liposomal mitoxantrone was given as a single dose following three doses of liposomal flavopiridol given at 5 mg/kg. These results are summarized in Table 4.5 and this dose was selected for the efficacy study which follows. 85 Table 4.1. Toxicity study of flavopiridol and mitoxantrone as single agents. Tumour-free mice were dosed with free mitoxantrone (Free-M), liposomal mitoxantrone (Lipo-M), free flavopiridol (Free-F), and liposomal flavopiridol (Lipo-F) using the indicated schedules. MWFx2 = Monday, Wednesday, Friday for two weeks. Treatment Dose (Schedule) Nadir Weight Loss Clinical Observations Free-M 10mg/kg (Single bolus) -23% Severe weight loss led to termination of entire group Lipo-M 10mg/kg (Single bolus) -4% Mild weight loss, moderate scruffiness, moderate skin tenting, mild paleness Free-M 1mg/kg (MWF x 2) No weight Loss Moderate scruffiness, mildly swollen tail, mild orbital tightening Lipo-M 2mg/kg (MWF x 2) -11% Mild to moderate weight loss, moderate scruffiness, moderate orbital tightening, mildly swollen tail Free-F 10mg/kg (MWF x 2) -8% Mild to moderate scruffiness, skin tenting, squinty eyes, one mouse euthanized due to high cumulative clinical score Lipo-F 5mg/kg (MWF x 2) No weight loss Mild scruffiness, mild skin tenting, mild hunching 86 Table 4.2. Toxicity study of flavopiridol and mitoxantrone in combinations. Tumour-free mice were dosed with free mitoxantrone (Free-M), liposomal mitoxantrone (Lipo-M), free flavopiridol (Free-F), and liposomal flavopiridol (Lipo-F) in various combinations. The components of the combinations were given at a dose of 75% of the maximum tolerated dose of the respective single agents. Mice were dosed Monday, Wednesday, and Friday for two weeks. Treatment Dose Nadir Weight Loss Clinical Observations Lipo-M + Lipo-F 1.5mg/kg + 3.75mg/kg -9% Moderate to severe weight loss, severe scruffiness, squinty eyes, orbital tightening, hunching, skin tenting, diarrhea, tail ulceration, entire group terminated due to dose-related toxicities Free-M + Free-F 0.75mg/kg + 7.5mg/kg -10% Moderate weight loss, severe diarrhea, hunching, moderate skin tenting, belaboured respiration, entire group terminated due to dose-related toxicities Lipo-M + Free-F 1.5mg/kg + 7.5mg/kg -18% Moderate to severe weight loss, severe diarrhea, hunching, moderate skin tenting, belaboured respiration, entire group terminated due to dose-related toxicities Free-M + Lipo-F 0.75mg/kg + 3.75mg/kg -12% Severe weight loss, severe diarrhea, scruffiness, skin tenting, hunching, squinty eyes, entire group terminated due to dose-related toxicities 87 Table 4.3. Toxicity study of flavopiridol and mitoxantrone in combinations. Tumour-free mice were dosed with free mitoxantrone (Free-M), liposomal mitoxantrone (Lipo-M), free flavopiridol (Free-F), and liposomal flavopiridol (Lipo-F) in various combinations. The components of the combinations were given at a dose of 50% or 25% of the maximum tolerated dose of the respective single agents. Mice were dosed Monday, Wednesday, and Friday for two weeks. Treatment Dose Nadir Weight Loss Clinical Observations Lipo-M + Lipo-F 1.0mg/kg + 2.5mg/kg (50% MTD) No weight loss Mild hunching, mild diarrhea, mild piloerection Free-M + Free-F 0.25mg/kg + 2.5mg/kg (25% MTD) No weight loss Mild hunching, mild hyperactivity Lipo-M + Free-F 0. 5mg/kg + 2.5mg/kg (25% MTD) No weight loss Mild piloerection, mild hyperactivity Free-M + Lipo-F 0.25mg/kg + 1.25mg/kg (25% MTD) No weight loss Mild tail bruising 88 Table 4.4. Toxicity study of liposomal flavopiridol and liposomal mitoxantrone in combination. Tumour-free mice were dosed with combinations of liposomal flavopiridol (Lipo-F) and liposomal mitoxantrone (Lipo-M). Mice were dosed Monday, Wednesday, and Friday for two weeks. On the first day of treatment, liposomal mitoxantrone was administered as a single bolus dose in combination with liposomal flavopiridol. For the rest of the treatment days, only liposomal flavopiridol was administered. Treatment Dose Nadir Weight Loss Clinical Observations Lipo-M + Lipo-F 7.5mg/kg + 5mg/kg -13% Severe weight loss, hyperactivity, moderate hunching, severe piloerection, moderate pale skin, skin tenting, entire group terminated due to dose-related toxicities Lipo-M + Lipo-F 5mg/kg + 5mg/kg -10% Moderate to severe weight loss, severe piloerection, skin tenting, pale skin, moderate hunching, entire group terminated due to dose-related toxicities Lipo-M + Lipo-F 2.5mg/kg + 5mg/kg -6% Moderate weight loss, moderate skin tenting, pale skin, moderate piloerection, moderate hunching, 50% of group terminated due to dose-related toxicities 89 Table 4.5. Toxicity study of liposomal flavopiridol and liposomal mitoxantrone in combination. Tumour-free mice were dosed with combinations of liposomal flavopiridol (Lipo-F) and liposomal mitoxantrone (Lipo-M). Mice were dosed on Days 1, 3, and 5 (post inoculation). Liposomal flavopiridol was administered on Days 1, 3 and 5 and on Day 5, liposomal mitoxantrone was administered in combination with liposomal flavopiridol. Although the combination of liposomal mitoxantrone and liposomal flavopiridol were surprisingly toxic, if significant improvements in efficacy are noted at well-tolerated doses, then the combination of liposomal flavopiridol and liposomal mitoxantrone will be worth pursuing. Initial studies focused on the P388 model and have been summarized in Figure 4.3. In this study liposomal flavopiridol given at 5 mg/kg (every other day for three days; treatment initiated one day after cell inoculation) was well tolerated (Figure 4.3A) with a drug induced weight loss that was equivalent to the saline control animals. However, at this dose, there was an insignificant 10% increase in median life span. This is in contrast to the results obtained with the single dose of liposomal mitoxantrone (10 mg/kg) which resulted in >60% survival 60 days after administration. Liposomal mitoxantrone administered at 10 mg/kg one time exhibited significant therapeutic effects. The combinations of liposomal flavopiridol (5mg/kg) and liposomal mitoxantrone (2.5, 5 and 7.5 mg/kg), where liposomal mitoxantrone was given with the third injection of liposomal flavopiridol resulted in significant therapeutic effects with median survival times of 15, 22, and 25 days for animals given 2.5, 5 and 7.5 mg/kg mitoxantrone with the liposomal flavopiridol (Figure 4.3B). These were all greater than what could be achieved with liposomal flavopiridol alone, but were significantly less than what could be achieved when using the well-tolerated 10 mg/kg dose of liposomal mitoxantrone alone Treatment Dose Nadir Weight Loss Clinical Observations Lipo-F + Lipo-M 5mg/kg + 7.5mg/kg No weight loss Mild piloerection, mild lethargy Lipo-F + Lipo-M 5mg/kg + 5mg/kg -2% Mild piloerection, mild hyperactivity, mild weight loss Lipo-F + Lipo-M 5mg/kg + 2.5mg/kg No weight loss Mild skin tenting, mild to moderate piloerection 90 (Figure 4.3B). The enhanced toxicity of the combination was not offset by significantly enhanced efficacy. Figure 4.3. Efficacy of liposomal flavopiridol and liposomal mitoxantrone in combination given intravenously to mice inoculated with P388 murine leukemia cells. Female BDF-1 mice were inoculated intravenously with P388 murine leukemia cells in a volume of 200µL of 0.5x106 cells/mL. 24 hours post-inoculation, the mice were treated with liposomal flavopiridol and liposomal mitoxantrone alone or in combination. For the single agents, liposomal flavopiridol was dosed at 5mg/kg on Days 1, 3, and 5 and liposomal mitoxantrone was dosed as a single bolus injection at 10mg/kg on Day 1 (A). For the combinations, liposomal flavopiridol was administered on Days 1 and 3 at 5mg/kg and on Day 5, 5mg/kg of liposomal flavopiridol was administered with liposomal mitoxantrone at 7.5, 5, and 2.5mg/kg (B). Percent survival, as defined by when the animals had to be terminated for humane reasons as defined by the IACC animal care protocol) was also plotted for each of the indicated treatment and control groups. 91 Since liposomal flavopiridol exhibited minimal therapeutic effects in the P388 model, combination treatments were also completed using the MOLM-13 model. This model exhibited significant benefits when treated with liposomal flavopiridol as a single agent (see Chapter 3). As illustrated in the results described in Table 4.3, the two drugs could be combined and tolerated, but the doses of the drugs required significant reductions, consistent with the fact that the combination was significantly more toxic than what would have been predicted on the basis of single agent activity. As shown in Figure 4.4B, at a tolerated dose of liposomal flavopiridol (2.5 mg/kg) and liposomal mitoxantrone (1 mg/kg), animals with established MOLM-13 tumours did not show significant tumour development until day 23/24, while control (saline treated) animals exhibited significant tumour growth at day 11/12. While effective, the results obtained with the tolerated combination were significantly less than what could be achieved with liposomal flavopiridol given as a single agent at the well tolerated dose of 5 mg/kg (Figure 4.4A). Similar to results obtained in the P388 model, the increase in toxicity was not offset by a significant increase in efficacy. 92 Figure 4.4. Efficacy of liposomal and free flavopiridol and mitoxantrone given intravenously to mice with established MOLM-13 tumours. Female NRG mice were inoculated subcutaneously with MOLM-13 acute myeloid leukemia cells with 50µL of 0.5x106 cells/mL. Tumour-bearing mice were then injected intravenously with liposomal and free flavopiridol and mitoxantrone formulations alone (A) or in various combinations (B) every Monday, Wednesday, and Friday for two weeks. Mean tumour volumes were measured three times per week and the results have been plotted as mean ± SEM. 93 4.4 Discussion Flavopiridol, or Alovocidib, is a pan-cyclin dependent kinase inhibitor that has shown clinical promise as a component of a combination regiment called FLAM for the treatment of acute myeloid leukemia (239, 241). Despite promising results from phase II clinical trial, the FLAM regimen failed to show improved overall survival when compared to the conventional “7+3” regimen (239). Through the use of nano-scaled drug delivery systems, it is possible that the therapeutic benefits expected in the FLAM regimen may be obtained and in the current study, flavopiridol/mitoxantrone combinations have been characterized. Flavopiridol has been shown to potentiate other chemotherapeutic agents in preclinical studies. In separate studies, flavopiridol enhanced the effects of docetaxel and doxorubicin in human gastric cancer cells and advanced sarcomas, respectively (256, 257). These benefits were achieved when using sequential dosing (256, 257). Docetaxel and doxorubicin both exert maximum cytotoxic effects on cells in S phase as docetaxel binds microtubules and is cell cycle specific, while doxorubicin is a DNA poison that binds DNA and inhibits topoisomerase II (258, 259). As such, the selection of cells into the S phase by flavopiridol was believed to improve the therapeutic activity of these drugs (260). This synchronization of cells to the S phase by flavopiridol was the basis of the FLAM regiment (238). In addition to these published results, we were able to demonstrate a synergistic relationship between flavopiridol and mitoxantrone, which is another S phase specific cytotoxic agent, through in vitro models of AML (Figure 4.1). In contrast to the previous studies, synergy was achieved without the use of a sequential schedule. Instead, the AML cells in culture were exposed to flavopiridol and mitoxantrone concomitantly for 72 hrs. This finding highlighted the fact that the effects of these drugs in combination were not ratio-dependent and suggested that this synergy could be achieved when using formulations that prolong drug exposure time. By demonstrating that concurrent administration and long exposure of the combinatory agents can result in synergy, the in vitro cytotoxicity data laid the groundwork for the development of “enFlaM”, a combination product consisting of liposomal flavopiridol and liposomal mitoxantrone. 94 Although the idea of achieving synergy was supported by the in vitro studies shown here, the potential to capture this synergy using liposomal formulations of flavopiridol and mitoxantrone was negated by a surprising, likely synergistic, toxicity. As shown in Tables 4.1 and 4.2, the simultaneous administration of liposomal flavopiridol and liposomal mitoxantrone on a multi-dose schedule generated dose-limiting toxicities that were not expected given the toxicity of the single agents. This enhanced toxicity would not have been a concern if the therapeutic effects were greater than what could be achieved with the single agents at well-tolerated does. This, however, was not the case and the therapeutic benefits that could be achieved with tolerated combinations were substantially less than what could be achieved with the single agents (see Figures 4.3 and 4.4). Despite that, the goal of combination therapy is to augment therapeutic efficacy by using multiple agents, it is not unprecedented that drugs in combinations could show more toxicity than single agents. In separate previous studies in different disease models, combination therapy generated greater toxicity when compared to the respective single-drug (261-264). Mitoxantrone formulations, liposomal and free, were administered in a single bolus dose when they were investigated back in 1997 by Lim et al. (253). A multi-dose liposomal mitoxantrone formulation could be tolerated as well as the single-bolus administration but it was not that effective in the MOLM-13 model (Figure 4.2). The therapeutic effects obtained may have been compromised by the models selected for these studies. While liposomal mitoxantrone exhibited significant activity in the P388 model, liposomal flavopiridol did not. Conversely, liposomal flavopiridol exhibited significant activity in the MOLM-13 model, but liposomal mitoxantrone did not (see Figure 4.4). In both models, the drugs had to be administered soon after tumour cell administration because the tumour grew so quickly (control animals had to be terminated in 9-12 days after tumour cell administration). As described in the Results, the current versions of liposomal and free mitoxantrone were less effective than what was reported previously. In Lim et al.‘s 1997 study, the rodents were housed in a “dirty”, open facility. In contrast, the rodents in the current study were housed in a Specified Pathogen Free barrier facility, where the allowable pathogens are strictly enforced. Compelling evidence has shown the microbiome of the animals can impact treatment response (265), particularly in the syngeneic immune competent models, such as the P388 model used here. 95 The combination studies reported here clearly caution the development of enFlaM as a combination product. It appears, based on murine models, to be more toxic and less effective than what could be achieved with the single agents given at their maximum tolerated doses. These results help to identify the challenges in the development of combination products. In order to attenuate dose-limiting toxicities as well as maximize the therapeutic efficacy of the enFlaM combination proposed here, the dosing schedule of the combination was modulated such that each agent would be administered using the most optimal scheduling established as single agents. As seen in Table 4.4, despite changing the liposomal mitoxantrone to single bolus administration, the dose-limiting toxicities prevented administration of a combination dose that would be better than the single agents. Even by adjusting the dosing schedule such that liposomal mitoxantrone was administered on the last day of treatment instead of the first day, where toxicity issues were better managed (see Table 4.5), the therapeutic effects were less than what could be achieved with single agent liposomal mitoxantrone. This may have been due to the fact that liposomal flavopiridol was not effective in the P388 model and that the liposomal mitoxantrone was given to animals at a time point where the tumour burden was substantially greater than what would have been present 24 hours after tumour cell inoculation. High disease burden is often associated with poor treatment outcomes and prognosis (266). This could explain the decreased effectiveness of the combination highlighted in Figure 4.3 when compared to liposomal mitoxantrone as a single agent. More speculatively, the present studies used what was defined as the optimal liposomal mitoxantrone formulation: the DMPC/Chol formulation that exhibited faster drug release than a less effective formulation prepared with DSPC/Chol liposomes. In the context of the approach defined here, the reduced therapeutic effects achieved using the DSPC/Chol formulation which slowly released mitoxantrone may have been improved substantially when compared to the DMPC/Chol formulation, which release the drug more rapidly thereby promoting toxicity and limiting efficacy. As such, further modifications to the liposomal mitoxantrone formulation used would need to be done to determine how to better define an enFlaM combination product. 96 4.5 Conclusions In summary, the combination of flavopiridol and mitoxantrone generated dose-limiting toxicities in vivo that hindered the development of enflaM as a combination product. Through the various studies, the importance of dosing schedule and sequence of administration to the overall toxicity profile and efficacy of enFlaM has been highlighted. The focus is now on evaluating a different formulation of mitoxantrone and to continue modulating the doses, schedules, and sequence of administration to determine an improved method to administer an enFlaM product that achieves therapeutic benefits in multiple murine models of AML. 97 5. Summarizing discussion 5.1 Summary of results In Chapter 2, liposomal formulations of quercetin were developed by exploiting quercetin’s ability to bind copper. Quercetin powder was added directly to pre-formed copper-containing liposomes (2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol (CHOL) (55:45 molar ratio)). Using this methodology, solutions containing quercetin at concentrations of >5mg/mL were attained, representing at least a >100-fold increase in apparent solubility. The resulting formulation was suitable for intravenous dosing with no overt toxicities when administered at doses of 50mg/kg in mice. Pharmacokinetic studies demonstrated that the copper-quercetin formulations had an AUC0-24H of 8382.1µg*hr/mL when administered to mice at 50mg/kg (see Figure 2.8). The resulting formulation is suitable for further development and also serves as a proof-of-concept for formulating other flavonoids and flavonoid-like compounds. Given that quercetin exhibits an IC50 of >10µM when tested against cancer cell lines, there was not sufficient justifications to move the formulation forward to further in vivo development as a single agent. Quercetin’s value may be best realized as part of a combination treatment as discussed in Chapter 2 Given quercetin’s general lack of therapeutic potential as a single agent, the approach developed for formulating quercetin was explored to reformulation of another water-insoluble flavonoid called flavopiridol (Chapter 3). Flavopiridol is defined as an aqueous-insoluble compound (< 1mg/ml in water) and some have argued that its therapeutic potential could be limited due to its affinity to serum proteins (See Chapter 3). Binding of serum proteins such as albumin, lipoproteins, and globulins is common for most drugs and these bindings affect the pharmacokinetics and pharmacodynamics of these drugs (267, 268). Additionally, protein-bound drugs are hindered from extravasating into extravascular space, which could negatively impact the drugs’ therapeutic effects (267). As such, in order to remedy these aforementioned therapeutic limitations, the research in Chapter 3 evaluated whether the copper-binding capabilities of flavopiridol could be used to prepare an injectable formulation that would exhibit enhanced exposure and improved efficacy. Similar to quercetin, liposomal loading of 98 flavopiridol relied on a powder-based method where flavopiridol powder was added directly to preformed copper-containing liposomes (DSPC:Chol or DSPC:DSPE-PEG2000). Pharmacokinetics and efficacy studies were then conducted with tumour-free and tumour-bearing mice, respectively. The liposomal flavopiridol formulations were well-tolerated in mice following i.v. administration at a dose of 5mg/kg with no apparent acute or chronic toxicities. In vivo pharmacokinetics of the optimized DSPC/DSPE-PEG2000 liposomal flavopiridol formulation demonstrated a 30-fold increase in AUC (0.804µg-hr/mL versus 26.92µg-hr/mL) compared to the free flavopiridol formulation (see Figure 3.5). The resultant liposomal formulation also demonstrated significant therapeutic activity in MV4-11 and MOLM-13 subcutaneous AML models (see Figure 3.7). MV4-11 and MOLM-13 AML models were selected based on their similarities on the genetic level as both cell lines are known to carry FLT3-internal tandem duplication mutations (269). Although an improved formulation of flavopiridol has significant therapeutic potential as a single agent, the therapeutic potential of flavopiridol as a component of a combination regimen called FLAM (sequential administration of flavopiridol, cytarabine, and mitoxantrone) inspired the development of a combination product comprising flavopiridol and mitoxantrone. In vitro cytotoxicity data showed significant synergistic activity when flavopiridol and mitoxantrone were used in combination against AML cell lines (MV4-11 and MOLM-13). Subsequently, the combination of liposomal flavopiridol and liposomal mitoxantrone (referred to as enFlaM) was examined in vivo. Surprisingly, when the two test agents were administered concurrently using a multi-dose schedule, there was significant toxicities that severely limited the dose of the combination product that could be administered. At tolerated, albeit reduced doses, there were therapeutic effects (see Chapter 4). However, at the MTDs of the single agents, better antitumour effects were observed with the enFlaM combination. For this reason, instead of administering both agents on a multi-dose schedule, enFlaM uses liposomal flavopiridol on a multi-dose schedule and liposomal mitoxantrone as a single bolus injection (see Figure 4.4). As shown in Chapter 4, this treatment was also associated with significant toxicities, which suggested that the EnFlaM combination would not be useful in the context of intravenous administration for the treatment of AML. This finding may have been due to the fact that liposomal flavopiridol and liposomal mitoxantrone were not effective in the P388 and 99 MOLM-13 models respectively. The lack of activity of the single agents in the respective models likely accounted for the decreased effectiveness of the combination highlighted in Figures 4.3 and 4.4. The differences in activity of flavopiridol and mitoxantrone in different cell lines are likely the result of varying tissue origin and genetic profiles between the cell lines that were used. Continued efforts will be spent to examine other AML cell lines that would best illustrate and highlight the synergism between flavopiridol and mitoxantrone in an in vivo setting. The enFlaM concept highlighted the challenges of developing combination therapy. In the sections below, the challenges in the development of experimental drug combination in the context of cancer as well as opportunities that are inspired by enFlaM are discussed. 5.2 Nanoparticle-based combination therapy – the road ahead As discussed in the Introduction, the genetic complexity and heterogeneity of AML has limited the effectiveness of an array of treatment options (10). As such, the use of multi-agent or combination therapy allows for the modulation of different mechanistic pathways in cancer cells, thereby maximizing the therapeutic benefits of the treatment regimen (270). Advancements in nanotechnology, like the use of liposomes, have opened up opportunities for new combination strategies. In addition to the previously described Vyxeos (see Chapter 1), various other nanoparticle drug delivery platforms have shed new light on the potential of nanoparticle-based combination therapy. In 2000, Soma et al. loaded a poly(alkylcyanoacrylate) (PACA) nanoparticle systems with doxorubicin and a p-glycoprotein inhibitor, cyclosporin A for the treatment of doxorubicin-resistant leukemia in vitro (271). The co-encapsulation of cyclosporine A and doxorubicin resulted in nearly a 2-fold improvement in cytotoxicity compared to cells treated with doxorubicin-only nanoparticles (271). In another study, van Vlerken et al. prepared a polymeric micelle formulation to co-deliver exogenous ceramide and paclitaxel to treat a paclitaxel-resistant cell line (SKOV-3TR) (272). The combination sensitized the resistant cells to the same level as the non-resistant variants (272). In 2008, Saad et al. prepared cationic liposomes for the co-delivery of MRP1 (a multi-drug resistance (MDR)-associated protein) and Bcl2 (an anti-apoptotic protein) siRNA in combination with doxorubicin to treat multi-drug resistant breast cancer, lung cancer, colon cancer, and ovarian cancer (273). 100 The formulation was able to mitigate the MDR effects through inhibition of drug efflux activity and promotion of apoptosis (273). 5.3 Challenges of developing combination therapy The success of these platforms reiterated the robustness of nanoparticles in the context of combination therapy and served as cornerstones for new nanoparticle-based therapies such as the enFlaM combination product considered in Chapter 4. However, the development of combination products and the road to clinical trials and approval are quite treacherous. Despite the evidence for improved benefits when anticancer agents are combined, the majority of anticancer drugs are developed as single agents (274). When considering single agents, between 1993 and 2002, roughly 90% of the oncology drugs that entered the clinical testing pipeline did not meet clinical trial endpoints and/or did not receive approval through the US Food and Drug Administration (FDA) (275). This is not unexpected, since the power of most therapeutics to treat cancer often requires their use in combination and activity of the single agent is often limited. With better efficacy and treatment outcomes expected, combination therapy can potentially improve on that dismal approval rate. However, the barriers preventing the development of combination products remain and include: (i) the desire to demonstrate activity of each as a single agent (a cost prohibitive requirement), (ii) the loss of control of dosing normally exercised by the treating physician who will want to dose- reduce one agent, but not the other, and (iii) the CMC (Chemistry Manufacturing and Control) requirements when developing carriers particularly when using two or more active ingredients. For a regimen like enFlaM, each component of the combination (liposomal flavopiridol and liposomal mitoxantrone) would need to be approved separately as single agents. For this reason, combination strategies will always be easier to develop if one of the components is already approved for use as a single agent (274). The overall process of taking a drug from preclinical development to approval by the FDA can take around 8 to 12 years (276). As such, it would be around 10 years before EnFlaM can even enter the clinical approval process. In addition to the time component, there are also economic barriers. The cost of developing a 101 cancer drug from discovery to approval would cost around $600-800 million USD (277-279). To exacerbate this problem, the cost is continuing to rise at a rate of 7.4% above overall price inflation (279). Thus, there is a significant financial hurdle that needs to be cleared before enFlaM can reach a stage where it can be reviewed by regulatory bodies. On top of regulatory and financial barriers, legal issues can also impede the development of enFlaM. For a formulation like enFlaM, where the therapeutic agents come from different sources, intellectual properties and the struggle to define who owns the rights to invention can further hinder and delay the development of enFlaM. In the case of this combination, both drugs being considered are generic, but flavopiridol has orphan drug status for treatment of elderly patients with AML: a status earned by Tolero, the company developing the drug. It is believed, however, that if a combination product exhibits significant improvements in therapeutic activity at well tolerated doses the hurdles will be reduced. Although the in vitro data (3-day cytotoxicity data shown in Chapter 4) provide compelling evidence of therapeutic synergy, the in vivo results demonstrate that the synergy is also observed from a toxicity perspective. The results suggest that an enFlaM combination product is likely not worth pursuing. This does not mean that combinations of liposomal mitoxantrone and liposomal flavopiridol will not be very effective when given in an appropriate sequence; but this would require each agent to be approved as a single product, which would be a challenge. Despite the challenges, the liposomal flavopiridol formulation described in Chapter 4 is still worth developing as a single agent. The formulation described is for copper flavopiridol, which is chemically distinct from the parent compound and therefore it can be protected through intellectual property law (IP). The agent is a pan-CDK inhibitor and there is a great deal of interest in the development of pan CDK inhibitors in general (217). Finally, both liposomal mitoxantrone and liposomal flavopiridol have a potential unique way of killing cancer cells: a cell death process that engages the immune system. The potential of these agents to trigger immunotherapeutic activity is novel and the immunotherapeutic landscape of AML is considered below. 102 5.4 Small molecule immunotherapy for treatment of AML Advances in cancer immunotherapy over the past decade have revolutionized treatment strategies for both solid tumours and hematologic malignancies (280, 281). Since 2011, six immune checkpoint inhibitors (ICPIs) have been approved by the FDA to treat various solid tumours including metastatic melanoma and advanced non-small cell lung cancer (NSCLC): illnesses that were considered untreatable at diagnosis until recent years (280). ICPIs have been associated with promising and durable responses extending survival, for the first time, for months or even years in patient populations that would otherwise succumb to their diseases within a year’s time. The use of genetically modified lymphocytes, specifically chimeric antigen receptor (CAR) T cells, as an alternative, has achieved great successes in the treatment of blood cancers, primarily B-cell malignancies such as B-cell acute lymphoblastic leukemia (ALL), chronic lymphoblastic leukemia (CLL), and non-Hodgkin’s lymphoma (50, 281, 282). The different immunotherapies that have been considered for treating AML are described in the following sections and summarized in Figure 5.1 below, but would note that these therapies are expensive costing patients or health care providers $100 to $500K. While reviewing these therapeutic strategies, the potential to use small molecules to achieve immunotherapeutic effects are discussed as a better, less expensive, and potentially more effective alternatives. 103 Figure 5.1. Immunotherapy strategies for AML treatment 5.4.1 Hematopoietic stem cell transplantation (HSCT) A small population of poor-risk AML patients may be given the option for allogeneic hematopoietic stem cell transplantation (HSCT) based on factors such as comorbidities, age, and availability of donors (283, 284). HSCT is one of the oldest and most successful immunotherapeutic approaches in the treatment of AML, which involves ablation of the bone marrow using radiation or chemotherapy followed by re-implantation of stem cells from the host (autologous) or a donor (allogeneic) (50). Unfortunately, the majority of elderly patients are not eligible for HSCT, which is the last curative option for r/r patients (50, 285). Even after HSCT, 40-60% patients develop graft-versus-host disease (GVHD) and approximately 20-50% 104 patients invariably relapse and are left with very limited treatment options, underscoring the importance of developing novel immunotherapeutic approaches to improve this dire scenario (286, 287). In recent years, efforts are being placed on optimising HSCT procedures, some of which include the use of reduced-intensity conditioning or myeloablative conditioning prior to HSCT (NCT02626715 (National Clinical Trial Identifier)), infusion of donor natural killer cells (NCT03300492), and prophylactic use of IL-2 in combination with HSCT (NCT01517347), all of which are designed to lower the incidence of GVHD, reduce toxicities, extend the application of HSCT to elderly and frail patients, and/or to prevent relapse (288-290). 5.4.2 Antibody-drug conjugates Novel, non-transplant immunotherapeutic modalities are also being heavily explored for AML. In addition to the CD33-targeting agent, Mylotarg™, which was discussed in Chapter 1 above, Vadastuximab talirine (SGN-CD33A) is another antibody-drug conjugate (ADC) that has been tested in human clinical trials in recent years. While results from phase I study were promising, a phase III trial evaluating the combination of SGN-CD33A with the hypomethylating agents azacitidine or decitabine in newly diagnosed elderly AML patients was discontinued by Seattle Genetics in 2017 due to increased treatment-related mortality (291, 292). CD123 is another AML-target of interest particularly due to its more restricted expression on normal hematopoietic stem cells, suggesting the potential to reduce “on-target off-leukemia toxicities”(47, 50, 293, 294). To date, talacotuzumab (NCT02472145) and SGN-CD123A (NCT02848248) have been tested in human clinical trials, of which both were halted in development due to unfavourable risks/benefits profiles. Taken together, additional studies are warranted to validate the feasibility and utility of targeting CD123 in the treatment of AML. 5.4.3 AML vaccines Vaccination is an attractive strategy for patients who are not eligible for HSCT or who relapse following HSCT. To date, three main types of vaccines are being tested in humans with AML: peptide vaccines, granulocyte macrophage colony stimulating factor (GM-CSF), and dendritic cell (DC) vaccines. Peptide vaccines involve the use of AML-associated antigens to stimulate a 105 cytotoxic T cell response. Two common targets are Wilms tumor 1 (WT1) and PR1 (peptide derived from proteinase 3), both of which are overexpressed in AML. Recent results from a phase II clinical trial investigating the multivalent WT1 peptide vaccine galinpepimut-S (NCT01266083) suggest that the peptide vaccine is tolerated and may contribute to increase in overall survival (295). On the other hand, a PR1 peptide vaccine has been shown to induce antigen-specific immune responses in patients based on a Phase I/II trial (NCT00004918) (296, 297). However, it is unclear when and how the vaccine should be used to produce optimal treatment efficacy (297). Another vaccination strategy is the use of gene-transduced tumor cell vaccine (GAVX) where cells are modified to express GM-CSF to recruit DCs to the site of intradermal vaccination to elicit an anti-tumour immune response (298). A phase II trial (NCT01773395) is being conducted to evaluate the efficacy of GVAX following allogeneic HSCT. Dendritic cell vaccination, which has the potential of presenting multiple tumour-derived antigens, is of particular interest for the treatment of AML due to the poor immunogenic nature of the disease (50, 299). DCP-001 (DCPrime), a vaccine developed through the differentiation of the AML cell line DCOne into mature DCs, is currently recruiting for a phase II clinical trial (NCT03697707) after demonstrating both safety and induction of immune responses in elderly AML patients in its phase I study (NCT01373515) (300). This strategy was designed to replace conventional DC vaccination approaches which involve administration of mature DCs that were isolated from the patients and matured ex vivo: an approach that is known to be costly, cumbersome, and logistically complex (26). Another DC-based treatment being tested in humans involves the vaccination with a hybridoma consisting of patient-derived AML cells fused with autologous DCs (301). In that study, 12/17 patients who had achieved remission experienced no recurrence after 57 months and showed expansion of disease-specific T cells which may help protect against relapse (301). This vaccination approach is being evaluated in combination with PD-1 blockade in a phase II clinical trial (NCT01096602). 5.4.4 Chimeric antigen receptor T (CAR-T) cell therapy CAR-T therapy has been greatly successful in the treatment of some hematologic malignancies but this accomplishment has yet to transfer to AML. Briefly, CAR-T cells are patient-derived T cells that have been genetically modified to recognize antigens expressed on the cancer cell’s 106 surface (302). The development of CAR-T therapy for AML, however, has been much more challenging compared to that for B-cell malignancies due to poor immunogenicity stemming from low mutation burden, the lack of AML-specific antigens leading to the risk of generating on-target off-leukemia toxicities, and the heterogeneous biology of the disease arising from various myeloid progenitors (50, 282, 302, 303). Numerous targets are being explored in the preclinical and/or clinical development of CAR-T treatments for AML including CD33, CD123, FLT3(CD135), CD7, NKG2D, CD133, FRβ, LeY, and CLL-1(302, 304-311). While CAR-T cells are attractive due to their demonstrated success in other hematologic malignancies, CAR Natural Killer (NK) cells are also being investigated for AML as they are believed to be associated with lower risks of generating severe clinical toxicities such as cytokine release syndrome (312). 5.4.5 Immune checkpoint inhibitors (ICPIs) ICPIs have been associated with remarkable treatment outcomes in various solid tumours including NSCLC and melanoma (50). ICPIs involve the removal of immunosuppressive signals that are often used as a mechanism by cancer cells to evade immune detection. Although no ICPI has been granted approval for the treatment of AML, over 30 clinical trials are underway to evaluate the efficacy of clinically approved ICPIs as single agents or in combinations for newly diagnosed patients as well as post-remission and r/r AML patients. The role of ICPIs in AML will become clearer when results become available from these clinical trials. 5.4.6 Small molecules selected to achieve immunogenic cell death and the immunotherapeutic potential of liposomal flavopiridol and/or liposomal mitoxantrone Immunogenic cell death (ICD) has emerged in recent years as a popular immunotherapeutic concept that is being investigated pre-clinically and in early clinical trials. ICD is a form of cell death wherein cancer cells, in the process of treatment-induced death, emit certain molecular signals in a specific spatiotemporal pattern that result in the recruitment of immune cells, presentation of tumour-specific antigens, and activation of an adaptive immune response that consequently leads to tumour eradication and generation of immunological memory against future re-challenges (313-315). This “therapeutic vaccination” approach has garnered much 107 attention as it has been shown that several existing chemotherapeutics are capable of inducing ICD, characterized by cell surface expression of calreticulin (CRT) and secretion of ATP and mobilization of HMGB1 (313, 316, 317). Traditionally, chemotherapeutics are tested in immune-compromised pre-clinical models to ensure that the agents exert cytotoxic and not immunogenic effects. It is now known that certain chemotherapeutics (e.g. doxorubicin, oxaliplatin, mitoxantrone) induce ICD as a secondary mechanism, providing long-term immune protection that could only be observed in immune competent syngeneic models of cancer (315, 316, 318-320). While ample evidence suggest that ICD induction may provide cures and vaccination in pre-clinical models, evidence of ICD in humans are still being explored. Currently, over 20 clinical trials are investigating the immunological profile of existing chemotherapeutic regimens in various types of cancer (319). In a recent report, CRT exposure on leukemic blast cells has been correlated with increased AML-specific immune response, superior relapse-free survival and overall survival, suggesting that ICD-associated markers may be clinically relevant in the AML setting (321). While ICD has not been widely considered as an AML treatment strategy, it is important to note that anthracyclines such as doxorubicin and daunorubicin are used as SOC chemotherapy for AML patients while mitoxantrone, also a known bona fide ICD inducer, is an approved chemotherapeutic for r/r AML patients. Currently, there are over 40 trials involving the use of mitoxantrone to treat AML, suggesting that there are great interests in determining how to best use this potent chemotherapeutic. Additionally, a recent paper by Goel et al, highlighted that cyclin-dependent kinase (CDK) 4/6 inhibitors did not only induce tumour cell cycle arrest, but also promoted anti-tumour immunity (322). As a well-established CDK inhibitor, flavopiridol could very well have similar immunity-inducing properties. As a result, the use of EnFlaM as an immune-based treatment may prove to be an effective therapeutic strategy. However, this would have to be further tested and validated in immune-competent cancer models. 5.5 Conclusions and future perspectives The standard of care for AML has remained almost unchanged for more than 40 years. Only recently in 2017 and 2018, a series of new treatments that provide therapeutic benefits especially to patients of older age or those who have recurrent disease have been approved for 108 clinical use. The emergence of these treatments, namely midostaurin, enasidenib, Vyxeos®, gemtuzumab ozogamicin, venetoclax, and glasdegib, provide new hopes for patients in the r/r setting. Through the lessons learned from reviewing the clinical challenges of AML and the breakthroughs with recently approved treatments, it is expected that the most effective treatments, as in the current standard of care of AML and other cancers, would rely on multi-modal therapeutics comprising combinations of chemotherapy, targeted therapy, and/or immunotherapeutic modalities. It is therefore anticipated that many more clinical trials will be designed to focus on combining two or more drugs with immunotherapeutic agents. For instance, there are clinical trials combining various immunotherapeutic strategies with standard treatments (e.g. NCT03286114) and exploring combinations of different ICPIs (e.g. NCT02397720). The FLAM regimen, which is based on timed sequential therapy, relies on flavopiridol, mitoxantrone, and cytarabine, has shown favourable outcomes with remarkable improvements in complete remission rates in AML. However, the FLAM regimen has some limitations and failed to improve overall survival. Thus, this thesis research proposed the use of nanomedicine to circumvent these limitations by introducing enFlaM. “EnFlaM” consists of a liposomal combination product of mitoxantrone and flavopiridol that can potentially improve the pharmacokinetics and biodistribution and increase the therapeutic efficacy of the drugs. However, as described in the thesis, despite significant therapeutic effects achieved with the liposomal formulations as single agents, the implementation of liposomal mitoxantrone and liposomal flavopiridol as enFlaM engendered synergistic toxicities. Further modifications to doses and schedules would be necessary to devise a combination scheme that would be optimal for the administration of enFlaM, and sequencing would be the key to improving therapeutic results as it is unlikely that enFlaM will be developed as a single combination product. This may, however, change if the combination is to be considered as an immunotherapeutic treatment where optimal results may not require the use of systemic administration. In this case, regional or intratumour administration may be required. With the development of novel immunotherapeutics, a plethora of clinical trials are being conducted to improve the prognosis 109 of AML patients and to overcome the inherent challenge of activating an anticancer immune response against a heterogeneous disease that is poorly immunogenic. However, AML still presents a number of clinical challenges and novel therapeutic strategies are necessary to provide additional treatment options for patients with high-risk disease. With novel nanoformulations such as Vyxeos® becoming standard treatment for subpopulations of AML patients and the potential of nanoparticles to enhance immunotherapeutic effects, it will be of great interests to evaluate the immunotherapeutic potential of enFlaM and perhaps explore combinations of enFlaM with approved immunotherapeutic treatments to determine whether or not improved immunotherapeutic effects and extended survival can be achieved. 110 6. References 1. Estey E, Dohner H. Acute myeloid leukaemia. Lancet. 2006;368(9550):1894-907. 2. Lowenberg B, Downing JR, Burnett A. Acute myeloid leukemia. The New England journal of medicine. 1999;341(14):1051-62. 3. Kumar CC. Genetic abnormalities and challenges in the treatment of acute myeloid leukemia. Genes & cancer. 2011;2(2):95-107. 4. 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Thesis/Dissertation
eng
Interdisciplinary Oncology
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Development of copper-flavonoid complexes for treatment of acute myeloid leukemia (AML)
Text